B cell immunotherapy

文档序号：277133 发布日期：2021-11-19 浏览：7次中文

阅读说明：本技术 B细胞免疫疗法 (B cell immunotherapy ) 是由马克·C·波兹南斯基鲁克桑德拉·F·西尔布列斯库于 2020-01-23 设计创作，主要内容包括：通常,本发明的特征在于一种治疗有需要的对象(例如人)中的神经退行性疾病(例如肌萎缩侧索硬化)或创伤性脑损伤的方法,所述方法包括向所述对象施用治疗有效量的分离的B细胞(例如自体或同种异体或异种B细胞)。(In general, the invention features a method of treating a neurodegenerative disease (e.g., amyotrophic lateral sclerosis) or traumatic brain injury in a subject (e.g., a human) in need thereof, the method comprising administering to the subject a therapeutically effective amount of isolated B cells (e.g., autologous or allogeneic or xenogeneic B cells).)

1. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of isolated B cells.

2. The method of claim 1, wherein the neurodegenerative disease is selected from Amyotrophic Lateral Sclerosis (ALS).

3. The method of claim 1, wherein allogeneic B cells are administered.

4. The method of claim 1, wherein autologous B cells are administered.

5. The method of claim 1, wherein xenogenic B cells are administered.

6. The method of claim 1, further comprising: a second therapeutic composition is administered.

7. The method of claim 5, wherein the second therapeutic composition is edaravone or riluzole.

8. The method of claim 6, wherein the second therapeutic composition is an immunomodulatory composition.

9. The method of claim 1, wherein the B cells are mature naive B cells.

10. The method of claim 1, wherein the B cells are stimulated ex vivo.

11. The method of claim 1, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist.

12. The method of claim 1, wherein the B cell is B_regA cell.

13. The method of claim 11, wherein said B_regThe cells express the immunomodulatory cytokine IL-10.

14. The method of claim 11, wherein said B_regThe cells comprise at least 80% CD19+ B cells.

15. The method of claim 11, wherein said B_regThe cells contained less than 10% CD138+ plasma B cells.

16. The method of claim 1, wherein the B cells are formulated for topical administration.

17. The method of claim 1, wherein the B cells are formulated for systemic administration.

18. The method of claim 1, wherein the B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration.

19. The method of claim 1, wherein the B cells are administered once a day, once a week, twice a week, once every 14 days, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or once a year.

20. The method of claim 1, wherein the therapeutically effective amount comprises at least 0.5x 10 per administration⁷And (4) B cells.

21. The method of claim 1, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁸And (4) B cells.

22. The method of claim 1, wherein the therapeutically effective amount comprises at least 2x 10 per administration⁸And (4) B cells.

23. The method of claim 1, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁹And (4) B cells.

24. A method of treating a subject having traumatic brain injury, TBI, comprising administering to the subject a therapeutically effective amount of isolated B cells.

25. The method of claim 22, wherein TBI is caused by a head injury or cerebral contusion.

26. The method of claim 22, wherein the subject suffers from one or more of a plurality of physical, cognitive, social, emotional, and/or behavioral disorders.

27. The method of claim 22, wherein allogeneic B cells are administered.

28. The method of claim 22, wherein autologous B cells are administered.

29. The method of claim 22, wherein xenogenic B cells are administered.

30. The method of claim 22, further comprising administering a second therapeutic composition.

31. The method of claim 22, wherein the second therapeutic composition is an antibiotic or a corticosteroid.

32. The method of claim 22, wherein the B cell is a mature naive B cell.

33. The method of claim 22, wherein the B cells are stimulated ex vivo.

34. The method of claim 22, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist.

35. The method of claim 22, wherein the B cell is B_regA cell.

36. The method of claim 32, wherein B is_regThe cells express the immunomodulatory cytokine IL-10.

37. The method of claim 32, wherein B is_regThe cell comprises at least80% CD19+ B cells.

38. The method of claim 32, wherein the B cells comprise less than 10% CD138+ plasma B cells.

39. The method of claim 22, wherein the B cells are formulated for topical administration.

40. The method of claim 22, wherein the B cells are formulated for systemic administration.

41. The method of claim 22, wherein the B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration.

42. The method of claim 22, wherein the B cells are formulated for administration via an intracranial pressure (ICP) monitoring catheter.

43. The method of claim 22, wherein the B cells are administered once a day, once a week, twice a week, once every 14 days, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or once a year.

44. The method of claim 22, wherein the therapeutically effective amount comprises at least 0.5x 10 per administration⁷And (4) B cells.

45. The method of claim 22, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁸And (4) B cells.

46. The method of claim 22, wherein the therapeutically effective amount comprises at least 2x 10 per administration⁸And (4) B cells.

47. The method of claim 22The method, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁹And (4) B cells.

48. The method of any one of the preceding claims, wherein the subject is a human.

Background

Degenerative diseases are medical conditions that lead to the degeneration of cells, tissues or organs. For example, Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), Alzheimer's Disease (AD), and Huntington's Disease (HD) belong to a number of neurodegenerative diseases that involve degeneration of central nervous system regions. Traumatic Brain Injury (TBI) is also an example of a disorder that may increase the risk of developing degenerative brain diseases such as PD or AD. Rheumatoid arthritis and osteoarthritis remain other examples of degenerative diseases involving inflammation. For most of these, no effective treatment is available. Treatments for some of these diseases or disorders are under investigation. However, the suggested treatments are often very expensive and involve significant risks and complications. Thus, there is a need for better methods of treating degenerative diseases, including neurodegenerative diseases, such as ALS and TBI.

Disclosure of Invention

Provided herein are compositions comprising B cells (e.g., isolated, purified, or modified B cells, or a combination thereof) and their use for treating diseases (e.g., neurodegenerative diseases, Traumatic Brain Injury (TBI), Spinal Cord Injury (SCI), and inflammatory and immune diseases as described herein).

In a first aspect, the invention features a method of treating a neurodegenerative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an isolated B cell.

In some embodiments, the neurodegenerative disease is selected from Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, alzheimer's disease, Chronic Traumatic Encephalopathy (CTE), frontotemporal dementia, huntington's disease, infantile axonal dystrophy, progressive supranuclear palsy, lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease. In a preferred embodiment, the neurodegenerative disease is ALS (e.g., sporadic or familial ALS). In yet another preferred embodiment, the neurodegenerative disease is parkinson's disease.

In another aspect, the invention features a method of treating a subject with ALS, the method including administering to the subject a therapeutically effective amount of B cells, where the therapeutically effective amount is an amount sufficient to reduce or ameliorate one or more symptoms of ALS.

In another aspect, the invention features a method of treating a subject exhibiting one or more symptoms of ALS, the method including administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount that results in alleviating or ameliorating one or more symptoms of ALS; monitoring one or more symptoms in the subject; and administering a second dose of B cells when one or more symptoms begin to worsen.

In some embodiments, the one or more symptoms of ALS include difficulty in lifting the front of the foot; difficulty in lifting the toes; weakness of one or both legs; weakness of one or both feet; weakness of single or double ankles; hand weakness; the hands are clumsy; muscle spasm; fasciculations (muscle twitches) in one or both arms, one or both legs, one or both shoulders or the tongue; muscle spasm; spasticity (tight and stiff muscles); and difficulty chewing or swallowing (dysphagia), difficulty speaking or forming words (dysarthria), and difficulty breathing (dyspnea).

In some embodiments, the method comprises monitoring one or more symptoms of ALS 1 to 7 days post-administration, 7 to 28 days post-administration, 1 to 28 weeks post-administration, 1 to 2 months post-administration, 2 to 6 months post-administration, 2 to 9 months post-administration, or 6 months to one year or more post-administration.

In some embodiments, the invention features a method of monitoring responsiveness of a patient having a neurodegenerative disease to treatment with a therapeutically effective amount of isolated B cells by determining a level of a molecular marker of disease progression (e.g., determining a level of a molecular marker of neurodegenerative disease progression before and after treatment with a therapeutically effective amount of B cells). The level of the molecular marker can be determined according to methods known to those skilled in the art.

In some embodiments, the level of the molecular marker may be determined from a sample from the subject being treated, e.g., a sample of plasma or cerebrospinal fluid (CSF) from the subject being treated. Exemplary molecular markers of progression of neurodegenerative diseases described herein are known in the art. Exemplary markers include T- τ (total τ), P- τ (hyperphosphorylated τ), A β 42 (amyloid β 42), the ratio of A β 42/A β 40, YKL-40 (chitinase-3-like protein 1), VLP-1 (cone-like protein 1), NFL (neurofilament L), pNFH (phosphorylated neurofilament heavy subunit), Ng (neurogranule protein) and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43(TAR DNA binding protein 43), reduced α -synuclein and/or reduced levels of 3, 4-dihydroxyphenyl acetate (see, e.g., Robey and Panegyres, Cerebrospinal fluidized biomakers in neurodegrederiders [ Cerebrospinal fluid biomarkers in neurodegenerative disorders ], Future Neurol [ 14. ] 2011. (2019), which is incorporated by reference in its entirety).

In another aspect, the invention features a method of treating an inflammatory disease or an immune disease in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of an isolated B cell.

In some embodiments, the inflammatory or immune disease is selected from the group consisting of cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratospheric, osteoarthritis, osteoporosis, pulmonary hypertension, retinitis pigmentosa, and rheumatoid arthritis.

In another aspect, the invention features a method of treating a subject having a Central Nervous System (CNS) injury, the method including administering to the subject a therapeutically effective amount of isolated B cells. In some embodiments, the CNS injury is Traumatic Brain Injury (TBI) or Spinal Cord Injury (SCI). In some embodiments, the CNS injury comprises both TBI and SCI.

In another aspect, the invention features a method of treating a subject having Traumatic Brain Injury (TBI), the method including administering to the subject a therapeutically effective amount of isolated B cells.

In another aspect, the invention features a method of treating a subject having Spinal Cord Injury (SCI), the method including administering to the subject a therapeutically effective amount of isolated B cells.

In some embodiments, the TBI is damage to the brain caused by external mechanical forces. In some embodiments, SCI involves damage to the spinal cord caused by external mechanical forces. In some embodiments, the TBI and/or SCI is caused by a head injury or brain contusion (e.g., caused by a fall, firearm wound, sporting accident, construction accident, vehicular accident, or injury that penetrates the skull or brain of the subject). In some embodiments, a subject with TBI and/or SCI suffers from one or more of a variety of physical, cognitive, social, emotional, and/or behavioral disorders. TBI and SCI may occur simultaneously and may be caused by the same lesion.

In another aspect, the invention features a method of treating a subject exhibiting one or more symptoms of TBI with TBI, the method comprising administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount that results in alleviation or amelioration of one or more symptoms of TBI; monitoring one or more symptoms in the subject; and administering a second dose of B cells when one or more symptoms begin to worsen.

In another aspect, the invention features a method of treating a subject having SCI (exhibiting one or more symptoms of SCI), the method including administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount that results in alleviating or ameliorating one or more symptoms of SCI; monitoring one or more symptoms in the subject; and administering a second dose of B cells when one or more symptoms begin to worsen.

In some embodiments, one or more symptoms of TBI and/or SCI include failure to recall traumatic events, confusion, difficulty learning and remembering new information, emotional and executive dysfunction, difficulty speaking coherent, instability, lack of coordination, and problems of vision or hearing; cognitive problems (e.g., amnesia, inability to speak or understand language, confusion, difficulty concentrating, difficulty thinking and understanding, inability to create new memory, or inability to recognize common things); behavioral problems (e.g., abnormal laughing and crying, aggression, impulsivity, irritability, lack of continence (impulsivity), or continuously repeating speech or behavior); emotional problems (e.g., anger, anxiety, apathy, or loneliness); systemic problems (e.g., blackout, dizziness, fainting, or fatigue); ocular problems (e.g., dilated pupils, raccoon eyes, or unequal pupils); muscle problems (e.g., instability or muscle stiffness); gastrointestinal problems (e.g., nausea or vomiting); speech problems (e.g., difficulty speaking or slurred speech); visual problems (e.g., blurred vision or sensitivity to light); bruising, depression, olfactory loss, nerve damage, post-traumatic seizure, tinnitus, sensitivity to sound, vertigo.

In some embodiments, the method comprises monitoring one or more symptoms of TBI and/or SCI from 1 to 7 days post-administration, 7 to 28 days post-administration, 1 to 28 weeks post-administration, 1 to 2 months post-administration, 2 to 6 months post-administration, 2 to 9 months post-administration, or 6 months to one year or more post-administration.

In some embodiments, the invention features a method of monitoring responsiveness of a patient with TBI and/or SCI to treatment with a therapeutically effective amount of isolated B cells by determining the level of a molecular marker of disease progression (e.g., determining the level of a molecular marker of neurodegenerative disease progression before and after treatment with a therapeutically effective amount of B cells). The level of the molecular marker can be determined according to methods known to those skilled in the art.

In some embodiments, the level of the molecular marker of TBI and/or SCI may be determined from a sample of the treated subject, e.g., a sample of plasma or cerebrospinal fluid (CSF) from the treated subject. Exemplary molecular markers of TBI progression described herein include, but are not limited to, protein biomarkers of: neuronal cell body injury (UCH-L1, NSE), astrocytic injury (GFAP, S100B), neuronal cell death (α II-spectrin breakdown product), axonal injury (NF protein), white matter injury (MBP), post-injury neurodegeneration (total τ and phospho- τ), post-injury autoimmune response (brain antigen targeting autoantibodies) (see, e.g., Wang et al, An update on diagnostic and diagnostic biomakers for the study of the prognostic biomarkers of traumatic brain injury, Expert Rev Mol diagnostics review [ 18(2):165 and 180(2018), which is incorporated by reference in its entirety).

In some embodiments, allogeneic B cells are administered. In some embodiments, the allogeneic B cells are haploid allogeneic B cells, HLA-matched allogeneic B cells, or genetically modified B cells (e.g., B cells that have been genetically modified, e.g., by CRISPR, to reduce the immunogenicity of the B cells).

In some embodiments, autologous B cells are administered.

In some embodiments, xenogenic B cells are administered.

In some embodiments, the method comprises administering a second therapeutic composition.

In some embodiments, wherein the disease or disorder is ALS, the second therapeutic composition is edaravone, riluzole, or an immunomodulatory composition (e.g., an anti-CD 14 antibody, an anti-CDL 40 antibody, or including T)_regA composition of cells).

In some embodiments, wherein the disease or disorder is TBI and/or SCI, the second therapeutic composition is an antibiotic or a corticosteroid (e.g., prednisone).

In some embodiments, the B cell is a mature naive B cell.

In some embodiments, the B cells are stimulated ex vivo.

In some embodiments, the B cells are stimulated ex vivo with a Toll-like receptor (TLR) agonist.

In some embodiments, the TLR agonist is an endogenous ligand selected from the group consisting of: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, lung surfactant protein a, Low Density Lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronic acid, alpha a-crystallin (alpha a-crystallin), and CpG chromatin-IgG complexes.

In some embodiments, the TLR agonist is an exogenous ligand selected from the group consisting of: pam3CSK4, triacylated lipopeptides, Glycosylphosphatidylinositol (GPI) -anchored proteins, lipoarabinomannans, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acids, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, Poly (I). Poly (c), paclitaxel, flagellin, regulatory proteins, imidazoquinolines (e.g., imiquimod, resiquimod, loxoribine, bromopirimid), antiviral compounds, unmethylated CpG oligodeoxynucleotides, and inhibitory proteins.

In some embodiments, B cells are stimulated ex vivo with an immunomodulatory cytokine (e.g., a proinflammatory cytokine, e.g., a proinflammatory cytokine selected from the group consisting of IL-1 β, IL-2, IL-4, IL-6, TNF α, or IFN γ).

In some embodiments, the B cell is B_regA cell. In some embodiments, B_regThe cells express the immunomodulatory cytokine IL-10. In some embodiments, B_regThe cell further expresses one or more additional immunomodulatory cytokines selected from the group consisting of: IL-2, IL-4, IL-6, IL-35, TNF- α, TGF β, PD-L1 FasL, and TIM 1. In some embodiments, B_regThe cell expresses one or more cell surface markers selected from the group consisting of: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD. In some embodiments, B_regThe cells express B220, CD19, CD20, CD24. CD138, IgM, and IgD. In some embodiments, B_regThe cells express CD25 and CD 71. In some embodiments, B_regThe cells do not express CD 73. In some embodiments, B_regThe cells comprise at least 80% (e.g., at least 85%, 90%, 95%, or 98%) CD19+ B cells. In some embodiments, B_regThe cells comprise less than 10% (e.g., less than 5%) CD138+ plasma B cells.

In some embodiments, the B cells have a neuroprotective effect, an anti-inflammatory effect, and/or an immunomodulatory effect.

In some embodiments, the B cells are formulated for local or systemic administration. In some embodiments, the B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration. In some embodiments, the B cells are formulated for administration by intravenous infusion or bolus injection. In some embodiments, the B cells are formulated for administration via an intracranial pressure (ICP) monitoring catheter.

In some embodiments, B cells are administered once a day, once a week, twice a week, once every 14 days, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or once a year.

In some embodiments, the B cells are administered at least two, three, four, five, six, seven, eight, nine, or ten times.

In some embodiments, a therapeutically effective amount of B cells comprises at least 0.5X 10 per administration⁶B cells, 0.5X 10 per administration⁷B cells, 1X 10 per administration⁸B cells, at least 2x 10 per administration⁸B cells, or at least 1x 10 per administration⁹And (4) B cells. In some embodiments, a therapeutically effective amount of B cells comprises 1x 10 per administration⁸B cell to 1x 10⁹B cells, 1X 10 per administration⁸B cells to 5x 10⁸Individual B cells, or 2x 10 per administration⁸B cells to 4x 10⁸And (4) B cells.

In another aspect, the invention features a pharmaceutical composition comprising a modified B cell and one or more pharmaceutically acceptable excipients, wherein the modified B cell has been stimulated ex vivo with a Toll-like receptor (TLR) agonist and/or an immunomodulatory cytokine.

In some embodiments, the modified B cell is a primary cell.

In some embodiments, the pharmaceutically acceptable excipient is an aqueous solution (e.g., saline solution).

In another aspect, the invention features a method of treating a disease or disorder in a subject in need thereof, the method including administering to the subject a pharmaceutical composition including modified B cells and one or more pharmaceutically acceptable excipients, wherein the modified B cells have been stimulated ex vivo with a Toll-like receptor (TLR) agonist and/or an immunomodulatory cytokine.

In some embodiments, the disease or disorder is selected from abnormal wound healing (e.g., diabetic wound healing), neurodegenerative disease, TBI, or SCI. Or an immune or inflammatory disease.

