B cell immunotherapy

Abstract

To provide better approaches for treatment of degenerative diseases including neurodegenerative disease such as ALS and TBI.SOLUTION: Provided is a method for treating a neurodegenerative disease (such as amyotrophic lateral sclerosis) or a 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 B cells, allogeneic B cells, or xenogeneic B cells).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 798,629, filed January 23, 2019, U.S. Provisional Patent Application No. 62 / 837,765, filed April 24, 2019, and U.S. Provisional Patent Application No. 62 / 965,032, filed January 23, 2020, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Background of the Invention Degenerative diseases are medical conditions that cause the deterioration of cells, tissues, or organs. Many neurodegenerative diseases involving the degeneration of regions of the central nervous system include, for example, amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease (HD). Traumatic brain injury (TBI) is also an example of a disorder that can increase the risk of developing a degenerative brain disease, such as PD or AD. Rheumatoid arthritis and osteoarthritis are other examples of degenerative diseases involving inflammation. For most of these, there is no effective treatment. Treatments for some of these diseases or disorders are under research. However, proposed treatments are often expensive and involve significant risks and complications. Therefore, there is a need for better approaches to treat degenerative diseases, including neurodegenerative diseases such as ALS and TBI. Summary of the Invention

[0003] SUMMARY OF THE INVENTION Provided herein are compositions comprising B cells (e.g., isolated B cells, purified B cells, or modified B cells, or a combination thereof), and their use for the treatment of diseases (e.g., neurodegenerative diseases, traumatic brain injury (TBI), spinal cord injury (SCI), and inflammatory and immune diseases, as described herein).

[0004] In a first aspect, the invention features a method of treating a neurodegenerative disease in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of isolated B cells.

[0005] In some embodiments, the neurodegenerative disease is selected from the following: amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia, Huntington's disease, infantile neuroaxonal dystrophy, progressive supranuclear palsy, dementia with Lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease. In preferred embodiments, the neurodegenerative disease is ALS (e.g., sporadic ALS or familial ALS). In yet another preferred embodiment, the neurodegenerative disease is Parkinson's disease.

[0006] In another aspect, the invention features a method of treating a subject having ALS, the method including administering a therapeutically effective amount of B cells to the subject, wherein the therapeutically effective amount is sufficient to reduce or ameliorate one or more symptoms of ALS.

[0007] In another aspect, the invention features a method of treating a subject with ALS who exhibits one or more symptoms of ALS, the method including the steps of: administering a therapeutically effective amount of B cells to a subject, wherein the therapeutically effective amount results in a reduction or amelioration of 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.

[0008] In some embodiments, one or more symptoms of ALS include: difficulty lifting the front of the foot; difficulty lifting the toes; weakness in one or both legs; weakness in one or both feet; weakness in one or both ankles; weakness in the hands; clumsiness in the hands; muscle cramps; fasciculations (muscle twitching) in one or both arms, one or both legs, one or both shoulders, or the tongue; muscle spasms; spasticity (tight, stiff muscles); and difficulty chewing or swallowing (dysphagia), difficulty speaking or forming words (dysarthria), and difficulty breathing (dyspnea).

[0009] In some embodiments, the method includes monitoring one or more symptoms of ALS for 1 to 7 days after administration, for 7 to 28 days after administration, for 1 to 28 weeks after administration, for 1 to 2 months after administration, for 2 to 6 months after administration, for 2 to 9 months after administration, or for 6 months to 1 year or longer after administration.

[0010] In some embodiments, the invention features methods for monitoring the responsiveness of a patient with a neurodegenerative disease to treatment with a therapeutically effective amount of isolated B cells by determining the level of a molecular marker of disease progression (e.g., by 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 a molecular marker can be determined according to techniques known to those of skill in the art.

[0011] In some embodiments, the level of the molecular marker can be determined from a sample from the subject receiving treatment, for example, from a sample that is plasma or cerebrospinal fluid (CSF) of the subject receiving treatment. Exemplary molecular markers of progression of the neurodegenerative diseases described herein are known in the art. Exemplary markers include: T-tau (total tau), P-tau (hyperphosphorylated tau), Aβ42 (amyloid beta 42), the ratio of Aβ42 / Aβ40, YKL-40 (chitinase 3-like protein 1), VLP-1 (visinin-like protein 1), NFL (neurofilament light chain), pNFH (phosphorylated neurofilament heavy chain subunit), Ng (neurogranin), and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43 (TAR DNA-binding protein 43), decreased alpha-synuclein, and / or decreased levels of 3,4-dihydroxyphenylacetic acid (see, e.g., Robey and Panegyres. Cerebrospinal fluid biomarkers in neurodegenerative disorders. Future Neurol. 14(1). (2019), incorporated by reference in its entirety).