In some embodiments, the immune or inflammatory disease is selected from the group consisting of cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratospheric, osteoarthritis, osteoporosis, pulmonary hypertension, retinitis pigmentosa, and rheumatoid arthritis.

In some embodiments, the modified B cell is an allogeneic B cell. In some embodiments, the allogeneic B cells are haploid allogeneic B cells, HLA-matched allogeneic B cells, or genetically modified B cells (e.g., B cells that have been genetically modified, e.g., by CRISPR, to reduce the immunogenicity of the B cells).

In some embodiments, the modified B cells are autologous B cells.

In some embodiments, the modified B cell is a xenogenic B cell.

In another aspect, the invention features a method of generating a modified B cell, the method including:

i) isolating mature naive B cells from the subject, and

ii) ex vivo stimulation of B cells with Toll-like receptor (TLR) agonists and/or immunomodulatory cytokines,

thereby producing a modified B cell.

In some embodiments, step i) further comprises isolating CD19+ mature naive B cells. In some embodiments, CD19+ mature naive B cells are isolated by immunoprecipitation with a CD19 antibody or antigen-binding fragment thereof. In some embodiments, the CD19 antibody or antigen binding fragment thereof remains bound to the modified B cell.

In some embodiments, the immunomodulatory cytokine is a proinflammatory cytokine (e.g., a proinflammatory cytokine selected from the group consisting of IL-1 β, IL-2, IL-4, IL-6, TNF α, or IFN γ).

In some embodiments, the modified B cell is B_regA cell. In some embodiments, B_regThe cells express the immunomodulatory cytokine IL-10. In some embodiments, B_regThe cell further expresses one or more additional immunomodulatory cytokines selected from the group consisting of: IL-2, IL-4, IL-6, IL-35, TNF- α, TGF β, PD-L1 FasL, and TIM 1. In some embodiments, B_regThe cell expresses one or more cell surface markers selected from the group consisting of: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD. In some embodiments, B_regThe cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. In some embodiments, B_regThe cells express CD25 and CD 71. In some embodiments, B_regThe cells do not express CD 73.

In some embodiments, the modified B cell has a neuroprotective effect, an anti-inflammatory effect, and/or an immunomodulatory effect.

In still other embodiments, the invention features B cells that can be used directly as anti-inflammatory and pro-regenerative cell-based therapeutics in a variety of disease contexts, including skin wounds and ulcers, muscle and heart injuries, brain and spinal cord injuries, and lesions of various internal organs. Genetically modified autologous, allogeneic or xenogeneic cells or cells primed with factors from the damaged microenvironment are useful because of their improved efficiency in promoting regeneration. Factors derived from B cells under these unique conditions, including antibodies, cytokines and growth factors, as well as micrornas and other small molecules, can also be purified and applied directly to damaged tissues to accelerate healing.

Thus, B cells may be used as a therapeutic strategy for patients with neurodegenerative diseases, TBI or SCI (e.g., caused by cerebral contusion), inflammatory disorders, or various immune diseases. Unlike any other existing cell-based therapies, B cells can be readily obtained from peripheral blood or other blood bank products, which is an important advantage for the development of rapid, ready-to-use therapeutics. In fact, rapid, minimally-manipulated B cell therapies (allogeneic or autologous or xenogeneic) are highly transferable to clinical settings. This is particularly true not only in the treatment of neurodegenerative diseases (e.g., ALS and PD), but also in severe brain pathologies where surgery is typically performed to remove hematomas or penetrating bone fragments and intraparenchymal or intraventricular catheters are placed to monitor intracranial pressure, thereby providing a convenient route of administration for B cells to enter the damaged brain.

Our results illustrate the first proof-of-principle observation that mature, peripherally isolated B cells represent a safe, rapid, and effective cell-based therapeutic strategy for several diseases disclosed herein (including ALS, PD, TBI, and SCI) for which there are currently no treatment options that improve neurological outcomes.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to those skilled in the art from this detailed description.

Some embodiments of the techniques and methods described herein are defined in accordance with any of the following numbered paragraphs.

1. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of isolated B cells.

2. The method of paragraph 1, wherein the neurodegenerative disease is selected from Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, alzheimer's disease, Chronic Traumatic Encephalopathy (CTE), frontotemporal dementia, huntington's disease, infantile axonal dystrophy, progressive supranuclear palsy, lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

3. The method of paragraph 1 wherein the allogeneic B cells are administered.

4. The method of paragraph 1 wherein autologous B cells are administered.

5. The method of paragraph 1, further comprising administering a second therapeutic composition.

6. The method of paragraph 5, wherein the second therapeutic composition is edaravone or riluzole.

7. The method of paragraph 6, wherein the second therapeutic composition is an immunomodulatory composition.

8. The method of any one of paragraphs 1-7, wherein the B cells are mature naive B cells.

9. The method of any of paragraphs 1-8, wherein the B cells are stimulated ex vivo.

10. The method of any of paragraphs 1-9, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist.

11. The method of paragraph 10, wherein the TLR agonist is an endogenous ligand selected from the group consisting of: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, lung surfactant protein a, Low Density Lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronic acid, alpha a-crystallin, and CpG-IgG complexes.

12. The method of paragraph 10, wherein the TLR agonist is an exogenous ligand selected from the group consisting of: pam3CSK4, triacylated lipopeptides, Glycosylphosphatidylinositol (GPI) -anchored proteins, lipoarabinomannans, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acids, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, Poly (I).

13. The method of any one of paragraphs 1-12, wherein the B cell is B_regA cell.

14. The method of paragraph 13 wherein said B_regThe cells express the immunomodulatory cytokine IL-10.

15. The method of paragraph 14 wherein said B_regThe cell further expresses one or more additional immunomodulatory cytokines selected from the group consisting of: IL-2, IL-4, IL-6, IL-35, TNF- α, TGF β, PD-L1 FasL, and TIM 1.

16. The method of any of paragraphs 13-15, wherein said B_regThe cell expresses one or more cell surface markers selected from the group consisting of: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.

17. The method of paragraph 16 wherein said B_regThe cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD.

18. The method of paragraph 16 wherein said B_regThe cells express CD25 and CD 71.

19. The method of any of paragraphs 13-18, wherein the B_regThe cells do not express CD 73.

20. The method of any of paragraphs 13-15, wherein said B_regThe cells comprise at least 80% CD19+ B cells.

21. As in any of paragraphs 13-15 or 20The method of (a), wherein B_regThe cells contained less than 10% CD138+ plasma B cells.

22. The method of any one of paragraphs 1-21, wherein the B cells are neuroprotective.

23. The method of any one of paragraphs 1-22, wherein the B cells are anti-inflammatory.

24. The method of any one of paragraphs 1-23, wherein the B cells are immunomodulatory.

25. The method of any one of paragraphs 1-24, wherein the B cells are formulated for topical administration.

26. The method of any of paragraphs 1-24, wherein the B cells are formulated for systemic administration.

27. The method of any of paragraphs 1-24, wherein the B cells are formulated for intravenous, intraarterial, subcutaneous, intrathecal, or intraparenchymal administration.

28. The method of paragraph 27, wherein the B cells are formulated for administration by intravenous infusion or bolus injection.

29. The method of any of paragraphs 1-28, wherein the B cells are administered once daily, once weekly, twice weekly, once every 14 days, once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, or once annually.

30. The method of any one of paragraphs 1-29, wherein the B cells are administered at least two, three, four, five, six, seven, eight, nine, or ten times.

31. The method of any of paragraphs 1-30, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁸And (4) B cells.

32. The method of any of paragraphs 1-30, wherein the therapeutically effective amount comprises at least 2x 10 per administration⁸And (4) B cells.

33. The method of any of paragraphs 1-30, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁹And (4) B cells.

34. A method of treating a subject having Traumatic Brain Injury (TBI), comprising administering to the subject a therapeutically effective amount of isolated B cells.

35. The method of paragraph 34, wherein the TBI is caused by a head injury, or a cerebral contusion.

36. The method of paragraph 34, wherein the subject suffers from one or more of a plurality of physical, cognitive, social, emotional, and/or behavioral disorders.

37. The method of paragraph 34 wherein allogeneic B cells are administered.

38. The method of paragraph 34 wherein autologous B cells are administered.

39. The method of paragraph 34, further comprising administering a second therapeutic composition.

40. The method of paragraph 39, wherein the second therapeutic composition is an antibiotic or a corticosteroid.

41. The method of any of paragraphs 34-40, wherein the B cells are mature naive B cells.

42. The method of any one of paragraphs 34-41, wherein the B cells are stimulated ex vivo.

43. The method of any of paragraphs 34-42, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist.

44. The method of paragraph 43, wherein the TLR agonist is an endogenous ligand selected from: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, lung surfactant protein a, Low Density Lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronic acid, alpha a-crystallin, and CpG-IgG complexes.

45. The method of paragraph 43, wherein the TLR agonist is an exogenous ligand selected from: pam3CSK4, triacylated lipopeptides, Glycosylphosphatidylinositol (GPI) -anchored proteins, lipoarabinomannans, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acids, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, Poly (I).

46. The method of any one of paragraphs 34-45, wherein the B cell is B_regA cell.

47. The method of paragraph 46 wherein said B_regThe cells express the immunomodulatory cytokine IL-10.

48. The method of paragraph 47, wherein said B_regThe cell further expresses one or more additional immunomodulatory cytokines selected from the group consisting of: IL-2, IL-4, IL-6, IL-35, TNF- α, TGF β, PD-L1 FasL, and TIM 1.

49. The method of any one of paragraphs 46-48, wherein said B_regThe cell expresses one or more cell surface markers selected from the group consisting of: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.

50. The method of paragraph 49 wherein said B_regThe cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD.

51. The method of paragraph 49 wherein said B_regThe cells express CD25 and CD 71.

52. The method of any one of paragraphs 46-51, wherein said B_regThe cells do not express CD 73.

53. The method of any one of paragraphs 46-48, wherein said B_regThe cells comprise at least 80% CD19+ B cells.

54. The method of any one of paragraphs 46-48 or 53, wherein the B cells comprise less than 10% CD138+ plasma B cells.

55. The method of any one of paragraphs 34-54, wherein the B cells are neuroprotective.

56. The method of any one of paragraphs 34-55, wherein the B cells are anti-inflammatory.

57. The method of any one of paragraphs 34-56, wherein the B cells are immunomodulatory.

58. The method of any one of paragraphs 34-57, wherein the B cells are formulated for topical administration.

59. The method of any of paragraphs 34-57, wherein the B cells are formulated for systemic administration.

60. The method of any of paragraphs 34-57, wherein the B cells are formulated for intravenous, intraarterial, subcutaneous, intrathecal, or intraparenchymal administration.

61. The method of paragraph 60 wherein the B cells are formulated for administration by intravenous infusion or bolus injection.

62. The method of any of paragraphs 34-57, wherein the B cells are formulated for administration by an intracranial pressure (ICP) monitoring catheter.

63. The method of any one of paragraphs 34-62, wherein the B cells are administered once per day, once per week, twice per week, once per 14 days, once per month, once per two months, once per three months, once per four months, once per five months, once per six months, or once per year.

64. The method of any one of paragraphs 34-63, wherein the B cells are administered at least two, three, four, five, six, seven, eight, nine, or ten times.

65. The method of any of paragraphs 34-63, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁸And (4) B cells.

66. The method of any of paragraphs 34-63, wherein the therapeutically effective amount comprises at least 2x 10 per administration⁸And (4) B cells.

67. The method of any of paragraphs 34-63, wherein the therapeutically effective amount comprises at least 1x 10 per administration⁹And (4) B cells.

68. A pharmaceutical composition comprising modified B cells and one or more pharmaceutically acceptable excipients, wherein the modified B cells have been stimulated ex vivo with a Toll-like receptor (TLR) agonist and/or an immunomodulatory cytokine.

69. The pharmaceutical composition of paragraph 68 wherein the TLR agonist is an endogenous ligand selected from: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, lung surfactant protein a, Low Density Lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronic acid, alpha a-crystallin, and CpG-IgG complexes.

70. The pharmaceutical composition of paragraph 68 wherein the TLR agonist is an exogenous ligand selected from: pam3CSK4, triacylated lipopeptides, Glycosylphosphatidylinositol (GPI) -anchored proteins, lipoarabinomannans, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acids, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, Poly (I).

71. The pharmaceutical composition of paragraph 68, wherein said immunomodulatory cytokine is a pro-inflammatory cytokine.

72. The pharmaceutical composition of paragraph 71, wherein said proinflammatory cytokine is selected from the group consisting of: IL-1 beta, IL-2, IL-4, IL-6, TNF alpha, or IFN gamma.

73. The pharmaceutical composition of any of paragraphs 68-72, wherein the modified B cells are primary cells.

74. The pharmaceutical composition of any one of paragraphs 68-73, wherein the modified B cell is B_regA cell.

75. The pharmaceutical composition of paragraph 74 wherein said B_regThe cells express the immunomodulatory cytokine IL-10.

76. The pharmaceutical composition of paragraph 75 wherein said B_regThe cell further expresses one or more additional immunomodulatory cytokines selected from the group consisting of: IL-2, IL-4, IL-6, IL-35, TNF- α, TGF β, PD-L1 FasL, and TIM 1.

77. The pharmaceutical composition of any one of paragraphs 74-76, wherein said B_regThe cell expresses one or more cell surface markers selected from the group consisting of: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.

78. The pharmaceutical composition of paragraph 77, wherein said B_regThe cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD.

79. The pharmaceutical composition of paragraph 77, wherein said B_regThe cells express CD25 and CD 71.

80. The pharmaceutical composition of any one of paragraphs 74-79, wherein said B is_regThe cells do not express CD 73.

81. The pharmaceutical composition of any one of paragraphs 68-80, wherein the modified B cell is neuroprotective.

82. The pharmaceutical composition of any one of paragraphs 68-81, wherein the modified B cell is anti-inflammatory.

83. The pharmaceutical composition of any one of paragraphs 68-82, wherein the modified B cell is immunomodulatory.

84. The pharmaceutical composition of any one of paragraphs 68-83, wherein the pharmaceutically acceptable excipient is an aqueous solution.

85. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition of any one of paragraphs 68-84.

86. The method of paragraph 85 wherein said disease or disorder is abnormal wound healing.

87. The method of paragraph 85, wherein said disease or disorder is a neurodegenerative disease.

88. The method of paragraph 87, wherein the neurodegenerative disease is selected from Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, alzheimer's disease, Chronic Traumatic Encephalopathy (CTE), frontotemporal dementia, huntington's disease, infantile axonal dystrophy, progressive supranuclear palsy, lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

89. The method of paragraph 85 wherein said disease or disorder is Traumatic Brain Injury (TBI).

90. The method of paragraph 85 wherein said disease or condition is selected from the group consisting of cystic fibrosis, cardiovascular disease, keratoconus, keratospheric, osteoarthritis, osteoporosis, pulmonary hypertension, retinitis pigmentosa, and rheumatoid arthritis.

91. The method of any one of paragraphs 85-90, wherein the modified B cells are allogeneic B cells.

92. The method of any of paragraphs 85-91, wherein the modified B cells are autologous B cells.

93. A method of producing a modified B cell, the method comprising:

i) isolating mature naive B cells from the subject, and

ii) ex vivo stimulation of B cells with Toll-like receptor (TLR) agonists and/or immunomodulatory cytokines,

thereby producing a modified B cell.

94. The method of paragraph 93 wherein step i) further comprises isolating CD19+ mature naive B cells.

95. The method of paragraph 94, isolating CD19+ mature naive B cells by immunoprecipitation with a CD19 antibody or antigen-binding fragment thereof.

96. The method of paragraph 95, wherein said CD19 antibody or antigen binding fragment thereof remains bound to modified B cells.

97. The method of paragraph 93, wherein the TLR agonist is an endogenous ligand selected from the group consisting of: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, lung surfactant protein a, Low Density Lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronic acid, alpha a-crystallin, and CpG-IgG complexes.

98. The method of paragraph 93, wherein the TLR agonist is an exogenous ligand selected from: pam3CSK4, triacylated lipopeptides, Glycosylphosphatidylinositol (GPI) -anchored proteins, lipoarabinomannans, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acids, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, Poly (I).

99. The method of paragraph 93 wherein said immunomodulatory cytokine is a proinflammatory cytokine.

100. The method of paragraph 99, wherein the proinflammatory cytokine is selected from the group consisting of: IL-1 beta, IL-2, IL-4, IL-6, TNF alpha, or IFN gamma.

101. The method of any of paragraphs 93-100, wherein the modified B cell is B_regA cell.

102. The method of paragraph 101 wherein said B_regThe cells express the immunomodulatory cytokine IL-10.

103. The method of paragraph 102 wherein said B_regThe cell further expresses one or more additional immunomodulatory cytokines selected from the group consisting of: IL-2, IL-4, IL-6, IL-35, TNF- α, TGF β, PD-L1 FasL, and TIM 1.

104. The method as described in any of paragraphs 101-103, wherein B is_regThe cell expresses one or more cell surface markers selected from the group consisting of: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA. And IgD.

105. The method of paragraph 105 wherein said B_regThe cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD.

106. The method of paragraph 105 wherein said B_regThe cells express CD25 and CD 71.

107. The method as described in any of paragraphs 101-106, wherein B is_regThe cells do not express CD 73.

108. The method of any of paragraphs 93-107, wherein the modified B cell is neuroprotective.

109. The method of any of paragraphs 93-108, wherein the modified B cell is anti-inflammatory.

110. The method of any one of paragraphs 93-109, wherein the modified B cell is immunomodulatory.

Drawings

Fig. 1A-1B show that B cell application induces complex changes in the molecular microenvironment of the wound. FIG. 1A is a graphical representation of the average duration of the major phases of wound healing in a wild type murine wound model. Fig. 1B shows a heat map summarizing the expression dynamics over time of proteins that significantly alter expression in response to B cell application. A total of 213 proteins, which represent aggregates of significantly altered proteins associated with B cell therapy (n 111; p <0.05, unpaired t-test), and those with high fold changes at each time point (the first 20 upregulated or downregulated proteins), were classified according to the process associated with wound healing, regardless of the level of significance (n 112). The heat maps show fold change expression after B cell treatment at days 0, 1, 4, 10 post injury. Red-up; green-down-regulation. Of particular note is the down-regulation of a variety of proteins associated with inflammation and inflammatory cells at day 4 post-injury, and the massive up-regulation of proteins associated with cell proliferation, prevention of apoptosis (cell death) and oxidative stress, and tissue remodeling (formation of hair follicles and muscle) at days 4-10 post-injury.

Fig. 2A-2H show the average expression of proteins of functional families over time in wounds after saline (control, conventional wound healing) or B-cell treatment. This analysis demonstrates the overall effect of B cells as homeostatic agents, rather than inducers or inhibitors of protein expression. B cell use is associated with maintaining stable protein expression levels (these proteins typically decrease or increase during injury and healing processes), which significantly reduces the inflammatory peaks observed during normal healing processes, prevents the reduction of anti-apoptotic factors (arrows) and oxidative stress protectors, and increase proliferation (FIG. 2A-FIG. 2B), reduce decrease in antioxidant stress protectant and cell proliferation, and maintaining cell migration at a lower level (FIGS. 2C-2D), maintaining a steady level of proteins associated with remodeling and secondary skin structure (FIGS. 2E-2F), reducing the level of protein degradation and autophagy observed at the early stage of injury in controls, and increasing the levels of proteins associated with angiogenesis and nerve regeneration at the later stages of healing (fig. 2G-fig. 2H).

Figure 3 shows an experimental example of the in vivo evaluation of B cells for use in acute wound healing. A total of 4 full-thickness lesions were generated in the dorsal skin of wild-type C57Bl6 mice, and mature primary B cells purified from syngeneic animals were applied directly to the wound bed. Control animals received saline application. As an internal control, also under intact skin, B cells or saline controls were injected subcutaneously to provide a similar microenvironment without injury. After a defined survival time, wound or skin undamaged tissue is collected, dissociated, and processed for flow cytometry analysis. The scatter plot on the right shows a typical distribution of cell suspension for each treatment class. The wound samples showed a characteristic influx of leukocytes (white open arrows), which was essentially absent in the undamaged tissue. Although B cells are typically rarely present at any one location, they are easily detected in large numbers after experimental application (red arrows).

Figure 4 shows gating strategies and analysis of B cell treated and control wound cell suspensions via flow cytometry. Viable cells are divided into 3 main categories: b cells (CD19+/B220+ lymphocytes); non-B cell leukocytes (CD140 a-/B220-leukocytes) comprising a mixture of neutrophils, monocytes and macrophages, dendritic cells and T cells; and fibroblasts (CD140a +/B220-). Markers for activation and cytokine production of these cell classes were assessed.

Figure 5 shows the dynamics of activation markers and key cytokines in B cells recovered from the wound bed after defined intervals of exposure to the wound microenvironment. B cells were exposed to the wound microenvironment in vivo, or injected under intact skin (control equivalent sites). For comparison, control B cells maintained on ice for the same duration immediately after isolation are shown. After time intervals including 18 hours, 2 days, 4 days and 10 days, the wounds were treated with brefeldin a for 4 hours to induce intracellular cytokine retention. B cells were then recovered by excision and dissociation of the tissue, and surface markers and intracellular cytokines were further characterized by flow cytometry. B cells exposed to the wound microenvironment transiently upregulate a variety of immunoregulatory cytokines, peaking at 2 days post-application. Some immunoregulatory cytokines, including TGF β and IL-6, remain elevated on day 4, and IL-10 remains elevated for up to 10 days. N-3-6 animals/group.

Figure 6 is a heat map summary of the average values of each marker in B cells exposed to the wound microenvironment, subcutaneous controls, or maintained on ice (unexposed).

Figure 7 shows the dynamics of activation markers and key cytokines in infiltrating non-B cell leukocytes collected in the wound. In conclusion, infiltrating leukocytes produce more of the anti-inflammatory cytokines IL-10, TGF β and IL-35, and less of the pro-inflammatory TNF α and IL-2 when B cells are present in the wound. This effect was most pronounced at day 4 after injury and B cell application and lasted for up to 10 days. N-3-6 animals/group.

FIG. 8 is a summary of the heatmap of the average values of each marker in infiltrating non-B cell leukocytes in the wound microenvironment, illustrating the pattern of increased anti-inflammatory cytokine (IL-10 and TGF β) production in the presence of B cells.

Figure 9 shows the dynamics of activation markers and key cytokines in the CD140a + fibroblast population of wounds and subcutaneous tissues. When the wound was exposed to B cells, fibroblasts in the wound produced significantly more IL-10 and TGF β on day 10 post injury. In addition, wound fibroblasts produced less of the pro-inflammatory cytokine TNF α when B cells were applied at days 4 and 10 post-injury.

Fig. 10 is a heat map summary of the mean values of each marker in fibroblasts in wounds and subcutaneous tissue treated with B cells or saline solution. Fibroblasts are one of the most important sources of anti-inflammatory and pro-regenerative factors in wound healing and produce high levels of IL-10 and TGF β regardless of treatment. However, on days 4 and 10 post-injury, fibroblasts from wounds treated with B cells continue to produce higher levels of IL-10 and TGF β, while in saline-treated wounds the levels of these anti-inflammatory cytokines are reduced. Interestingly, a significant effect of B cell application was observed in the reduction of pro-inflammatory cytokines (including IL-6 and TNF α) in wound fibroblasts.

Figure 11 shows that functional TLR signaling and IL-10 production are essential components of the regenerative function of exogenous B cells in wound healing. Whole-thickness resection wounds (described here on day 6 of healing) were treated on day 0 with B cells lacking the common TLR signaling adaptor myeloid differentiation factor 88(MyD88), IL-10, or WT B cells as controls. Saline was also included as an internal control in each test animal. Although WT B cells continued to accelerate wound closure in WT animals for 2-3 days, MyD 88-/-or IL-10-/-B cells had no benefit on wound closure, similar to saline application.

Figure 12 shows unsupervised hierarchical cluster analysis of identified proteins expressed in skin wound samples. Only the identified proteins that were consistently present in all samples were included in the analysis. (A) Using 3809 proteins (rows) that are consistently expressed in the wounds of all B-cells and saline-treated animals, graded clusters were made at 4 different time points (columns) post-injury. The pseudo-color scale depicts the normalized, log-fold-transformed change expression values for each protein. The dendrogram shows 15 protein clusters resulting from this analysis, where the color of each cell in (a) maps to the mean expression value of the cluster at the corresponding time point. Proteins are clustered according to their expression pattern over time. (B) Heatmap from hierarchical clustering of (a), showing all 3809 proteins. (C) Gene ontology analysis of 15 hierarchical clusters. A list of mouse GOslim genes from QuickGO (accessible from https:// www.ebi.ac.uk/QuickGO) was used to probe 15 hierarchical clusters. The bar graph shows the top biofunctional categories for each cluster.

Figure 13 shows the distribution of significantly altered proteins in response to B cell treatment at each of the assessed time points during wound healing.

Figure 14 shows an experimental paradigm for assessing the effect of B cell application on functional (behavioral) and histological recovery following contusion TBI. Adult male C57BL/6J mice were anesthetized and a 5-mm circular craniotomy was performed above the left parietal temporal cortex and the bone flap was removed. Just before being damaged, 2X 10⁶A single infusion of individual B cells was delivered intraparenchymally through the brain into the ipsilateral hemisphere. Mice were then treated with CCI or sham lesions. After recovery, various assays are used to assess motor function, motor and spatial learning and memory performance, anxiety, and depression-like behavior. At the end of the behavioral testing (day 35), animals were euthanized and brains were collected for assessment of total lesion volume.

Figure 15 shows the effect of acute B cell processing on vestibular motor function and striatal learning. (A) Rotarod evaluation showed significant protection of B cells administered in CCI. Notably, in B cell treated mice as well as in pseudodiseased animals, the delay in falls for repeat experiments increased, indicating a component of motor learning. In controls treated with T cells or saline, no such improvement was observed. After the second trial, no significant differences were observed between CCI-injured mice receiving B cell treatment and sham-diseased B cell-treated animals. (B) Assessment of vestibular motor restoration following injury using the puller assay showed that injury had a significant effect compared to sham controls, however, no statistically significant differences were observed between the treatment conditions of injured mice. Data are mean ± SEM. P < 0.05; p < 0.01; p < 0.001; p < 0.0001. CCI + B cells, n ═ 12 mice; CCI + T cells, n ═ 12 mice; CCI + saline, n ═ 12 mice; mock-treated + B cells, n ═ 10 mice; sham-treated + saline, n ═ 10 mice.

Figure 16 shows the effect of a single acute B cell application on learning and memory. (A-D) Morris Water maze assessment. The learning curve shows that CCI mice treated with B cells are significantly improved (p <0.05) over saline-treated CCI animals. After the third trial, no significant difference (p >0.98) (a) was observed between the B cell treated wounded animals and either of the pseudopathological conditions. The visual plateau test showed no difference between the treatment conditions with or without damage (B). (C) Probing experiments showed that B cell treated CCI-injured animals spent more time than chance in the target quadrant, with no significant difference from sham mice. In contrast, control CCI-injured mice treated with T cells or saline only spent an opportunity level time to explore the target quadrant and differed significantly from the pseudodisease shift (p < 0.05). The dashed line represents the level of opportunity. (D) Representative swim path tracking during the probe trial demonstrated spatial search patterns in CCI-B cell mice, and in both sham groups, while CCI mice employed a non-spatial strategy in the T cell and saline groups. (E) The Y maze assesses short term learning and memory. The alternation score of B cell treated CCI mice was significantly higher than that of injured T cells or saline treated groups, behaving similarly to sham operated groups. All data are shown as mean ± SEM. P < 0.05; p < 0.01; p < 0.001; p < 0.0001. CCI + B cells, n ═ 12 mice; CCI + T cells, n ═ 12 mice; CCI + saline, n ═ 12 mice; mock-treated + B cells, n ═ 10 mice; sham-treated + saline, n ═ 10 mice.

Figure 17 shows the effect of B cell treatment on post-CCI anxiety and depressive-like behavior. (A) Elevated plus maze assay for anxiety-like behavior. No overall significant differences were observed between treatment groups (p <0.05) except that the time spent on the closed arms was slightly different between CCI-injured mice receiving B cells at the time of injury and animals receiving the same number of T cells. (B) Forced swim determination of depressive-like behavior. No effect of the lesion or treatment was observed in this assay. All data are shown as mean ± SEM. P < 0.05. CCI + B cells, n ═ 12 mice; CCI + T cells, n ═ 12 mice; CCI + saline, n ═ 12 mice; mock-treated + B cells, n ═ 10 mice; sham-treated + saline, n ═ 10 mice.

FIG. 18 shows the effect of B cell treatment on histological results after CCI. (A) Representative coronal sections through the lesion site at day 35 post-injury. In B cell treated animals, a portion of the hippocampus generally survived in the diseased hemisphere (arrow). The section shown is located approximately 2.2mm from bregma. (B) The total volume of brain lesions in B cell treated mice was significantly reduced by 40% -60% at day 35 post TBI compared to saline and T cell controls. (C) Lesion areas in transverse brain slices along the cranio-caudal axis of the brain. The results show that lesion size continues to decrease in diseased brain that received B cell treatment. (D) The total volume of the surviving hippocampus in the injured hemisphere was significantly higher in B cell treated animals compared to either CCI control. All data are shown as mean ± SEM. P < 0.05; p < 0.01; p < 0.001; p < 0.0001. CCI + B cells, n ═ 12 mice; CCI + T cells, n ═ 12 mice; CCI + saline, n ═ 12 mice; mock-treated + B cells, n ═ 10 mice; sham-treated + saline, n ═ 10 mice.

Figure 19 shows the effect of B cell treatment on gliosis and microglial activation. (A-D) confocal images show the immunological labeling of GFAP and CD68 in a summary of the medial aspect of the lesion at day 35, following CCI and treatment with any of saline (A), B cells (B), T cells (C), or in saline-treated sham-lesion control (D). (E) Quantitative analysis of the area covered by GFAP immunostaining showed a significant reduction in reactive astrocytosis in injured animals treated with B cells compared to saline or T cell treated CCI controls. (F) Quantitative analysis of CD68 immunostaining showed a significant reduction in the presence of CD68 in animals treated with T cells or B cells following CCI compared to saline-treated CCI controls. n-4 imaging fields per animal. All data are shown as mean ± SEM. P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

FIG. 20 shows the brainB cell survival and persistence. (A) WT C57Bl6/J mice harbored a 5X 10 in CCI and brain parenchyma⁶B is^lucRepresentative examples of in vivo imaging at multiple time points after the cell. (B) Luminescence from the CCI lesion in the head (n ═ 6 mice) indicated that cells survived in situ for up to approximately 14 days after application, with a significant reduction in the number of viable cells after day 7. [ p/s ]]Photons per second.

Fig. 21 shows B cell localization at the injury site after CCI. (A) Pre-labeled B cells can be visualized at the site of injury immediately after intraparenchymal injection and CCI. Black squares indicate the imaged areas in B. (B) Confocal microscopy images of coronal sections through the lesion showed that B220+ B cells clustered at the injection site (arrow). Ki67 immunolabeling showed that no cell proliferation was observed immediately after injury. (C) Four days after B cell injection and CCI, labeled B cells could still be observed at the site of injury, although the intensity of vital staining at this time point had diminished compared to that seen immediately after injection. The black squares indicate the imaged areas in D. (D) Confocal images of coronal sections through the injection site four days after injury and B cell administration. A large number of B220+ B cells clustered at the site of injury can still be found. Extensive cell proliferation was observed throughout the area, however no co-staining of B220 and Ki67 was observed. (E) D enlarged view of the framed area. (F) In sham animals, summarized by the astroglial scar, stitches through the cortex could still be found 35 days after treatment. No B220+ B cells were observed at the original injection site at this time. (G) F high magnification confocal image of the framed area. In all confocal images, nuclei were counterstained with DAPI. n-4 animals per time point.

Fig. 22 shows an overview of the experimental design. Body weight and neurological score assessments were performed twice weekly, at the same time of day, by an experimenter blinded to treatment conditions.

Figure 23 shows the normalized body weight (percentage of the value on day 76 for each individual animal) over time in the B cell and saline treated groups measured twice weekly. The figure shows a combined measure of normalized body weight and survival, where the dead individual received a body weight value of 0. We observed the expected difference in body weight between the non-carrier control animals and the SOD1 transgenic animals in each treatment group. In non-carrier control animals, regardless of treatment, a gradual weight gain was observed over the course of the study. The results also indicated a delay in the decline of transgenic SOD1 animals that received B cells (arrows). N is 32/treatment condition.

FIGS. 24A and 24B show analysis of peak body weight in SOD1-G93A animals. A. The survival plot shows the time point at which the animal reached its peak body weight. B. The time to peak body weight indicates that treatment with B cells significantly delayed the onset of the paralytic symptoms. Counting: a: Gehan-Breslow-Wilcoxon test; b: unpaired t-test. N-32 animals/group.

Figure 25 shows the neural score values as a function of time. A composite plot of nervous system score and survival, where dead individuals, after reaching a neurological score of 4, were further assigned a value of 4 for the remaining duration of the study. In animals treated with B cells, the increase in neurological score values typically observed in transgenic SOD1 animals slowed, particularly in the early stages of disease progression (orange squares). Counting: two-way ANOVA with multiple comparisons using Tukey post correction.

FIG. 26 shows survival analysis of transgenic SOD1-G93A animals. Animals with a neurological score of 4 (complete paralysis) were considered to die from ALS. Treatment with naive B cells significantly prolonged survival compared to saline control treatment (N ═ 32 animals/group). Counting: (left): Gehan-Breslow-Wilcoxon test; (right): one-way ANOVA. N-32 animals/group.

Figure 27 shows endpoint motor neuron assessment in the lumbar spinal cord. A, B: lumbar spinal cord sections were stained with H & E and all motor neurons (large cell bodies, at least one nucleolus) as well as damaged abnormal neurons showing morphological features of injury/degeneration (arrows) were counted by experimenters blinded (blind) to treatment. C: the total number of motor neurons in the transgenic animals was significantly reduced, but there was no difference from the treatment within this group. D: the significant benefit of B cell processing becomes apparent when the percentage of degenerated, pycnotic motor neurons is specifically analyzed. Counting: (left): two-way ANOVA; (right): unpaired t-test. N-19-24 animals/group. Note that it is not possible to collect tissue samples in all test animals.

Detailed Description

The invention will now be described in detail by way of reference using only the following definitions and examples. All patents and publications cited herein are expressly incorporated by reference. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numerical ranges include the numbers defining the range.

The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

Definition of

As used herein, the term "neurodegenerative disease" refers to a neurological disease, disorder, or condition characterized by progressive loss of structure or function of neurons, including, for example, death of neurons in the Central Nervous System (CNS). There are many similarities that have been made to correlate these diseases at the sub-cellular level. In addition, there are many similarities between different neurodegenerative disorders, including atypical protein assembly and induced cell death. Neurodegeneration can be found in many different levels of neuronal circuits ranging from molecules to systems. Neurodegeneration can be characterized by the following molecular markers of disease progression: for example: t-tau (total tau), P-tau (hyperphosphorylated tau), Abeta 42 (amyloid beta 42), Abeta 42/Abeta 40 ratio, YKL-40 (chitinase-3-like protein 1), VLP-1 (cone-like protein 1), NFL (neurofilament L), pNFH (phosphorylated neurofilament heavy subunit), Ng (neurotrypsin) and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43(TAR DNA binding protein 43), reduced alpha-synuclein and/or reduced levels of 3, 4-dihydroxyphenylacetate (see, e.g., Robey and panagyres, cererospinal fluidized biomakers in neurogenetic disorders Cerebrospinal fluid biomarkers in neurodegenerative disorders, Future Neurol 14(1) (2019), which is incorporated by reference in its entirety). Exemplary neurodegenerative disorders include Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, alzheimer's disease, Chronic Traumatic Encephalopathy (CTE), frontotemporal dementia, huntington's disease, infantile axonal dystrophy, progressive supranuclear palsy, lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

As used herein, the term "Central Nervous System (CNS) injury" refers to an injury that disrupts the normal function of the brain and/or spinal cord. As described herein, CNS injury may be caused by external mechanical forces. CNS injury includes Traumatic Brain Injury (TBI) and/or Spinal Cord Injury (SCI).

As used herein, the term "Traumatic Brain Injury (TBI)" refers to the disruption of normal function of the brain due to external mechanical forces. For example, TBI may be caused by head injury or cerebral contusion (e.g., caused by a fall, firearm wound, sporting accident, construction accident, vehicular accident, or injury that penetrates the skull or brain of a subject). TBI is diagnosed according to clinical guidelines known to those skilled in the art. TBI can be further characterized by molecular markers of disease progression, such as protein biomarkers of: neuronal cell body injury (UCH-L1, NSE), astrocytic injury (GFAP, S100B), neuronal cell death (α II-spectrin breakdown product), axonal injury (NF protein), white matter injury (MBP), post-injury neurodegeneration (total τ and phospho- τ), post-injury autoimmune response (brain antigen targeting autoantibodies) (see, e.g., Wang et al, An update on diagnostic and diagnostic biomakers for the study of the prognostic biomarkers of traumatic brain injury, Expert Rev Mol diagnostics review [ 18(2):165 and 180(2018), which is incorporated by reference in its entirety). TBI may be concurrent with SCI and may be caused by the same injury or accident.