[0012] In another aspect, the invention features a method of treating an inflammatory or immune disease in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of isolated B cells.

[0013] In some embodiments, the inflammatory or immune disease is selected from the following: cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis.

[0014] In another aspect, the present invention features a method for treating a subject with central nervous system (CNS) injury, the method comprising administering a therapeutically effective amount of isolated B cells to the subject.In some embodiments, the CNS injury is traumatic brain injury (TBI) or spinal cord injury (SCI).In some embodiments, the CNS injury includes both TBI and SCI.

[0015] In another aspect, the invention features a method of treating a subject having a traumatic brain injury (TBI), the method including administering to the subject a therapeutically effective amount of isolated B cells.

[0016] In another aspect, the invention features a method of treating a subject having a spinal cord injury (SCI), the method including administering to the subject a therapeutically effective amount of isolated B cells.

[0017] In some embodiments, TBI is damage to the brain caused by external mechanical force. In some embodiments, SCI is related to damage to the spinal cord caused by external mechanical force. In some embodiments, TBI and / or SCI are caused by head trauma or brain contusion (for example, due to a fall, gunshot wound, sports accident, construction accident, traffic accident, or injury that penetrates the skull or brain of the subject). In some embodiments, the subject with TBI and / or SCI has one or more of several physical disorders, cognitive disorders, social disorders, emotional disorders, and / or behavioral disorders. TBI and SCI can occur simultaneously and can be caused by the same injury.

[0018] In another aspect, the invention features a method of treating a subject with a TBI who exhibits one or more symptoms of a TBI, the method including: administering a therapeutically effective amount of B cells to a subject, wherein the therapeutically effective amount is an amount that results in a reduction 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.

[0019] In another aspect, the invention features a method of treating a subject having an SCI (exhibiting one or more symptoms of an SCI), the method including the steps of: administering a therapeutically effective amount of B cells to a subject, wherein the therapeutically effective amount is an amount that results in a reduction or amelioration of 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.

[0020] In some embodiments, one or more symptoms of TBI and / or SCI include: inability to recall the traumatic event, confusion, difficulty learning and remembering new information, emotional and executive dysfunction, inability to speak coherently, unsteadiness, lack of coordination, and vision or hearing problems; cognitive problems (e.g., amnesia, inability to speak or understand language, confusion, difficulty concentrating, difficulty thinking and understanding, inability to remember new things, or inability to recognize mundane objects); behavioral problems (e.g., abnormal laughing and crying, aggression, impulsivity, irritability, lack of inhibitions (impulsivity), or persistent repetitive words or actions); mood problems (e.g., anger, anxiety, apathy, or feelings of loneliness); systemic problems (e.g., loss of consciousness, dizziness, fainting, or fatigue); eye problems (e.g., dilated pupils, periorbital bruising, raccoon eyes), or anisocoria); muscle problems (e.g., instability or stiff muscles); gastrointestinal problems (e.g., nausea or vomiting); speech problems (e.g., difficulty speaking or slurred speech); vision problems (e.g., blurred vision or sensitivity to light); bruising, depression, loss of smell, nerve damage, post-traumatic seizures, tinnitus, hyperacusis, vertigo.

[0021] In some embodiments, the method includes monitoring one or more symptoms of TBI and / or SCI for 1 to 7 days after administration, 7 to 28 days after administration, 1 to 28 weeks after administration, 1 to 2 months after administration, 2 to 6 months after administration, 2 to 9 months after administration, or 6 months to 1 year or longer after administration.

[0022] In some embodiments, the invention features methods for monitoring the responsiveness of patients 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., by 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 a molecular marker can be determined according to techniques known to those of skill in the art.

[0023] In some embodiments, the level of molecular markers of TBI and / or SCI can be determined from a sample for the subject receiving treatment, for example, from a sample that is plasma or cerebrospinal fluid (CSF) of the subject receiving treatment. Exemplary molecular markers for TBI progression described herein include, but are not limited to, protein biomarkers for neuronal cell body injury (UCH-L1, NSE), protein biomarkers for astroglial injury (GFAP, S100B), protein biomarkers for neuronal cell death (αII-spectrin breakdown products), protein biomarkers for axonal injury (NF proteins), protein biomarkers for white matter injury (MBP), protein biomarkers for post-injury neurodegeneration (total tau and phosphorylated tau), protein biomarkers for post-injury autoimmune response (autoantibodies targeting brain antigens) (see, e.g., Wang et al. An update on diagnostic and prognostic biomarkers for traumatic brain injury. Expert Rev Mol Diagn. 18(2): 165-180 (2018), incorporated by reference in its entirety).