As used herein, the term "Spinal Cord Injury (SCI)" refers to injury of the spinal cord due to external mechanical forces. For example, SCI may result from spinal cord injury or spinal cord contusion (e.g., from a fall, firearm wound, sports accident, construction accident, vehicular accident, or injury to the spinal cord of a subject). SCI is diagnosed according to clinical guidelines known to those skilled in the art. SCI may be concurrent with TBI and may be caused by the same injury or accident.

As used herein, the term "inflammatory disease" or "immune disease" refers to a disease, disorder, or condition whose etiology, pathogenesis, progression, or symptomatology has an inflammatory or immune component. For example, an inflammatory or immune disorder may include a dysregulation of an inflammatory or immune pathway and/or an aberrant inflammatory or immune response to a stimulus. Exemplary inflammatory or immune disorders include cystic fibrosis, cardiovascular disease, keratoconus, keratospheric, osteoarthritis, osteoporosis, pulmonary hypertension, retinitis pigmentosa, and rheumatoid arthritis.

As used herein, the term "neuroprotective" refers to a property that prevents, inhibits, or reduces neuronal cell death. For example, a neuroprotective composition or method can be characterized by an alteration (e.g., a decrease) in a symptom associated with a neurodegenerative disorder, TBI, or SCI. Alternatively, the neuroprotective composition or method may be characterized by its effect on molecular markers of disease (such as those described herein for neurodegenerative disorders, TBI, or SCI).

As used herein, the term "anti-inflammatory" refers to a property that prevents, inhibits, or reduces inflammation. For example, an anti-inflammatory composition or method can be characterized by an alteration (e.g., a reduction) in a symptom associated with an inflammatory disorder. Alternatively, an anti-inflammatory composition or method may be characterized by a decrease in an inflammatory marker (e.g., a decrease in a pro-inflammatory cytokine) or an increase in an anti-inflammatory marker (e.g., an increase in an anti-inflammatory cytokine).

As used herein, the term "immunomodulation" refers to the property of initiating or altering (e.g., increasing or decreasing) the activity of cells involved in an immune response. An immunomodulatory composition or method can increase the activity of a cell involved in an immune response, e.g., by increasing a pro-inflammatory marker, e.g., a cytokine, and/or can decrease the activity of a cell involved in an immune response, e.g., by decreasing a pro-inflammatory marker, e.g., a cytokine.

As used herein, the term "B cell" or "B lymphocyte," as used interchangeably herein, refers to a type of white blood cell of the small lymphocyte subtype. Unlike the other two types of lymphocytes, T cells and natural killer cells, B cells express a B Cell Receptor (BCR) on their cell membrane. BCR allows B cells to bind to specific antigens against which an antibody response is initiated. B cells play a role in the humoral immune component of the adaptive immune system by secreting antibodies. In addition, B cells present antigens (they are also classified as professional Antigen Presenting Cells (APCs)) and secrete cytokines. In mammals, B cells mature in the bone marrow.

As used herein, the term "mature B cell" refers to a B cell that has completed the B cell maturation process, e.g., in the bone marrow of a mammal. Mature B cells leave the bone marrow and migrate to secondary lymphoid tissues where they may interact with exogenous antigens and/or helper T cells. The stage of B cell maturation is well characterized in the scientific literature and is known to those skilled in the art.

As used herein, the term "naive B cell" refers to a B cell that has not been exposed to an antigen.

As used herein, the term "Breg cell" or "B regulatory cell" refers to a class of B cells that are involved in immune regulation and suppression of immune responses. Breg cells of the present disclosure are mature naive B cells, expressing characteristic cell surface markers. Breg cells may express one or more of the following: b220, CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71, CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD. In particular, Breg cells may express cell surface markers including, but not limited to, B220, CD19, CD20, CD24, IgM, IgD, and CD 138. Upon introduction into the damaging environment, Breg cells may produce immunomodulatory cytokines including, but not limited to, IL-2, IL-4, IL-6, IL-10, IL-35, TNF- α, TGF- β, interferon- γ. In particular, Breg cells are characterized by the production of IL-10.

As used herein, the term "cytokine" refers to a small protein involved in cell signaling. Cytokines may be produced and secreted by immune cells such as T cells, B cells, macrophages, and mast cells, and include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

As used herein, the term "pro-inflammatory cytokine" refers to a cytokine secreted from immune cells that promote inflammation. Immune cells that produce and secrete proinflammatory cytokines include T cells (e.g., Th cells), macrophages, B cells, and mast cells. Proinflammatory cytokines include interleukin-1 (IL-1, e.g., IL-1 β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, e.g., TNF α), interferon γ (IFN γ), and Granulocyte Macrophage Colony Stimulating Factor (GMCSF).

As used herein, the term "Toll-like receptor (TLR) agonist" refers to a ligand that binds to and activates a Toll-like receptor (TLR), resulting in downstream TLR cell signaling. TLR agonists are known to those skilled in the art and include both endogenous and exogenous ligands. Exemplary endogenous ligands that are TLR agonists include heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, lung surfactant protein a, Low Density Lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronic acid, alpha a-crystallin, and CpG chromatin-IgG complexes. Exemplary exogenous ligands that are TLR agonists include Pam3CSK4, triacylated lipopeptides, Glycosylphosphatidylinositol (GPI) -anchored proteins, lipoarabinomannans, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acids, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, Poly (I) · Poly (c), paclitaxel, flagellin, regulatory proteins, imidazoquinoline, antiviral compounds, unmethylated CpG oligodeoxynucleotides, and inhibitory proteins.

As used herein, the term "treatment" (and variants thereof, such as "treating" or "treatment") refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and may be used to prevent or progress through the course of clinical pathology.

As used herein, the term "administering" refers to a method of administering a dose to a subject. The compositions for use in the methods described herein may be administered, for example, in the following manner: intravitreal (e.g., by intravitreal injection), by eye drop, intramuscular, intravenous, intradermal, transdermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraparenchymal, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, topical, intratumoral, peritoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, oral, topical, transdermal, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local infusion, by direct bathing of target cells, by catheter, by lavage, in cream, or in a lipid composition. The compositions for use in the methods described herein may also be administered systemically or locally. For topical administration, administration is in the form of a lotion, cream, ointment, or gel. The method of administration may vary depending on various factors (e.g., the composition administered, and the severity of the condition, disease, or disorder of the immune disorder being treated).

The subject treated according to the invention is a mammal. The mammal can be, for example, a primate (e.g., a human), a rodent (e.g., a rat or mouse), or another species of mammal (e.g., a farm or other domesticated animal). In each of the above methods, the mammal may be a mammal having any of the diseases or disorders disclosed herein. In a preferred embodiment, the subject is a human.

A mammal in "need of treatment" may include, but is not limited to, a mammal with a neurodegenerative disorder, TBI, SCI, an immune disorder, a mammal with an immune disorder, or a mammal with symptoms of an immune disorder or a mammal with an inflammatory disorder or disease. Exemplary disorders are disclosed herein.

An "effective amount" of a pharmaceutical agent (e.g., a pharmaceutical formulation) is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result or purpose for which a particular statement is made. An "effective amount" can be determined empirically and by known methods relevant to the stated purpose.

The term "isolating" refers to the physical identification and isolation of a cell or group of cells from a cell culture or biological sample. The separation is carried out by applying appropriate cell biology techniques based on the examination of the cell culture and on the characterization of the cells corresponding to the standard (and physical separation if possible and desired), or can be based on automatic sorting of the cells according to characteristics such as the presence/absence of antigens and/or the size of the cells (for example by FACS). In some embodiments, the term "isolating" may comprise a further step of physical separation and/or quantification of the cells, in particular by performing flow cytometry. Physical separation also includes enriching a particular characteristic of a cell or cell population. An "isolated" cell or population of cells is a cell or population of cells that has been identified and/or isolated as described above.

The term "population of cells" or "population of cells" generally refers to a group of cells. Unless otherwise specified, the term means consisting essentially of or comprising cells as defined hereinA group of cells of (a). The population of cells may consist essentially of cells having a common phenotype or may comprise at least a portion of cells having a common phenotype. Cells are considered to have a common phenotype when they are substantially similar or identical in terms of one or more demonstrable characteristics, including, but not limited to, morphological appearance, expression levels of particular cellular components or products (e.g., RNA or protein), activity of certain biochemical pathways, proliferative capacity and/or kinetics, differentiation potential during in vitro culture, and/or response to differentiation signals or behavior. Thus, such demonstrable features may define a population of cells or a portion thereof. A population of cells may be "substantially homogeneous" if a vast majority of the cells have a common phenotype. A "substantially homogeneous" cell population can comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, or even at least 99% of cells having a common phenotype (e.g., B cells)_regCell) specific phenotype). Furthermore, a population of cells can consist essentially of cells having a common phenotype (e.g., B cells (e.g., B) if any other cells present in the population do not alter or have a substantial effect on the overall characteristics of the population of cells_regCells) and thus they can be defined as a cell line. Thus, an isolated cell population (or e.g., isolated B cells) typically comprises at least 60%, or 60% to 99%, or 70% to 90% B cells (or subpopulations of B cells, e.g., B cells)_regA cell).

Collection and isolation of B cells

B cells of any origin, also known as B lymphocytes, can be used for collection purposes. As known to those of ordinary skill in the art, such B cells may be derived from bone marrow, spleen, lymph nodes, blood or other allogeneic tissue as a source of B cells. Preferred sources of B cells are bone marrow and blood. Preferably, autologous or allogeneic or xenogeneic B cells are collected.

Using sterile techniques, in one embodiment, bone marrow is preferably obtained from the posterior superior iliac bone. The obtained B cells may be used immediately after isolation and relative purification, may be stored for later use, or may be cultured for a period of time before use. The B cell population in bone marrow contains pre-pro B cells, pre B cells, immature B cells and some mature B cells.

In the present application, the term B cell population encompasses preprobb cells, pro B cells, pre B cells, immature B cells and mature B cells. B cells can be isolated from blood or other tissues using standard techniques known to those of ordinary skill in the art.

Methods for obtaining B cells or e.g. precursor B cells from heterogeneous cell populations are known. Many of these techniques employ primary antibodies that recognize molecules on the surface of desired B cells or B cell precursors, and use these antibodies to actively select these cells and separate them from undesired cells. This technique is called positive selection.

Other commonly used techniques use primary antibodies that recognize molecules on the cell surface to separate the cells from the desired B cells or B cell precursors. In this way, molecules on the unwanted cells bind to the antisera and remove the cells from the heterogeneous cell population. This technique is called negative selection.

A combination of positive and negative selection techniques may be employed to obtain a relatively pure population of B cells or precursor B cells. Such populations are referred to as isolated B cells. As used herein, relative purity refers to a purity of at least 60%, 65%, 70%, 75%, 80%, 85%, 88%, or a higher degree, e.g., at least 90%, at least 95%, at least 97%, or at least 98% to 99% purity.

The skilled person can use a variety of techniques to isolate the cell-bound antibody. Antibodies can be linked to various molecules that provide labels or tags to facilitate separation. In one embodiment, the primary antibody may be attached to magnetic beads that allow separation in a magnetic field. In another embodiment, the primary antibody may be linked to a fluorescent molecule that allows for separation in a fluorescence activated cell sorter. Fluorescent and magnetic labels are typically used for the primary and/or secondary antibodies to effect separation. The secondary antibody bound to the primary antibody may be labeled with a fluorescent molecule that allows for isolation of cells in a fluorescence activated cell sorter. Alternatively, the metal microbeads may be linked to a primary or secondary antibody. In this way, a magnet can be used to separate these antibodies and the cells bound to them.

To achieve positive or negative selection, the heterogeneous cell population is incubated with a primary antibody for a sufficient time to achieve binding of the antibody to the antigen on the cell surface. If the primary antibody is labeled, separation may occur at this step. If a secondary antibody is used, the secondary (anti-primary) antibody is incubated with the cells that bind the primary antibody for a sufficient time to effect binding of the secondary antibody to the primary antibody. If the secondary antibody has a fluorescent label, the cells will be sent through a fluorescence activated cell sorter, thereby isolating the labeled antiserum that binds to the desired cells. If the secondary antibody has a magnetic label, the selected cells form complexes with the primary and secondary antibody-labeled microbeads, which remain behind when passed over the magnet, while other unlabeled cells are removed with the cell culture medium. The positively labeled cells are then eluted and ready for further processing. Negative selection is a collection of unlabeled cells that have been passed through a magnetic field.

Various products were developed by american whirlwind (Miltenyi Biotec) for direct magnetic separation of B cells and different B cell subsets. B cells can be isolated directly from whole blood or buffy coat without density gradient centrifugation or red blood cell lysis, or from Peripheral Blood Mononuclear Cells (PBMCs) after density gradient centrifugation. B cells can be directly isolated and depleted using positive selection and depletion strategies according to standard methods.

Thus, in one working embodiment, patients and potential donors are tested for HLA, for example by the American Red Cross (A, B and DR-B1). Finding potential donors that are haploid matched to the recipient is considered a useful allogeneic donor. Donors were then apheresed to isolate and collect B cells. The B cell product was then prepared for infusion.

After receiving donor allogeneic monocyte-MNC (A), America and whirlpool were usedCD19 selection for enriched apheresis productsB cells. After platelet washing, the product was paired with CD19 microbeads (up to 4x 10 CD19 CliniMACS reagent per vial) separated on LS column¹⁰Total cell and up to 5x 10⁹Individual CD19+ cells) were subjected to CD19+ cell enrichment treatment. The target fraction was washed and then the infusion medium was Plasma-Lyte a supplemented with 25% HSA (1% final concentration).

These methods generally include:

day 1

a. Donor apheresis product is received and sampled for sterility assessment, cell counting, viability assessment, and flow cytometry.

b. The product was refrigerated overnight.

Day 2

a. The product was removed from the refrigerator, mixed well, and allowed to equilibrate to ambient temperature for 30 minutes. Samples were removed for sterility evaluation, cell counting, viability evaluation and flow cytometry (DuraClone panel with CD 20).

b. Platelet washing was performed according to standard CliniMacs procedure.

c. Beads were added according to standard CliniMacs procedures (except incubation at 4 ℃) and incubated on a shaker for 30 minutes.

d. After bead incubation, 1 antibody wash was performed using chilled (4 ℃) media and the product was loaded onto a CliniMacs LS column according to standard CliniMacs procedures:

e. the separation run was performed using the CliniMacs Enrichment 1.1 program.

f. Sampled CD 19-enriched target fraction for cytometric, flow cytometry, stability and sterility assessment

CD19 depleted (non-target fraction) sampling for cytometry and flow cytometry

The isolation of B cells from heterogeneous and stem cell populations may also involve a negative selection process in which the bone marrow is first lysed by placing the bone marrow in a hypotonic buffer and centrifuging the red blood cells out of the buffer. The red blood cell debris remained in the supernatant removed from the tube. Bone marrow-derived cells are then resuspended in a buffer with appropriate conditions for binding the antibody. Alternatively, the bone marrow may be subjected to density gradient centrifugation. After centrifugation, the buffy coat containing bone marrow-derived cells was removed from the gradient. Cells were washed and resuspended in antibody binding buffer and then incubated with primary antibodies against stem cells, T cells, granulocytes and monocytes/macrophages (referred to as lineage depletion) followed by positive selection using antibodies against B cells.

Different B cell subsets can be distinguished based on differential expression of various surface markers and collected accordingly.

Ex vivo stimulation of B cells

Once isolated, B cells may be treated or stimulated by exposing them to one or more TLR agonists or immunomodulatory cytokines, as described herein. Use of such ex vivo stimulation to generate IL-10 producing B_regCells, are considered useful in the methods and treatment strategies described herein.

Administration of

The number of cells to be administered will be related to the area or volume of the affected area to be treated and the method of delivery.

A non-limiting range of B cell numbers for administration is 10⁴To 10¹⁴B cells, depending on the volume of the tissue or organ to be treated. Other ranges include 10⁵To 10¹²B cells and 10⁶To 10¹⁰And (4) B cells. Comprising B cells (e.g., B)_regCells) may comprise 10 in a single dose⁴To 10¹⁴B cell, 10⁵To 10¹²B cell, or 10⁶To 10¹⁰And (4) B cells.

A single injection volume may include non-limiting ranges from 1. mu.l to 1000. mu.l, 1. mu.l to 500. mu.l, 10. mu.l to 250. mu.l, or 20. mu.l to 150. mu.l. The total injection volume per animal ranges from 10 μ l to 10ml, depending on the species, method of delivery and volume of tissue or organ to be treated.

Pharmaceutical composition

The B cells described herein can be incorporated into a vehicle for administration to a patient (e.g., a human patient having a disease or disorder described herein). B cell containing pharmaceutical compositions can be prepared using methods known in the art. Such compositions can be prepared, for example, using physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacology [ Remington: Science and Practice of Pharmacology ], 22 nd edition, Allen, L. ed (2013); incorporated herein by reference) and in The desired form, for example, in The form of an aqueous solution.

The B cells described herein can be administered in any physiologically compatible carrier, such as a buffered saline solution or a solution containing one or more electrolytes, such as one or more of sodium chloride, magnesium chloride, potassium chloride, sodium gluconate, or sodium acetate trihydrate. For example, B cells may be administered in boehmeria force (PlasmaLyte) infusion buffer. Bovieni is a family of balanced crystal solutions in which a variety of different formulations are provided worldwide, according to regional clinical practice and preference. It is very similar to human plasma in terms of electrolyte content, osmotic pressure and pH. The Bo Mai solution has additional buffering capacity and contains bicarbonate convertible to CO₂And water anions such as acetate, gluconate, and even lactate. Advantages of bovines include correction of volume and electrolyte deficiencies while addressing acidosis. In a preferred embodiment, the infusion buffer is Boehmeria force A. Boehmeria is a sterile, pyrogen-free isotonic solution for administration by injection (e.g., intravenously). Every 100mL of Boehmeria force A contains 526mg of sodium chloride (NaCl); 502mg of sodium gluconate (C)₆H₁₁NaO₇) (ii) a 368mg sodium acetate trihydrate, (C)₂H₃NaO₂·3H₂O); 37mg of potassium chloride (KCl); and 30mg of magnesium chloride (MgCl)₂·6H₂O). It does not contain an antimicrobial agent. The pH was adjusted with sodium hydroxide. The pH is about 7.4 (e.g., 6.5 to 8.0).

Other pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid media such as Dartbuck Modified Eagle's Medium (DMEM), sterile saline, sterile phosphate buffered saline, Leibovitz's medium (L15, Invitrogen, carlsbad, ca), dextrose in sterile water, and any other physiologically acceptable liquid.

Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as oils. The carrier may be a solvent or dispersion medium containing: for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. The solution is preferably sterile and fluid to the extent that it is easily injectable. Preferably, the solutions are stable under the conditions of manufacture and storage and prevent the contaminating action of microorganisms (e.g., bacteria and fungi) by using, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. As described herein, the solution of the present invention can be prepared by using a pharmaceutically acceptable carrier or diluent, and if necessary, the other ingredients enumerated above, followed by filter sterilization, and then incorporated into B cells.

For example, if necessary, a solution containing the pharmaceutical composition described herein may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, one skilled in the art will be aware of sterile aqueous media that may be employed in light of this disclosure. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the formulations are required to meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biologics standards.

The pharmaceutical composition may also include excipients to promote cell membrane stability. The infusion medium may be supplemented with, for example, a highly soluble osmotin, e.g., a highly soluble osmotin having a high molecular weight. Serum proteins, such as Human Serum Albumin (HSA), may be included in the pharmaceutical compositions described herein as a media supplement for maintaining cell membrane stability. HSA includes recombinant albumin. Alternatively, human serum can be used to stabilize pharmaceutical compositions comprising cells.

Examples of the invention

The present invention is described in further detail in the following examples, which are in no way intended to limit the scope of the invention as claimed. The drawings are intended to be considered as integral parts of the specification and description of the invention. All references cited are specifically incorporated by reference for all that is described herein. The following examples are provided to illustrate, but not to limit, the claimed invention.

Example 1: modulation of immune infiltration and response by exogenous B cells

This example demonstrates a large-scale analysis of the molecular impact of B cells in wound healing using isobaric labeling multiplex proteomics.

Our data show that B cell application has a significant homeostatic effect on the wound microenvironment, a significant reduction in proteins associated with inflammatory responses, and an increase in proteins associated with tissue growth and remodeling. By recovering applied exogenous B cells from the wound microenvironment at various time points after application, and examining the cell population by multi-color flow cytometry, we determined that mature naive B cells applied to the wound are transformed into a regulatory phenotype characterized by expression of CD138 and immunomodulatory cytokines IL-10, IL-35 and TGF- β. This Breg-like phenotype is transient, with a peak at day 2 post-application. In addition, the phenotype of monocytes and macrophages in the wound environment is significantly altered by B cell application, with reduced expression of pro-inflammatory cytokines including IL-2, IL-4, IL-6 and IFN- γ. Thus, naive B cells placed at the site of injury detect local inflammatory signaling and injury-associated molecular patterns (DAMPs) via TLR and B Cell Receptor (BCR) -dependent pathways, and adopt a regulatory phenotype associated with the production of anti-inflammatory cytokines (preferably IL-10, but also IL-4, IL-35, and TGF-. beta.) that act on neighboring immune and fibroblasts and bias their phenotype towards an anti-inflammatory, pro-regenerative phenotype. Indeed, wound healing studies have shown that B cells lacking the common TLR signaling adaptor myeloid differentiation factor 88(MyD88) or lacking IL-10 lose their pro-regenerative capacity.

Materials and methods

The following materials and methods were used for this study.

Animal(s) production

Wound healing studies were performed in 7-9 week old male wild-type C57Bl6/J mice (Jackson Laboratories). Male WT C57Bl6/J was used as an isogenic donor for B cell isolation. Animals were maintained under standard laboratory care conditions at a temperature range of 20-23 deg.C and were given access to food and acidified water ad libitum during a 12h to 12h light-dark cycle. All Animal procedures were conducted in accordance with Public Health Service policies regarding the humanistic Care of experimental Animals (Public Health Service Policy on human Care of Laboratory Animals) and approved by the Institutional Animal Care and Use Committee of Mass General Hospital. All efforts were made to reduce the number of animals used and to minimize the suffering of the animals.

Cell separation

In cold EasySep^TMMouse spleens were collected in buffer (stem cell Technologies) containing 2% Fetal Bovine Serum (FBS) and 1mM ethylenediaminetetraacetic acid (EDTA) in Phosphate Buffered Saline (PBS). Spleen was mechanically separated through a 40 μm cell filter and spleen cell suspensions were processed using a commercially available cell separation kit (stem cell technology) according to the manufacturer's instructions for negative B or T cell selection by immunomagnetic separation.

Wound model and tissue sampling

As previously described (Wang et al, (2013) Nat protocol. [ nature-experimental techniques ]]8(2):302-9.) induce full-thickness excision of the wound through the back skin. Briefly, mice were anesthetized with a mixture of ketamine (100mg/kg) and xylazine (10mg/kg), and the back skin was shaved and depilated. Analgesia was performed preoperatively by subcutaneous injection of 0.08mg/kg buprenorphine. The dorsal skin was stretched and a 5-mm biopsy punch was placed through the skin folds to create two symmetrical wounds on both sides of the back. Each wound having a diameter of about 20mm²The initial area of (a). Silicone splints (Sigma-Aldrich) with an internal diameter of 7mm were attached around the wound using Vetbond tissue adhesive (3M). Then using Tegaderm^TMA transparent dressing (3M) is covered over the splint wound. A suspension of cells in PBS or an equal volume of PBS solution (saline control) was applied directly to the wound bed using a manual pipette. Each mouse also received two local subcutaneous injections of B cells or saline solution with equal doses under the back skin. Each treated wound or subcutaneous site received 15-20X 10 in 20. mu.l PBS⁶And (4) B cells.

After a defined time interval (18 hours, 2 days or 4 days), O is used₂Mice were lightly anesthetized with 3% isoflurane and 10-20 μ l of a working solution of brefeldin a (golgiplug, BD Pharmingen) in PBS was applied to each of the treated wounds and subcutaneous sites to promote intracellular cytokine accumulation. After 4 hours of incubation, mice were euthanized and tissue biopsies including wounds and subcutaneous injection sites were collected. The product is prepared from 5% FBS, 0.5% L-glutamine, 0.5% penicillin-streptomycin, 1.5mg/ml 0.25U/mg collagenase D (Roche), 1.5mg/ml>400U/mg hyaluronidase from bovine testis (Millipore Sigma), 0.4mg/ml 400U/mg DNase I (Roche), 0.025mg/ml>Tissue biopsy was performed enzymatically at 37 ℃ for 30 minutes in 10U/mg of dispase I (Michibo Sigma) in RPMI medium with gentle shaking. The tissue was then mechanically minced into smaller pieces and then further enzymatically digested for 30 minutes in the same solution at 37 ℃ with gentle shakingA clock. Digested tissues from individual wounds and subcutaneous samples were then pooled for each mouse and passed through a 100 μm cell filter, then through a 40 μm cell filter, resulting in a single cell suspension.

Proteomics

Tissue sampling. As described above, full-thickness excision wounds were generated in the dorsal skin of mice. With a solution of purified B cells (2X 10 in 20. mu.l saline⁶Individual cells), or wound treated with saline control. After defined time intervals (0 days (approximately 10 min), 1 day, 4 days and 10 days), mice (n ═ 3-5/condition) were euthanized and the wound area (including wound margins and subcutaneous layers) were excised and snap frozen in liquid nitrogen, then stored at-80 ℃ until lysis.

Protein digestion and Tandem Mass Tag (TMT) labeling. Sample treatment was performed as described previously (Lapek et al (2017) Nat Biotechnol. [ Nature Biotechnology ]35(10): 983-. Protein concentration of cell lysates was determined by BCA assay (Thermo Scientific). The protein was then reduced with DTT and alkylated with iodoacetamide as previously described. The reduced and alkylated protein was precipitated via methanol-chloroform precipitation. The precipitated protein was reconstituted in 300. mu.L of 1M urea in 50mM HEPES (pH 8.5). Vortexing, sonication, and hand milling were used to aid in dissolution. The solubilized protein was digested in a two-step process, wherein first 3. mu.g of Lys-C (Wako, Japan and Wako pure chemical industries) was digested overnight at room temperature, and then digested with 3. mu.g of trypsin (sequencing grade, Promega) at 37 ℃ for 6 h. The digest was acidified with trifluoroacetic acid (TFA). The digested peptide was desalted by C18 Solid Phase Extraction (SPE) (Sep-Pak, Waters). The concentration of the desalted peptide solution was measured using the BCA assay, and the peptides were dried under vacuum to 50- μ g aliquots, stored at-80 ℃ until they were labeled with TMT reagent. TMT reagent (Thermo Scientific) was suspended in anhydrous Acetonitrile (ACN) at a concentration of 20. mu.g/. mu.L. The dried peptide (50 μ g) was resuspended in 30% ACN in 200mM HEPES, pH 8.5, and 5 μ L of the appropriate TMT reagent was added to the sample. The peptide was incubated with the reagents for 1h at room temperature. The labeling reaction was quenched by the addition of 6 μ L of 5% hydroxylamine. The labeled sample was then acidified by adding 50 μ L of 1% TFA, and the peptide mixture was combined into the TMT10plex sample. The combined samples were desalted on a Sep-Pak column via C18 SPE as described above.

Basic pH reverse phase liquid chromatography (bRPLC) sample fractionation. Sample fractionation was performed by bRPLC39, and fractions were combined for mass spectrometry analysis. Briefly, samples were resuspended in a solution of 5% formic acid and 5% ACN and purified by chromatography on an Agilent (Agilent)1260 HPLC system equipped with a fraction collector, degasser and variable wavelength detector, through a 4.6-mm x 250-mm ZORBAX extended C18 column (5 μm,agilent Technologies) for isolation. The separation was performed by applying a gradient from 22% to 35% ACN in 10mM ammonium bicarbonate at a flow rate of 0.5mL/min over 60 min. As described previously (Edwards et al, (2016) Methods Mol Biol. [ Methods of molecular biology ]]1394:1-13), a total of 96 fractions were combined. The combined fractions were dried under vacuum, reconstituted with a solution of 5% formic acid and 5% ACN, and then analyzed by LC-MS2/MS3 for identification and quantification.

Liquid chromatography was coupled with mass spectrometry. All LC-MS2/MS3 experiments were performed on Orbitrap Fusion (Thermo Fisher Scientific) in combination with Easy-nLC 1000 (Thermo Fisher Scientific) and a cooled autosampler. The peptides were separated on a microcapillary column (inner diameter, 100 μm; outer diameter, 360 μm) pulled out of the laboratory and filled in the laboratory. The column was first packed with about 0.5cm of Magic C4 resin (5 μm,michrom Bioresources company), then filled with about 0.5cm of Maccel C18 AQ resin (3 μm,nest Group company) and then treated with GP-C18(1.8 μm,setaritech Ltd (Sepax Technologies)) to a final length of 30 cm. The peptide was eluted with a linear gradient of 11% to 30% ACN in 0.125% formic acid over 165min at a flow rate of 300nL/min while heating the column to 60 ℃. Electrospray ionization was achieved by applying 1,800V through the PEEK T-joint at the entrance of the microcapillary column.

The Orbitrap Fusion runs in data dependency mode, in Orbitrap at 6 × 10⁴Is performed in the m/z range of 500-1,200. For a full spectrum scan of MS1, the Automatic Gain Control (AGC) is set to 5 × 10⁵Maximum injection time was set at 100ms and Radio Frequency (RF) of S-lens was set at 60. The largest amount of ions detected in a full spectrum scan is subjected to MS2 and MS3 experiments using a "highest velocity" setting that enables the largest number of spectra to be obtained within a 5-s experimental period, and then the next period begins, with another full spectrum full MS scan. For the MS2 analysis, a decision tree option is enabled, selecting precursors based on charge state and m/z range. The doubly charged ions were selected from the m/z range of 600-1,200 because the three-and four-charged ions had to be detected in the m/z range of 500-1,200. The ion intensity threshold was set at 5X 10⁵. In acquiring the MS2 spectra, ions were separated by applying a 0.5-m/z window using quadrupole rods and fragmented using Collision Induced Dissociation (CID) at 30% normalized collision energy. Fragment ions are detected in the ion trap at a fast scan rate. AGC target set to 1 × 10⁴And the maximum ion injection time is 35 ms.

MS3 analysis was performed using synchronized precursor selection (MultiNotch MS3) that maximizes the sensitivity of TMT reporter ion quantitation. Up to 10 MS2 precursors were simultaneously isolated and fragmented for MS3 analysis. The separation window was set to 2.5m/z and fragmentation was performed by HCD at 50% normalized collision energy. Fragment ions in the MS3 spectrum were detected in Orbitrap with a resolution of 60,000, and m/z ≧ 110. AGC target setting 5x 10⁴One ion and the maximum ion injection time reaches 250 ms. M/z in MS2 spectrum is lower than 40Fragment ions of m/z and 15m/z above the precursor m/z were excluded from the selection for MS3 analysis.

And (4) processing and analyzing data. Data were processed using a software suite developed in the laboratory (Huttlin et al, (2010) Cell [ cells ], 143(7): 1174-89). The RAW file is converted to mzXML format using a modified version of ReAdW. exe (http:// www.ionsource.com/functional _ reviews/readw/t2x _ update _ readw. htm). Spectrogram assignment of MS2 data was performed using the sequence algorithm to search Uniprot mouse protein sequence databases, including known contaminants such as trypsin.

The database is appended to include a bait database consisting of all protein sequences in reverse order. The search was conducted at a precursor mass tolerance of 50-p.p.m. Static modifications included the lysine residue and the TMT10plex tag at the N-terminus of the peptide (+229.162932Da), and carbamoylmethylation of cysteine (+57.02146 Da). Oxidation of methionine (+15.99492Da) was included as a variable modification. Data were filtered to a peptide and protein False Discovery Rate (FDR) of < 1% using a targeted bait search strategy (Elias et al, (2010) Methods Mol Biol [ molecular biology Methods ], 604: 55-71). This is achieved by first applying a linear discriminator analysis to filter peptide annotations (peptide-spectrum matching) using the combined scores from the following peptide and spectrum properties (XCorr, Δ Cn, missed trypsin cleavage, peptide mass accuracy and peptide length). The probability of a peptide profile match being correct is calculated using a posterior error histogram, the probability of all peptides assigned to one particular protein is combined by multiplication and the dataset is re-filtered to protein assigned FDR, the entire dataset for all proteins identified in all samples analyzed is FDR < 1%. According to the naive principle (law of parsimony), peptides matching more than one protein are assigned to the protein containing the most matching redundant peptide sequence.

For quantitative analysis, TMT reporter ion intensities were extracted from the MS3 spectra by selecting the strongest ion within a 0.003-m/z window centered on the predicted m/z value of each reporter ion, and signal-to-noise (S/N) values were extracted from the RAW file. If the sum of the S/N values of all reporter ions is ≧ 386, and the separation specificity of the precursor ion is ≧ 0.75, the spectra are used for quantitation. Protein intensity was calculated by summing the TMT reporter ions of all peptides assigned to the protein.

Flow cytometry

To assess cell viability after recovery from tissue digestion, the cell suspension was washed and resuspended in PBS and stained using the Zombie UV fixable viability kit (pocky) for 30 minutes in the dark with gentle shaking at 4 ℃. The stained cells were then washed and resuspended in PBS containing 1% FBS, 0.01% sodium azide (RICCA Chemical, argington, texas) and 5% FcR blocking agent (Miltenyi Biotec, Inc) at 4 ℃ for 10 minutes in the dark. The blocked cells were then incubated with the following fluorophore conjugated primary surface antibodies at 4 ℃ for 30 minutes in the dark: brilliant Violet 785 conjugated rat anti-mouse CD19 (clone 6D5), Alexa700 conjugated rat anti-mouse/human CD45R/B220 (clone RA3-6B2), APC/Cy7 conjugated rat anti-mouse CD138 (clone 281-2) (all from Bosch); brilliant Ultraviolet 395-conjugated hamster anti-mouse CD69 (clone H1.2F3), PE-CF 594-conjugated rat anti-mouse CD140a (clone APA5) (both from BD Biosciences, san jose, ca). The surface stained cells were washed and resuspended in fixation buffer (bolcke corporation (Biolegend, Inc.) for 30 minutes at 4 ℃ followed by permeabilization wash buffer (1X) (bolcke corporation (Biolegend, Inc.). The permeabilized cells were then incubated with the following fluorophore-conjugated primary intrabodies for 30 minutes at 4 ℃ in the dark: brilliant Violet 421 conjugated mouse anti-mouse TGF-beta 1 (clone TW7-16B4), Brilliant Violet 510 conjugated rat anti-mouse IFN-gamma (clone XMG1.2), Brilliant Violet 605 conjugated rat anti-mouse IL-4 (clone 11B11), Brilliant Violet 711 conjugated rat anti-mouse TNF-alpha (clone MP6-XT22), PerCP/Cy5.5 conjugated rat anti-mouse IL-2 (clone JES6-5H4), PE/Cy7 conjugated rat anti-mouse TGF-beta 1 (clone TW7-16B4), Brilliant Violet 510 conjugated rat anti-mouse IFN-gamma (clone XMG1.2), Brilliant Violet 605 conjugated rat anti-mouse IL-4 (clone 11B11), and PerCP/Cy 7 conjugated rat anti-mouse IL-2IL-10 (clone JES5-16E3), APC conjugated rat anti-mouse IL-6 (clone MP5-20F3) (all from Bosch); fluorescein-conjugated rat anti-mouse IFN-. beta.s (clone RMMB-1), PE-conjugated rat anti-mouse IL-27/IL-35 EBI3 subunit (clone 355022) (all from R)&D Systems, Inc., Minneapolis, Minn.). In a BD FACSDiva equipped^TMCells were analyzed on a LSRFortessa X-20 flow cytometer (BD Biosciences, san Jose, Calif.) with software and 355nm, 405nm, 488nm, 561nm, and 640nm lasers. At least 100,000 events were collected from each sample for analysis. Data were analyzed using FlowJo software version 10.3 (TreeStar, ashland, oregon).

Immunohistochemistry

Wound biopsies collected on day 0 (intact), day 1, day 4, day 10 and day 16 post-injury were fixed in 4% buffered paraformaldehyde for 24-48 hours at 4 ℃ followed by re-cryoprotection in 1M sucrose solution for 24-48 hours at 4 ℃ and embedded in tissue freezing medium (Electron Microscopy Sciences). A cross section through the wound bed was cut to a thickness of 10 μm using a cryostat (Leica Biosystems) and thaw mounted on SuperFrost Plus Gold glass slides (Fisher Scientific). For immunohistochemical detection of antigens, sections were washed by three portions of Tris Buffered Saline (TBS) pH 7.4, then permeabilized and blocked by incubation at room temperature for 1 hour with TBS containing 5% bovine serum albumin, 5% FBS and 0.3% Triton X-100. The sections were then incubated overnight at 4 ℃ with the following primary antibodies diluted in blocking solution: APC-conjugated rat anti-mouse CD45R/B220 (clone RA3-6B 2; Bosch Corp. (BioLegend, Inc.)), PE-conjugated rat anti-mouse CD31 (clone MEC 13.3; BD Biosciences), Alexa488-conjugated mouse anti-tubulin beta 3 (clone TUJ 1; Bosch Corp.), Alexa488-conjugated rat anti-mouse F4/80 (clone BM 8; Bosch), PE-conjugated rat anti-mouse CD11b (clone M1/70; Bosch), rabbit polyclonal anti-Ki 67 (Abcam), and rabbit monoclonal anti-activated caspase 3 (clone C92-605; BD Pharmingen). Unbound primary antibody was removed by washing 3 times in TBS, 5min each. If unconjugated primary antibody is used, the sections are incubated with Alexa FluorConjugated F (ab') 2-goat anti-rabbit IgG (Thermo Fisher Scientific) (1:200 diluted in blocking solution) were incubated together for 2 hours at room temperature to visualize the antigenic sites. Sections were counterstained by incubation with 2. mu.g/ml 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich) in PBS at room temperature for 3 min. Sections were washed 3 times in TBS for 7min and embedded using fluorocount (Novus Biologicals). Antibody controls included incubating tissue sections with isotype antibodies and omitting primary antibody when visualization was performed using secondary antibody. No non-specific signal was detected in the control sample.

The stained tissue sections were imaged using a Zeiss LSM 710 laser scanning microscope (Carl Zeiss) equipped with 20 x, 40 x and 63 x objective lenses. Confocal images were taken using Zen software (Carl Zeiss) at a resolution of 0.1-0.7 μm/pixel and an optical thickness of 0.5-2.2 μm.

Histology

Wound biopsies at the end of the wound healing time course were collected using a 10-mm biopsy punch and fixed in 4% paraformaldehyde in PBS for 24-48 hours at 4 ℃, after which the samples were dehydrated through graded ethanol and xylene washes and embedded in paraffin. The cross section through the wound bed was cut to a thickness of 5 μm and mounted on a microscope slide. Serial sections were stained with hematoxylin and eosin, and stained with metson's trichrome stain to visualize collagen fibers. The stained slides were digitized with an Aperio CS2 scanner (Leica Biosystems) at a resolution of 0.25 μm/pixel. Tissue regeneration was scored using digitized slides by experimenters blinded to treatment conditions (blinded).

Statistics of

The time-dependent effect of B-cell application on target cell marker expression was evaluated using linear mixing effect modeling in SPSS 23(IBM Corporation) for each cell population examined separately. For variance stabilization, the proportion of gated cells for each sample and examination marker was logarithmically (logarithmically of probability) transformed prior to analysis. Survival time (18, 45 or 93 hours), environment (wound, subcutaneous or B cell only, ice), markers and conditions (B cell treatment or saline) if applicable were included as fixed factors using a three-way (or four-way, if applicable) all-factor design. Technical (date of run) and biological replicates (mouse/sample ID) were included as random effects. The Dunn-Sidak method was used to adjust the post hoc comparisons between fixed factor levels for multiple comparisons. P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

Results

B cell application induces complex changes in the molecular microenvironment of a wound

Proteomic analysis of whole wound lysates identified up to 9125 proteins in all samples and treatments. For the analysis between treatment conditions and cross-time points, only the proteins present in all samples (n-30 animals) were considered. This resulted in a total of 3809 proteins (fig. 12).

A schematic of the average duration of the major phases of wound healing in the wild type murine wound model is shown in fig. 1A. A thermal map summarizing the expression dynamics over time of proteins that significantly change expression in response to B cell application is shown in fig. 1B. A total of 213 proteins, which represent aggregates of significantly altered proteins associated with B cell therapy (n 111; p <0.05, unpaired t-test), and those with high fold changes at each time point (the first 20 upregulated or downregulated proteins), were classified according to the process associated with wound healing, regardless of the level of significance (n 112). The heat maps show fold change expression after B cell treatment at days 0, 1, 4, 10 post injury. Red-up; green-down-regulation. Of particular note is the down-regulation of a variety of proteins associated with inflammation and inflammatory cells at day 4 post-injury, and the massive up-regulation of proteins associated with cell proliferation, prevention of apoptosis (cell death) and oxidative stress, and tissue remodeling (formation of hair follicles and muscle) at days 4-10 post-injury.

Expression of proteins by functional families

The average expression of proteins of the functional family over time in wounds after saline (control, conventional wound healing) or B-cell treatment is shown in figures 2A to 2H. This analysis demonstrates the overall effect of B cells as homeostatic agents, rather than inducers or inhibitors of protein expression. B cell use is associated with maintaining stable protein expression levels (these proteins typically decrease or increase during injury and healing processes), which significantly reduces the inflammatory peaks observed during normal healing processes, prevents the reduction of anti-apoptotic factors (arrows) and oxidative stress protectors, and increase proliferation (FIG. 2A-FIG. 2B), reduce decrease in antioxidant stress protectant and cell proliferation, and maintaining cell migration at a lower level (FIGS. 2C-2D), maintaining a steady level of proteins associated with remodeling and secondary skin structure (FIGS. 2E-2F), reducing the level of protein degradation and autophagy observed at the early stage of injury in controls, and increasing the levels of proteins associated with angiogenesis and nerve regeneration at the later stages of healing (fig. 2G-fig. 2H).

Unsupervised hierarchical cluster analysis of the identified proteins expressed in the skin wound samples is depicted in fig. 12. Only the identified proteins that were consistently present in all samples were included in the analysis. Hierarchical clustering of the well-linked 3809 proteins (rows) consistently expressed in the wounds of all B-cell and saline-treated animals at 4 different time points (columns) post-injury is shown in fig. 12A. The pseudo-color scale depicts the normalized, log-fold-transformed change expression values for each protein. The dendrogram shows 15 protein clusters derived from this analysis, where (fig. 12A) the color of each cell maps to the mean expression value of the cluster at the corresponding time point. Proteins are clustered according to their expression pattern over time. In fig. 12B, a heatmap showing hierarchical clustering of all 3809 proteins from (fig. 12A) is depicted. The gene ontology analysis of 15 hierarchical clusters is shown in fig. 12C. A list of mouse GOslim genes from QuickGO (accessible from https:// www.ebi.ac.uk/QuickGO) was used to probe 15 hierarchical clusters. The bar graph shows the top biofunctional categories for each cluster.

The distribution of significantly altered proteins in response to B cell treatment at each of the assessed time points during wound healing was determined and is shown in figure 13.

In vivo evaluation of B cell application in acute wound healing

An experimental example of the in vivo evaluation of B cells for use in acute wound healing is depicted in fig. 3. A total of 4 full-thickness lesions were generated in the dorsal skin of wild-type C57Bl6 mice, and mature primary B cells purified from syngeneic animals were applied directly to the wound bed. Control animals received saline application. As an internal control, also under intact skin, B cells or saline controls were injected subcutaneously to provide a similar microenvironment without injury. After a defined survival time, wound or skin undamaged tissue is collected, dissociated, and processed for flow cytometry analysis. The scatter plot on the right shows a typical distribution of cell suspension for each treatment class. The wound samples showed a characteristic influx of leukocytes (white open arrows), which was essentially absent in the undamaged tissue. Although there are typically few B cells at any one location, they are easily detected in large numbers after experimental application (solid red arrows).

Analysis of B cell treated and control wound cell suspensions via flow cytometry

To assess the change in B cells and cells of the wound environment between different conditions and time points, samples were analyzed using flow cytometry. We performed gating strategies and analyses on B-cell treated and control wound cell suspensions via flow cytometry are depicted in fig. 4. Viable cells are divided into 3 main categories: b cells (CD19+/B220+ lymphocytes); non-B cell leukocytes (CD140 a-/B220-leukocytes) comprising a mixture of neutrophils, monocytes and macrophages, dendritic cells and T cells; and fibroblasts (CD140a +/B220-). The activation markers and cytokine production of these cell classes were evaluated.

Dynamics of activation markers and key cytokines in B cells recovered from wound beds

The dynamics of activation markers and key cytokines in B cells recovered from the wound bed after defined intervals of exposure to the wound microenvironment are shown in figure 5. B cells were exposed to the wound microenvironment in vivo, or injected under intact skin (control equivalent sites). For comparison, control B cells maintained on ice for the same duration immediately after isolation are shown. After time intervals including 18 hours, 2 days, 4 days and 10 days, the wounds were treated with brefeldin a for 4 hours to induce intracellular cytokine retention. B cells were then recovered by excision and dissociation of the tissue, and surface markers and intracellular cytokines were further characterized by flow cytometry. B cells exposed to the wound microenvironment transiently upregulate a variety of immunoregulatory cytokines, peaking at 2 days post-application. Some immunoregulatory cytokines, including TGF β and IL-6, remain elevated on day 4, and IL-10 remains elevated for up to 10 days. N-3-6 animals/group.

Heatmap analysis

A summary of the heat maps of the mean values for each marker in B cells exposed to the wound microenvironment, subcutaneous controls, or maintained on ice (unexposed) is seen in fig. 6. The dynamics of activation markers and key cytokines in the aggregated infiltrating non-B cell leukocytes in the wound are shown in fig. 7. In conclusion, infiltrating leukocytes produce more of the anti-inflammatory cytokines IL-10, TGF β and IL-35, and less of the pro-inflammatory TNF α and IL-2 when B cells are present in the wound. This effect was most pronounced at day 4 after injury and B cell application and lasted for up to 10 days. N-3-6 animals/group.

A summary of the heatmap of the average values of each marker in infiltrating non-B cell leukocytes in the wound microenvironment (illustrating the pattern of increased production of anti-inflammatory cytokines (IL-10 and TGFb) in the presence of B cells) is shown in figure 8.

The dynamics of activation markers and key cytokines in CD140a + fibroblast population in wounds and subcutaneous tissues are shown in figure 9. When the wound was exposed to B cells, fibroblasts in the wound produced significantly more IL-10 and TGF β on day 10 post injury. In addition, wound fibroblasts produced less of the pro-inflammatory cytokine TNF α when B cells were applied at days 4 and 10 post-injury.

Additional heat maps summarising the mean values of each marker in fibroblasts in wounds and subcutaneous tissue treated with B cells or saline solution are shown in figure 10. Fibroblasts are one of the most important sources of anti-inflammatory and pro-regenerative factors in wound healing and produce high levels of IL-10 and TGF β regardless of treatment. However, on days 4 and 10 post-injury, fibroblasts from wounds treated with B cells continue to produce higher levels of IL-10 and TGF β, while in saline-treated wounds the levels of these anti-inflammatory cytokines are reduced. Interestingly, a significant effect of B cell application was observed in the reduction of pro-inflammatory cytokines (including IL-6 and TNF α) in wound fibroblasts.

TLR signaling and IL-10 production are essential components of the regenerative function of exogenous B cells in wound healing

Functional TLR signaling and IL-10 production are essential components of the regenerative function of exogenous B cells in wound healing as shown in figure 11. Whole-thickness resection wounds (described here on day 6 of healing) were treated on day 0 with B cells lacking the common TLR signaling adaptor myeloid differentiation factor 88(MyD88), IL-10, or WT B cells as controls. Saline was also included as an internal control in each test animal. Although WT B cells continued to accelerate wound closure in WT animals for 2-3 days, MyD 88-/-or IL-10-/-B cells had no benefit on wound closure, similar to saline application.

Example 2: results after B cell treatment to improve TBI

This example demonstrates that exogenously applied B cells significantly improved post-injury performance in the mouse TBI model.

Cerebral contusion leads to neurological dysfunction, mediated in part by an inflammatory response to injury. B lymphocytes are dynamic regulators of the immune system and have not been studied systemically in TBI. Using the mouse Controlled Cortical Impingement (CCI) model, we assessed the possible beneficial effects of exogenously applied B cells on histopathological and functional outcomes following TBI. Injecting 2X 10 to the brain parenchyma of mice at the lesion site⁶Mature, initially syngeneic splenic B cells are then subjected to CCI. Control CCI mice received equal amounts of T cells or saline, and sham-injured mice (craniotomy only) were given B cells or saline. The sham-injured group performed similarly in both exercise and learning tests. Injured mice administered B cells showed significantly improved post-injury rotarod, Y-maze, and Morris Water Maze (MWM) performance compared to the saline or T cell-treated CCI groups. Furthermore, lesion volume was significantly reduced by 40% and astrocyte proliferation and microglial activation were reduced in B cell-treated mice at day 35 post-TBI compared to saline and T cell controls. In vivo tracking of exogenous B cells showed that they had a limited life span in situ, approximately 14 days, and did not appear to proliferate. The data indicate that topical administration of B lymphocytes represents a treatment option for treating cerebral contusions, particularly when clinical management involves procedures that allow access to the site of injury.

Thus, the studies described below investigated the potential of mature primary B cells to prevent cognitive and histopathological deficits in the mouse CCI TBI model. Single dose B cells delivered by intraparenchymal brain injection at the time of injury were associated with significant improvement in hippocampus and striatal-dependent behavioral tasks compared to administration of splenic T cells or saline. The observed behavioral improvement was associated with a significant reduction in lesion volume in animals treated with B cells, while retaining hippocampal structure. In vivo tracking of exogenously applied B cells following intraparenchymal injection of the brain revealed limited in situ survival of these cells, approximately 2 weeks, suggesting that they would represent a safe, viable option for treating acute and subacute contusion TBI.

Materials and methods

The following materials and methods were used for this study.

Animals: all animal procedures were performed in accordance with NIH laboratory animal care and instructions and public health services policies for laboratory animal humanity care. All protocols were approved by the Institutional Animal Care and Use Committee of Mass Institutional of Mass General Hospital. The study was conducted in adult 12-14 week old male C57Bl6/J mice weighing 25-32g (Jackson Laboratories, Balport, Maine). Male C57Bl6/J and FVB-Tg (CAG-luc-GFP) L2G85Chco/J (both from Jackson Laboratories, Balport, Maine) were used as isogenic donors for B and T cell isolation. Animals were housed peacefully (4-5 individuals per cage) and maintained under standard laboratory care conditions at a temperature range of 20-23 ℃ with food and acidified water available ad libitum under a 12h light-dark cycle. Animals were age matched and randomly assigned to experimental conditions. To avoid bias, animals from different treatment arms were housed together.

Lymphocyte separation: cell isolation Using negative immunomagnetic selection, as described previously¹⁶. Briefly, mouse spleens were collected in ice-cold buffer containing 2% Fetal Bovine Serum (FBS) and 1mM ethylenediaminetetraacetic acid (EDTA) in Phosphate Buffered Saline (PBS). Spleen was mechanically dissociated by a 40 μm cell filter and spleen cell suspensions were treated using a commercially available cell isolation kit (stem cell technologies, Vancouver, Canada) according to the manufacturer's instructions for negative B or T cell selection by immunomagnetic separation and retention of non-target cells. Validation of the B cell isolation procedure by flow cytometry analysis, and typically results>Mature primary CD45R of 98% purity⁺/CD19⁺B lymphocyte populations, in which contamination by other leukocytes is less than 1%, although some residual erythrocytes may be present.¹⁶Purified lymphocytes are 4X 10⁵The concentration of individual cells/. mu.l was resuspended in sterile PBS.

Controlled skin impact (CCI): all surgical procedures, including injury and application of cells or saline, were performed by experimenters unaware of treatment conditions (blinded), who were not involved in the preparation of treatment doses for injection. Fluotec3 evaporator (Colonial Medical, Wendham, N.) at 70% N₂O and 30% of O₂The mixture of (1) was anesthetized with 4.5% isoflurane (Baxter, dilfield, il) for 90s and placed in a stereotaxic frame. Anesthesia was maintained with 4.5% isoflurane. After an inside scalp incision, craniotomy was performed on the left parietal temporal cortex using a portable drill and 5-mm trephine, and the bone flap was discarded. Ipsilateral intraparenchymal injection was performed at approximately-1 mm from bregma, at +2mm medial/lateral on the anterior/posterior axis, through the left parietal cortex at a depth of 3 mm. A total of 5. mu.l of saline solution containing 200 million B cells, 200 million T cells, or no cells was injected using a 10-ul Hamilton syringe (Hamilton Company, Franklin, Mass.) with a 26s blunt needle. The selected cell dose has been previously optimized in a skin lesion model with similar lesion volume¹⁶. Cell application was performed just prior to CCI to ensure correct and consistent injection of cells while the brain structure was intact. Immediately thereafter, mice were subjected to CCI using a pneumatic cylinder with a 3-mm flat head impactor (impactor), at a speed of 6m/s, a depth of 0.6mm, and an impact duration of 100-ms. Sham-injured mice received anesthesia, craniotomy and intraparenchymal injection of equal amounts of B cells or saline, but no CCI injury. Craniotomy remained open and the skin was sutured to the skull using 6-0 nylon suture (Thermo Fisher Scientific), waltham, massachusetts).

Behavior test scheme: behavioral testing was performed during the light phase of the circadian cycle by experimenters unaware of the treatment conditions (blind). Mice were acclimated for at least 30min prior to each test. Mice were tested in a series of assays according to the protocol described in figure 14. Vestibular motility was assessed on days 1, 3 and 7 post-injury by a wire-puller assay. Rotarod testing was performed on days 7, 9,10, 13 and 14 post-injury. Animals were evaluated for anxiety using the elevated plus maze assay on day 17 post-injury. The Morris Water Maze (MWM) test was performed on days 20, 21, 22, 23 and 24 post-injury, with the probing test performed on day 27. On day 29 post-injury, mice received a forced swim test to determine depression-like behavior, and the Y maze was performed on day 30, a hippocampus-dependent working memory assay.

Testing a wire grip: vestibular motor function was assessed using the wire puller test (Bermpohl et al, (2007), J Cereb Blood Flow Metab [ J. cerebral Blood Flow and metabolism ]27, 1806-1818). The mice were placed on a 45-cm length of wire, which was suspended 45cm high from the ground and allowed to cross the wire for 60 s. The delay of a fall over a 60s interval was measured and the puller score was quantified using 5 scores. The tests were performed in triplicate and the average per mouse was calculated on each test day.

A rod rotating instrument: mice were placed on an automatic rotarod Apparatus (Harvard Apparatus, holliston, massachusetts) that was accelerated from 4r/min to 40r/min over 60 s. The longest duration of the test was 300s, or until the mice fell off the rotarod. Each mouse was evaluated five times per day with a rest interval of 5 min. The average decay delay and the average r/min velocity achieved in five trials were recorded each day of the test.

MWM: MWM was performed as described previously with minor modifications (Mannix et al, (2013) Ann neuron [ neurological annals ]74, 65-75). Spatial learning is evaluated at approximately the same time each day. Each mouse was subjected to seven hidden platform trials (one to two trials per day) using a random set of starting positions in any of the four quadrants. One test consisted of: average delay from each of the four start positions. If the mouse fails to find a platform within 90s, it is placed on the platform for about 10 s. The probe test was performed 24h after the last hidden platform test, allowing the mice to swim in the water tank without the platform for 30s, and recording the time spent in the target quadrant.

Boswell forced swimming test: the mice were placed in a cylindrical transparent glass jar of 30cm (height). times.20 cm (diameter) filled with water (25 ℃) to a height of 20 cm. A white foam box provides visual shielding on three sides. The mice were placed in water for 6min and swimming movements were recorded. The total activity time (swimming, paw scratching/climbing the beaker wall) versus the inactivity time (passive flotation) was quantified in the last four minutes of the test.

Spontaneous alternation test of Y maze: the Y-maze test was performed in a device composed of white opaque acrylic, connected by three 40-cm long arms at 120 ° angle, with a wall height of 15 cm. Each arm is marked with a different contrasting visual cue (black and white squares, circles, stars). The mouse was placed in the center of the device and allowed to explore the maze for 10 min. Their movements were recorded using a webcam directly overhead and Photo Booth software (ANY-maze). Normal exploratory behavior of rodents includes a preference to enter a maze arm with fewer recent visits (spontaneous alternation). The alternation score is calculated by dividing the number of three consecutive selections comprising one instance of each arm by the total number of arm entries (i.e., alternation opportunities). The device was cleaned with 70% ethanol between trials.

Elevated cross maze: the device consists of two 130 x 8cm platforms, the intersection of which has an 8 x 8cm square area that is 60cm above the ground. The closing arms of the platform have 10cm walls, while the opening arms have no walls. Each mouse was placed in the central area of the maze and video recorded for 5 min. The device was cleaned with 70% ethanol between trials. The average speed and time percentage of the closed and open arms of the video recording were analyzed by the ANY maze (stockling corporation, wood dall, illinois) software.

IVIS imaging: spleen B cells were isolated from CAG-luc-eGFP L2G85 transgenic homozygous mice showing extensive expression of firefly luciferase and enhanced green fluorescent protein under the CAG promoter (Jackson Laboratories, Balport, Maine). Approximately 500 million luciferase-expressing B cells in 5. mu.l PBS were injected into the left hemisphere of the recipient WT C57Bl6/J mouse as described above. Mice were imaged periodically on the day of surgery and thereafter for a total of 4 weeks using the IVIS luminea II system (Caliper Life Sciences, waltham, ma). For each imaging phase, anesthesia was induced using 3% isoflurane in oxygen and maintained using 2% -3% isoflurane at 1l/min throughout the imaging phase. To visualize luciferase activity, 100 μ l of 30mg/ml aqueous D-luciferin solution (Regis technologies, Mo. Grov., Ill.) was injected subcutaneously proximal to the lesion site at least 6 minutes prior to imaging. Mice were imaged for 10min and the same parameters were maintained for each repeat of imaging.

Tissue sampling: on day 35 post CCI and treatment, mice were deeply anesthetized with ketamine (100mg/kg) and xylazine (10mg/kg), perfused through the heart with 10-15ml heparinized PBS to remove blood, and the head removed. The brain was rapidly extracted on ice, frozen in liquid nitrogen vapor, and stored at-80 ℃. For frozen sections, brains were embedded in M-1 embedding matrix (Thermo Fisher Scientific, waltham, massachusetts) and coronal sections were performed at a thickness of 16 μ M using a cryostat. Sections were collected at 500 μm intervals along the head-to-tail axis and thawed and mounted on SuperFrost Plus Gold slides (Fisher Scientific, Waltham, Mass.).

Immunohistochemistry: tissue treatment for immunohistochemical analysis tissue treatment was performed as previously described (Et al, (2017) Wound Repair Regen [ Wound Repair and regeneration],25,774-791). Briefly, sections were washed with PBS, then permeabilized and blocked by incubation with PBS containing 5% bovine serum albumin, 5% fetal bovine serum, and 0.3% Triton X-100 for 1 hour at room temperature. The sections were then incubated overnight at 4 ℃ with the following primary antibodies diluted in blocking solution: alexa594 conjugated rat anti-mouse CD45R/B220 (clone RA3-6B 2; Boche, san Diego, Calif.), Alexa488-conjugated mouse anti-mouse CD45.1 (clone A20; Bochki, san Diego, Calif.), Alexa488-conjugated mouse anti-Glial Fibrillary Acidic Protein (GFAP) (clone 2E1. E9; Bocke, san-Chi, Calif.), Alexa647 conjugated rat anti-mouse CD68 (clone FA-11; Bosch Corp., san Diego, Calif.) and rabbit polyclonal anti-Ki 67 (Ebos Corp., Cambridge, Mass.). Unbound primary antibody was removed by washing 3 times in PBS. If unconjugated primary antibody is used, the sections are incubated with Alexa FluorConjugated F (ab') 2-goat anti-rabbit IgG (Thermo Fisher Scientific, Waltherm, Mass.) (1:200 diluted in blocking solution) were incubated together at room temperature for 2 hours to visualize the antigenic sites. Sections were counterstained by incubation with 2. mu.g/ml 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich). Antibody controls included incubating tissue sections with isotype antibodies and omitting primary antibody when visualization was performed using secondary antibody. No non-specific signal was detected in the control sample. The stained tissue sections were imaged using a Zeiss LSM 710 laser scanning microscope (Carl Zeiss) and confocal images were collected using Zen software (Carl Zeiss).

And (3) measuring the volume of the lesion: staining the sections with hematoxylin and collectingHigh resolution overview pictures of slides. Morphometric image analysis in ImageJ (NIH, besiesda, maryland) was used to determine the area of each hemisphere. For each section, the area of the injured hemisphere (left) was subtracted from the area of the uninjured hemisphere, and the difference was multiplied by 0.5 to obtain the volume of brain tissue lost in mm³And (4) showing.

Image analysis: to unbiased quantification of GFAP and CD68 immune markers, confocal images collected around The lesion site, including medial and lateral aspects of The lesion area (n-4 1400 × 1400 μm fields/animal) were analyzed using standard functions in MATLAB (meiswokwalk, The MathWorks, Inc.), nabeke, ma). For each image, the target region of analysis is semi-automatically defined by morphological closing, morphological opening, and interactive filling of a binary mask generated by applying a minimum intensity threshold of 10 to the maximum intensity projection of all imaging channels. The background level was determined by image smoothing using a 3x 3 pixel moving average, followed by separate morphological opening for each channel using a 10 pixel radius structuring element. Foreground objects were identified as a set of pixels with intensities exceeding the corresponding background levels, with GFAP exceeding 25 and CD68 immunolabeling exceeding 50, respectively. For each marker analyzed, the relative area of the marker is calculated as the fraction of pixels within the target region labeled as foreground, and the average marker intensity is calculated as the average intensity of all foreground pixels found within the target region. All image analyses were performed by experimenters who were unaware of the processing conditions (blind).

Statistical analysis: prior to statistical testing, the normal distribution of all datasets was assessed using the agostio Pearson (D' Agostino & Pearson) normality test and found to pass (p > 0.2). Statistical significance of differences between experimental groups of repeated behavioral tests, including wire puller assay, rotameter, MWM hidden and visible platform, and elevated plus maze, was assessed using a two-way (treatment x time) repeated measures ANOVA (with trial/time point as repeated measure factor) with matched subjects, followed by multiple comparisons post hoc using the graph-based or sidack method. Single time point measurements, including MWM probing, Y maze, forced swim, and histological comparisons, were evaluated using one-way ANOVA followed by a graph-based multiple comparison test. All reported descriptive statistics are estimated marginal means ± mean Standard Error (SEM). All statistical analyses were performed using GraphPad Prism 7(GraphPad Software, la, ca). P <0.05 was considered statistically significant.

Results

Cognitive function recovery after improving CCI by B lymphocyte application during injury

A total of 63 mice completed the study, of which 1 died. Treatment of brain parenchyma with B lymphocytes was significantly protective in rotarod assays (fig. 15A). Although all lesion groups performed worse than sham-treated variants, the B cell treated lesioned mice performed better than the lesioned T cell or saline treated groups (p <0.01 for this group) and their performance was not different from that of the sham B cell treated surgery. Interestingly, successive experiments showed that the performance of the CCI + B cell group, as well as the two sham control groups, continued to improve, indicating that programmed learning occurred in these groups. There were no significant differences between the CCI group and sham-injured mice for B cell treatment, except for trial 3, where B cell treated sham-injured mice performed better (p < 0.01). In contrast, CCI mice treated intraparenchymally with saline or 200 ten thousand T cells at the time of injury exhibited minimal daily improvement and delay in falls was significantly reduced (p <0.0001) compared to either B cell treated CCI injured or sham injured animals. There were no statistically significant differences between CCI-injured groups receiving T cells or saline.

All lesion groups performed worse than sham surgery in the puller test, but no group differences were observed between the CCI groups treated with B cells, saline, or T cells (fig. 15B).

In MWM (fig. 16A-16D), the performance of mock-lesioned mice was significantly better than all CCI groups in the hidden platform trial, indicating a strong effect of CCI (p <0.001 for this group), with no differences noted between the mock-lesioned groups. In the lesion group, B cell treated mice had a very significant treatment effect (p <0.0001) compared to saline treated mice in test 7, with a significant treatment x test interaction (p 0.02) (p 0.016, fig. 16A). The performance improvement rate during 7 hidden platform trials was significantly higher in injured animals receiving B cells (compared to saline control group) (p <0.01) as estimated by the difference in mean plateau time of arrival between trial 1 and trial 7. No significant differences between any of the groups were observed in the visual platform trial (fig. 16B). In the probe trial, the injured B cell-treated mice behaved similarly to the sham group (fig. 16C). In contrast, CCI-injured mice receiving T cells or saline performed worse than sham surgery (p < 0.05). Swimming patterns of injured B cell-treated mice demonstrated evidence that the spatial search strategy was similar to those of the sham-injured group, whereas the search strategy was non-spatial in the injured T cell and saline-treated groups (fig. 16D).

In the Y maze test (FIG. 16E), the alternating score for CCI injured mice receiving B cells within the brain parenchyma was 76.65. + -. 2.46, significantly higher than that of injured animals treated with T cells (46.86. + -. 2.41) or saline (38.92. + -. 2.56; p <0.0001), and similar to that of sham-injured B cell-treated mice. There was no significant difference between any of the lesions or treatment groups in terms of total number of maze arm entries (p >0.13) or total distance covered (p > 0.07).

In the elevated plus maze (fig. 17A), there was no significant difference in the time spent in the open arms (p >0.84) or the central starting region (p >0.94) between any treatment groups. There was significant treatment x positional interaction (p < 0.05). In the lesion group, mice receiving B cells spent significantly more time on the closed arms of the maze (p <0.05) than animals receiving the same number of T cells.

The sham and lesion groups did not differ from each other in the forced swim test, indicating that the lesion paradigm employed here did not affect the depression-like behavior as measured by this assay (fig. 17B).

B lymphocyte application at injury was associated with decreased lesion volume and glial scar 35 days after CCI

To assess whether the observed behavioral improvement associated with B cell processing was associated with neuropathology, brains of all animals receiving behavioral assessment were collected for histological examination on day 35 post-injury (fig. 18). Analysis of brain tissue damage in the CCI group showed cavitation of the lesion area in all mice receiving CCI, while the sham group showed no brain tissue loss (fig. 18A). Quantitative analysis of lesion volumes for all groups (fig. 18B) showed significant effect of CCI as expected (p < 0.0001). Brain tissue loss was significantly less in B cell treated CCI mice compared to the injured group treated with saline (p <0.001) or T cells (p < 0.0001). There was no significant difference in lesion volume between CCI lesion groups receiving T cells or saline. Analysis of lesion area distribution for serial sections throughout the brain (fig. 18C) showed that the most significant difference between treatment conditions was observed at 2/3 after the lesion, where the hippocampus was most involved. Indeed, targeted volume measurements showed that B cell treated CCI mice had a significantly greater proportion of surviving hippocampal tissue in the injured hemisphere compared to saline or T cell treated CCI controls (p < 0.0001; FIG. 18D).

To assess the effect of B cell treatment on the long-term reactive responses of the injured brain, including astrocytosis and microglial activation, tissue sections collected on day 35 post-injury were immuno-labeled for GFAP and activation-related marker CD68, respectively. Unbiased analysis of confocal images collected from locations adjacent to cavitation lesions formed at the site of initial CCI injury revealed significant effects of B cell treatment at injury compared to saline and T cell treatment controls (fig. 19). In B cell treated animals, glial scarring was significantly reduced, as indicated by the strong GFAP immune marker and extensive astrocyte hypertrophy, as was the immune marker for the microglial activation marker CD68 (fig. 19E, 19F).

B lymphocytes do not proliferate in situ and have a limited lifespan after application, approximately 2 weeks

To determine whether exogenously delivered B cells persist in vivo following injection into the brain parenchyma, we used purified B cells that constitutively express firefly luciferase under the CAG promoter. Since the biological half-life of luciferase is short, it must be produced continuously for detection. When luciferase-expressing cells die, the signal rapidly disappears, and thus this assay can be used to determine the long-term survival of experimentally introduced B cells (Zinn et al, (2008) ILAR J49, 103-115). This method allows non-invasive tracking of the viability and persistence of cells over time, up to hundreds of cells, since only exogenous living B cells will produce optical signals. The results show that injected B cells were clearly detectable in all animals examined immediately after application (fig. 20A). Quantitative analysis of the total photon flux in the head region showed that the active enzyme activity in the applied cells initially showed a modest increase, peaking at days 3-7, and then rapidly declining after day 7, reaching undetectable levels at day 17 post-injection (fig. 20B). These results support the notion that exogenous B cells have a limited lifespan of approximately 2 weeks in situ.

To investigate whether the introduced B cells could proliferate in situ, and thus potentially create a longer lived population within the brain parenchyma, coronal brain sections were collected across the injection site and the B cells and proliferation markers were immunolabeled. Labeling B cells ex vivo with toluidine blue prior to injection showed that cells were only distributed within a radius of about 1mm around the injection site after delivery (fig. 21A). Confocal imaging confirmed that injected B cells remained well localized at the application site up to 4 days after delivery with some dispersion throughout the lesion (fig. 21D). The immunological labeling of the proliferation marker Ki67 showed that B cells did not express this marker at the time points of day 0 or day 4, whereas neighboring non-B cells showed high levels of Ki67 immunopositivity at day 4 post-injury in all animals examined (n ═ 4 animals per time point; fig. 21D, 21E). This finding is consistent with the fact that B cells could not be observed on day 35 post-application in CD45R (B220) and CD45.1 immunolabeled tissue sections from injured or pseudodiseased animals (data not shown). Although the original injection site was cavitated at day 35 in CCI injured animals, it could be easily detected in sham controls due to needle-induced damage-surrounding gliosis (fig. 21F). By day 35, no B220-positive B cells were found at the injection site in the sham operation of B cell treatment (fig. 21F, 21G).

Conclusion

To our knowledge, this study describes for the first time the direct application of B lymphocytes to modulate structural and functional consequences after injury in preclinical TBI models. We found that a single delivery of purified (> 95%) mature naive B cells within brain parenchyma in CCI can significantly reduce learning and memory deficits following injury, and reduce brain tissue loss. The results indicate that endogenous B cells have a hitherto unknown protective effect in a model of cerebral contusion.

The CCI lesion model produces highly reproducible lesions and very low mortality (Xiong et al, (2013) Nat Rev Neurosci [ natural review of neurology science ]14,128- & 142). Although the impact is transmitted to the cortical surface with the dura intact, the neuropathological consequences of the injury are typically extensive and include cortical, hippocampal and thalamic degeneration (supra). These pathologies are associated with long-term cognitive deficits and alterations in emotional behavior (supra). In the current CCI injury paradigm, this area is truly and invariably denatured in all animals, regardless of treatment conditions, due to the impact force applied directly to the cortex, while differences between treatment conditions are observed in terms of retention of subcortical structures, particularly the hippocampus. The observed behavioral benefits of B cell processing are also closely related to the functions supported by these structures, suggesting a link between localization of the introduced lymphocytes, retention of tissue surrounding the injection site, and functional neuroprotection. In support of this hypothesis, B lymphocytes were injected approximately 1mm behind bregma and predominantly located between the caucasian nucleus and hippocampus in this model (Lein et al (2007) Nature [ Nature ],445,168-17).

In this study, confocal imaging of tissue sections collected from CCI sites showed that B cells clustered around the initial injection site, with a modest distribution throughout the injured tissue on day 4 post injury and application. These results suggest that injected B cells within the brain parenchyma may remain in situ at the lesion site throughout their lifetime. Although the underlying molecular mechanisms observed for B-cell neuroprotection are not completely understood, it is likely that cell-derived spreading factors are involved, reaching distal regions beyond the tight localization of the applied cells. This hypothesis is supported by the observation that in animals treated with B cells in CCI, lesion volume is significantly reduced, and potential adverse reaction phenomena at the site of injury, including astrocytosis and microglial activation, are significantly reduced, as compared to controls.

Thus, B cells can be used as a therapeutic strategy for patients with cerebral contusion. Unlike any other existing cell-based therapies, B cells can be readily obtained from peripheral blood or other blood bank products, which is an important advantage for the development of rapid, ready-to-use therapeutics. Indeed, a fast, minimally-operated autologous B cell therapy would be highly transformable into a clinical setting. This is particularly true in severe brain lesions where surgery is typically performed to remove hematomas or penetrating bone fragments and an intraparenchymal or intraventricular catheter is placed to monitor intracranial pressure (Stocchetti et al, (2017) Lancet Neurol [ Lancet neurology ], 16, 452-464; Galgano et al, (2017) Cell Transplant [ Cell Transplant ],26,1118-1130) to provide a convenient route of administration for B cells to enter the damaged brain.

Unlike other cell types used in therapy (e.g., stem cells), B lymphocytes are mature terminally differentiated cells with a limited natural life span in vivo of 5-6 weeks. Their use in a disrupted microenvironment of cerebral contusion is expected to eliminate transplanted cells in even shorter time. This is advantageous because longer survival of transplanted cells may represent an important safety concern, particularly given that the microenvironment of the central nervous system contains multiple B-cell trophic factors. We used in vivo visualization of luciferase-expressing B cells to monitor the presence and persistence of metabolically active transplanted cells over time. We observed a rapid decrease in cell signal after day 7 to undetectable levels by day 17. In the context of CCI, an initial modest increase in luciferase signal intensity 3 to 7 days after intraparenchymal injection may indicate that transplanted cells have undergone some period of adaptation and/or stimulation in the local microenvironment of CCI damage. The increase in signal intensity is unlikely to be due to in situ cell proliferation, since immunohistochemical examination of the injection site (performed immediately or up to 4 days after B cell administration and CCI) showed that the introduced B cells did not express markers of cell proliferation. Indeed, although B cells do enhance the proliferation of neighboring cells, their proliferation is not observed after application to a wound.

This study describes the first proof-of-principle observation that mature peripherally isolated B cells may represent a safe, rapid, and effective cell-based therapeutic strategy for acute and subacute treatment of contusion TBI for which no treatment options currently exist to improve neurological outcomes.

Example 3: evaluation of B cell immunotherapy in the SOD1-G93A mouse model of ALS

This example illustrates the efficacy of the enzyme in the ALS standard murine model (SOD 1)^G93AMice) safety and efficacy of intravenous (i.v.) administration of B cells.

Male and female transgenic SOD1^G93AMice and the same number of gender matched non-carrier controls (n ═ 32/condition) started on week 10 (day 72) of life on a weekly schedule of 5 × 10 in saline⁶Individual B cells (or saline control) were treated for a total of 10 weeks. We found that B cell treatment delayed SOD1^G93ASymptomatic attack in mice (p)<0.0001), as indicated by the peak body weight achieved, and significantly prolonged survival (p)<0.05). The relative number of injured/degenerated motor neurons in the lumbar spinal cord was significantly reduced at treatment with B cells and endpoint (p)<0.05) correlation, even though the total number of motor neurons did not change with treatment. In identically treated non-transgenic control wild-type littermates, B cell treatment was not associated with any observable adverse effects (behavior and phenotype) that continued to increase body weight during repeated treatments. Taken together, these findings demonstrate that B cell therapy may provide a viable approach to reduce neuroinflammation in ALS.

ALS is a fatal disease characterized by progressive degeneration of upper motor neurons in the motor cortex of the brain, and lower motor neurons in the brainstem and ventral horn of the spinal cord. To date, there is no cure for ALS, highlighting an urgent and unmet need in the field.

Design of research

To assess the efficacy of B Cell immunotherapy in ALS, we followed the design and interpretation guidelines for published results (Galgano (2017) Cell transplantation [ Cell transplantation ]]26,1118-; nordstrom et al, (2014) Ann neuron (annual book of neurology)]75,374-^G93ATransgenic mouse model. Mice transgenic for human SOD1 expressing the G93A mutant form exhibited phenotypes similar to human ALS. Their limb or limbs are progressively paralyzed and paralyzed due to the loss of spinal motor neurons. Transgenic mice also had shorter lifespan. Neurodegenerative symptoms typically begin to appear around 12-14 weeks of age, and mice die at approximately 20-24 weeks of age. Thus, this model shows a rapid, aggressive progression of the disease. This model is very stable and reproducible, allowing the evaluation of treatment options for this neurodegenerative disorder.

A total of 15-17 females and males per experimental condition (treatment) were used throughout the study (according to the recommended guidelines in the field (supra)) along with the same number of gender-matched non-littermate non-carrier controls (table 1). Starting at week 10 of life (day 72), all animals received a total of 10B cell (or saline control) intravenous infusions weekly delivered via retrobulbar injection (figure 22). For feasibility, the study was performed in 2 overlapping cohorts, as in table 1 (summary of genotypes, sex, and animals used for treatment for each cohort).

TABLE 1

Materials and methods

The following materials and methods were used for this study.

Animals: b6SJL-Tg (SOD 1G 93A)1Gur/J (SOD 1G 93A) transgenic mice were purchased from Jackson Laboratories (Jackson Laboratories) (Balport, Myene; stock number: 002726). Jackson Laboratories reports that this founder line (commonly known as G1H) has a high SOD1 transgene copy number. All animals were genotyped separately by Jackson Laboratories (Jackson Laboratories) prior to shipment, and only heterozygous animals with high SOD1 transgene copy number (the upper third of the distribution) were commercialized. The control animals were littermates without the SOD1 transgene (non-carrier).

The donor animals used for B cell isolation were C57BL/6J mice, also purchased from Jackson Laboratories (Jackson Laboratories) (stock number: 000664).

All animal procedures were performed in accordance with NIH laboratory animal care and instructions and public health services policies for laboratory animal humanity care. All protocols were approved by the Institutional Animal Care and Use Committee of Mass Institutional of Mass General Hospital (Institutional Animal Care and Use Committee of Mass General Hospital), protocol No.: 2019N 000004.

B cell separation: all cell isolation procedures were performed under sterile conditions in a clean biosafety cabinet and the resulting cell suspension was delivered into sterile Phosphate Buffered Saline (PBS). Cells were isolated and purified from the spleen of C57BL6 wild-type donor animals, sharing half the genetic background of the recipient (similar to siblings). Spleens were isolated as splenic cell suspensions and all B cells were isolated by negative immunomagnetic selection using a commercially available kit (american day whirlpool (Miltenyi) B cell isolation kit, mouse; 130-873, american day whirlpool (Miltenyi Biotec) as we published previously (DeKosky et al (2013) Nat Rev Neurosci [ natural reviews of nervous system ]9, 192-. The cells produced were > 98% CD19+ B cells, typically more than 85% -90% CD19+/B220+/IgM +/IgD +, including approximately 5% CD138+ plasma cells, and < 1% other cell populations as demonstrated after isolation by flow cytometry analysis (DeKosky et al, (2013) Nat Rev Neurol [ natural reviews of nervous system ]9,192-. This represents the initial B cell fraction (treatment) and was injected into the animals the same day after isolation.

And (3) treatment: starting at week 10 of life (day 72), all animals received a total of 10B cell (or saline control) intravenous infusions weekly, delivered via retrobulbar injection. Animals were anesthetized with 3% isoflurane in oxygen and a 100 μ l saline bolus containing 500 ten thousand primary B cells (treatment) or no cells (saline control) was injected into the retrobulbar sinus. An eye ointment is then applied to the treated eye. This method of administration was chosen because it has a much lower risk of failure compared to tail vein injection for cell transplantation, especially in case of repeated administration.

And (3) safety evaluation: all animals were evaluated for health three times a week to assess the potential adverse effects of treatment according to IACUC guidelines (weight loss, apathy, poor physical condition, fur erection, hunched posture).

And (3) evaluating the efficacy: body weight and nervous system score (NeuroScore) were assessed twice weekly by experimenters unaware of treatment conditions (blinded) and used to assess disease progression as described in, for example: hatzipetros, t. et al (2015), j.vis. exp. [ journal of visual experiment ] (104), e53257, doi: 10.3791/53257; mashkouri et al, (2016) Neural Regen Res [ nerve regeneration research ]11, 1379-1384:

neurological score 0 (pre-symptom): when the mouse is suspended by the tail, the hind limb assumes a normal flare, i.e. it extends completely away from the lateral midline and stays in this position for 2 seconds or more. When the mice were allowed to walk, a normal gait was observed.

Neurological score 1 (first symptom): when the mouse is suspended by the tail, the hind limb appears abnormally open, i.e. it collapses or partially collapses towards the lateral midline, or it shivers or retracts/clasps during tail suspension. When the mice were allowed to walk, a normal or slightly slow gait was observed.

Neurological score 2 (local)Paralysis attack): when the mouse is suspended by the tail, the hind limbs are partially or completely collapsed and do not stretch much. (and possibly also articulation). When the mouse is allowed to walk, the hind limbs are used to move forward, however the toes curl at least twice down during 90cm of walking, or any part of the foot is dragging. When the center of gravity of the mouse is shifted to the left and right, it can be adjusted by itself from both sides within 10 seconds.

Neurological score 3 (paralysis): when the mice are suspended by the tail, stiff paralysis of the hind limbs or minimal joint movement occurs. When the mouse is allowed to walk, there is forward movement, however the hind limbs are not used for forward movement. When the center of gravity of the mouse is shifted to the left and right, it can be adjusted by itself from both sides within 10 seconds.

Nerve score 4 (humanitarian endpoint): when mice are suspended by the tail, stiff paralysis of the hind limbs occurs. When the mouse is allowed to walk, there is no forward motion. When the mouse center of gravity was shifted to the left and right, it could not adjust itself from either side within 10 seconds, i.e., there was no orthostatic reflection.

Body weight was used as a reliable and unbiased assessment of disease progression. The age of the maximum body weight, which is a reliable and objective measure of the onset of muscle denervation, was used retrospectively to determine the occurrence of disease episodes as described previously (Turner et al (2014) Neurobiol Aging 35, 906-915). Histology: at euthanasia, lumbar spinal cords were collected from all animals, stored in fixative, and processed histologically. The spinal cord was longitudinally sectioned at a horizontal plane, with a thickness of 10 μm, and stained with hematoxylin and eosin (H & E) to visualize motor neurons in the ventral horn. Motor neurons, which are easily identifiable based on large size and unique morphology, were counted in 3-6 target areas of at least 500x 500 μm, collected randomly from 2-3 longitudinal sections per animal. We also quantified healthy motor neurons (large and round cell bodies; nuclei with single prominent nucleoli; presence of nisel material; see fig. 27A) and damaged/degenerated motor neurons (cell body atrophy, hyperphagocytomia, strong aggregation of chromatin, pyknosis; see fig. 27B), respectively. All counts were performed by experimenters who were unaware of the treatment conditions (blind).

Results

The following results summarize data collected from a complete animal study. The study was terminated at postnatal day 150 when the last transgenic SOD1 animal was euthanized.

Safety feature

No observable (behavioral and phenotypic) deleterious effects (weight loss, apathy, poor physical condition, fur erection, hunched posture) were found in control non-carrier animals receiving naive B cells (at a dose of approximately 2 hundred million cells/kg). Control animals continued to gain weight throughout the study, independent of B cell treatment (figure 23). In addition, in transgenic SOD1-G93A animals, such detrimental effects attributable to cell infusion treatment were not observed during the pre-symptomatic period (days 72-90 of treatment).

Treatment of symptoms of ALS progression

a. Peak body weight (shown in figure 24). Peak body weight is defined as the time point after which the measured body weight of the individual animal shows a sustained decrease. This information was used to perform survival analysis, where peak body weight time represents "survival". This assay used only transgenic SOD1-G93A animals, since the control non-carrier mice showed no weight loss.

Peak body weight (i.e., onset of symptoms, associated with weight loss) was delayed by an average of approximately 28 days (p <0.0001, one-way ANOVA with post-sida correction) for multiple comparisons under the initial B-cell treatment conditions compared to the saline-treated controls (see Turner et al, neurobiol. aging, 35,906-915 (2014)).

b. Neurological score assessment (shown in figure 25). For this analysis, disease onset was defined as the time point at which animals had a neurological score of 1 for 3 consecutive assessments and then had no decrease in neurological score. A gradual increase in neurological score over time was observed in all animals, however B cell treatment was associated with a slower rate of progression (figure 25).

c. Survival analysis (shown in figure 26). As described above, animals in a fully paralyzed state failed to self-adjust within 10 seconds after either side of the shift in center of gravity, were classified as having a neurological score of 4, and were humanely euthanized. These animals were scored as dying from ALS. Death analysis as a result of disease progression demonstrated significant overall survival benefit associated with intravenous administration of naive B cells (p ═ 0.0286, one-way ANOVA with sidak post-correction for multiple comparisons; fig. 26).

d. Histological analysis of spinal cord motor neurons (shown in figure 27). All histological samples were processed and examined by experimenters who were not aware of the processing conditions (blind). As expected, a very significant reduction in the total number of motor neurons in the ventral horn of the lumbar spinal cord was observed in transgenic SOD1 animals compared to WT controls. Although no significant difference was observed in the total number of neurons found in the spinal cord between the B cell treated transgenic SOD1 animals and the control transgenic SOD1 animals, a significantly lower percentage of these motor neurons that were dead or dying was found in the B cell treated animals compared to the saline control.

Conclusion

In this study against the ALS transgenic SOD1-G93A mouse model, intravenous injection of purified allogeneic mature naive B cells into mice 10 weeks continuously per week was associated with: (i) a statistically significant delay of 28 days in symptom onset, (ii) a significant prolongation of survival, and (iii) a significant reduction in the presence of damaged, dead, or dying neurons in the lumbar spinal cord of treated animals compared to saline-treated controls. No observable adverse side effects associated with treatment were noted in wild type or transgenic animals receiving an injected B cell dose of 200,000 cells/gram (or 2 hundred million B cells/Kg).

EXAMPLE 4 treatment of Parkinson's disease with B cells

A composition comprising a therapeutically effective amount of B cells, e.g., any of the compositions described herein, can be administered to a subject having parkinson's disease. The methods described herein can be used to treat a subject with a therapeutic B cell (e.g., B)_regCells) are administered to an appropriate animal model of Parkinson's disease to evaluate treatment of Parkinson's disease (see, e.g., Bobela w. et al, Overview of mouse models of Parkinson's disease [ review of mouse models of Parkinson's disease ]]Curr Protoc Mouse Biol. [ current protocol for Mouse biology](2014) And the effect of the treatment is monitored according to methods known to those skilled in the art. Methods for monitoring responses include Assessment of motor function, pain, neuroinflammation, and substantia nigra neuronal death (see, e.g., Peng Q. et al, The Rodent Models of Dyskinesia and The hair Behavior Association [ Rodent Models of Dyskinesia and Behavioral Assessment thereof ]]Front neuron, [ neurological frontier ]](2019))。

Responsiveness to treatment can be monitored by a decrease in the rate of disease progression (e.g., a decrease in the rate of progression as measured by the severity of symptoms associated with parkinson's disease). Alternatively, responsiveness to treatment may be monitored by determining the levels of the following molecular markers of disease progression associated with neurodegenerative diseases, such as: t- τ (total τ), P- τ (hyperphosphorylated τ), A β 42 (amyloid β 42), A β 42/A β 40 ratio, YKL-40 (chitinase-3-like protein 1), VLP-1 (cone-like protein 1), NFL (neurofilament L), pNFH (phosphorylated neurofilament heavy subunit), Ng (neuropil protein) and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43(TAR DNA binding protein 43), reduced α -synuclein and/or reduced levels of 3, 4-dihydroxyphenyl acetate (see, e.g., Robey and Panegyres, Cerebrospinal fluidized biomakers in neuroneegerids [ Cerebrospinal fluid biomarkers in neurodegenerative disorders ], Future Neurol [ Future neurology ]14 (2011) (2019)).

Example 5 administration of B cells to treat additional neurodegenerative diseases

Other neurodegenerative diseases can be assessed using the methods described herein by administering B cells (e.g., Breg cells) to an appropriate animal model. Additional neurodegenerative diseases include alzheimer's disease, Chronic Traumatic Encephalopathy (CTE), frontotemporal dementia, huntington's disease, infantile axonal dystrophy, progressive supranuclear palsy, dementia with lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

Exemplary animal models for studying such neurodegenerative diseases have been described in the art, for example in the following documents:

alzheimer's Disease (see, e.g., esparda-Canals g. et al, Mouse Models of Alzheimer's Disease [ Mouse model of Alzheimer's Disease ], J Alzheimer's Disease [ journal of Alzheimer's Disease ] (2017));

chronic Traumatic Encephalopathy (CTE) (see, e.g., dauul HR et al, focused input therapy for or after controlled clinical examinations histopathology and functional output in a mixed traumatic brain injury model in mice where Concussive injury before or after controlled cortical impact exacerbates histopathology and functional outcomes; J neurotraumatology a. [ journal of neural trauma ],30(5):382-91 (2013)); and

huntington's Disease (see, e.g., Farshim PP, et al, Mouse Models of Huntington's Disease [ Mouse model for Huntington's Disease ], Methods Mol Biol [ Methods of molecular biology ] (2018)).

EXAMPLE 6 administration of B cells to treat inflammatory or immune diseases

The B cells described herein can be administered to treat an inflammatory or immune disorder, such as cystic fibrosis, a cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratospheric, osteoarthritis, osteoporosis, pulmonary hypertension, retinitis pigmentosa, or rheumatoid arthritis. These treatments for treating inflammatory or immune disorders can be evaluated using the methods described herein by administering therapeutic B cells (e.g., Breg cells) to an appropriate animal model and monitoring the effect of the treatment according to methods known to those skilled in the art.

Exemplary animal models for studying such inflammatory or immune diseases have been described in the art, for example in the following documents:

cystic fibrosis (see, e.g., Dreano, e.g., Characterization of the two rat cystic fibrosis models KO and F508del CFTR-Generated by critical by criprpr-Cas 9[ two rat models of cystic fibrosis KO and F508del CFTR Generated by criprpr-Cas 9 ], Animal Model Exp Med [ Animal models and experimental medicine ]2(4):297-311 (2019));

cardiovascular disease (Goodchild, T.T. et al, Bone marrow-derived B cells previous ventricular function after cardiac infarction [ B cells of myeloid origin retain ventricular function after acute myocardial infarction ], JACC cardiovascular Interv [ J. American society of cardiology & cardiovascular intervention ], 2(10):1005-16 (2009));

keratoconus (see, e.g., Tachibana M. et al, android-dependent heredity mouse keratoconus: ligation to MHC region, Invest Ophthalmol Vis Sci [ research ophthalmology and Vision ], 43(1):51-7 (2002));

osteoarthritis (see, e.g., Kuyinu. E.L. et al, Animal models of osteo-arthritis: classification, update, and measurement of results in Animal models of osteoarthritis ], J ortho Surg Res. [ J. orthopedics and Res. ], 11:19 (2016));

osteoporosis (see, e.g., Komori t., Animal models for osteoporosis, [ Animal model for osteoporosis ], Eur J Pharmacol, [ european journal of pharmacology ], 759:287-94 (2015));

pulmonary hypertension (see, e.g., Sztuka K. and Jasi ń ska-Stroscein M., Animal models of pulmonary arterial hypertension: A systematic review and meta-analysis of data from 6126 animals, Pharmacol Res. [ pharmacological study ]125(Pt B):201-214 (2017));

retinitis pigmentosa (see, e.g., Tsubura a. et al, Animal models for retinitis pigmentosa induced by MNU: disease progression, mechanisms, and therapeutic trials; histo Histopathol. [ histology and histopathology ], 25(7):933-44 (2010)); and

rheumatoid arthritis (see, e.g., acquisition d.l. et al, Animal models of rhematoid arthritis [ Animal models of rheumatoid arthritis ], Eur J Immunol [ european journal of immunology ], 39(8):2040-4 (2009)).

All publications (including patents and patent applications, including U.S. provisional application serial nos. 62/795,629, 62/837,765, and 62/965,032) mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Other embodiments

From the foregoing, it will be apparent that variations and modifications may be made to the invention described herein for various uses and conditions. Such embodiments are also within the scope of the following claims.

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