[0024] In some embodiments, allogeneic B cells are administered. In some embodiments, the allogeneic B cells are haploidentical allogeneic B cells, HLA-matched allogeneic B cells, or genetically modified B cells (e.g., B cells genetically modified, such as by CRISPR, to reduce the immunogenicity of the B cells).

[0025] In some embodiments, autologous B cells are administered.

[0026] In some embodiments, heterologous B cells are administered.

[0027] In some embodiments, the method includes administering a second therapeutic composition.

[0028] In some embodiments, where the disease or disorder is ALS, the second therapeutic composition is edaravone, riluzole, or an immunomodulatory composition (e.g., an anti-CD14 antibody, an anti-CDL40 antibody, or a T reg a composition comprising cells.

[0029] In some embodiments where the disease or disorder is TBI and / or SCI, the second therapeutic composition is an antibiotic or a corticosteroid (eg, prednisone).

[0030] In some embodiments, the B cells are mature naive B cells.

[0031] In some embodiments, the B cells are stimulated ex vivo.

[0032] In some embodiments, B cells are stimulated ex vivo with a Toll-like receptor (TLR) agonist.

[0033] In some embodiments, the TLR agonist is an endogenous ligand selected from the following: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, uric acid crystals, mRNA, β-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, α A-crystallin, and CpG chromatin-IgG complexes.

[0034] In some embodiments, the TLR agonist is an exogenous ligand selected from the following: Pam3CSK4, triacylated lipopeptides, glycosylphosphatidylinositol (GPI)-anchored proteins, lipoarabinomannan, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acid, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinolines (e.g., imiquimod, resiquimod, loxoribine, bropirimine), antiviral compounds, unmethylated CpG oligodeoxynucleotides, and profilin.

[0035] In some embodiments, B cells are stimulated ex vivo with an immunomodulatory cytokine (e.g., a pro-inflammatory cytokine, such as a pro-inflammatory cytokine selected from IL-1β, IL-2, IL-4, IL-6, TNFα, or IFNγ).

[0036] In some embodiments, the B cells are B reg In some embodiments, B reg The cells express the immunomodulatory cytokine IL-10. regThe cells further express one or more additional immunomodulatory cytokines selected from the following: IL-2, IL-4, IL-6, IL-35, TNF-α, TGFβ, PD-L1 FasL, and TIM1. reg The cells express one or more cell surface markers selected from 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. reg The cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. reg The cells express CD25 and CD71. reg In some embodiments, the B cells do not express CD73. reg The cells comprise at least 80% (e.g., at least 85%, 90%, 95%, or 98%) CD19+ B cells. In some embodiments, the B reg The cells comprise less than 10% (eg, less than 5%) CD138+ plasma B cells.

[0037] In some embodiments, the B cells are neuroprotective, anti-inflammatory, and / or immunomodulatory.

[0038] In some embodiments, the B cells are formulated for local or systemic administration. In some embodiments, the B cells are formulated for intravenous, intraarterial, subcutaneous, intrathecal, or intraparenchymal administration. In some embodiments, the B cells are formulated for administration by intravenous infusion or intravenous bolus. In some embodiments, the B cells are formulated for administration through an intracranial pressure (ICP) monitoring catheter.

[0039] In some embodiments, 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 yearly.

[0040] In some embodiments, the B cells are administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0041] In some embodiments, the therapeutically effective amount of B cells is at least 0.5 X 10 per administration. 6 B cells or 0.5 X 10 per dose 7 B cells or 1 x 10 per dose 8 B cells or at least 2 x 10 per dose 8 B cells or at least 1 x 10 per dose 9 In some embodiments, a therapeutically effective amount of B cells comprises 1 x 10 B cells per administration. 8 B cells ~1 x 10 9 B cells or 1 x 10 per dose 8 B cells ~5 x 10 8 B or 2 x 10 per dose 8 B cells ~4 x 10 8 Contains B cells.

[0042] In another aspect, the invention features a pharmaceutical composition that includes 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.

[0043] In some embodiments, the modified B cells are primary cells.

[0044] In some embodiments, the pharmaceutically acceptable excipient is an aqueous solution (eg, saline).

[0045] 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.

[0046] In some embodiments, the disease or disorder is selected from abnormal wound healing (e.g., diabetic wound healing), a neurodegenerative disease, TBI, or SCI, or is an immune or inflammatory disease.

[0047] 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 neuroaxonal dystrophy, progressive supranuclear palsy, dementia with Lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

[0048] In some embodiments, the immune or inflammatory disease is selected from the following: cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis.

[0049] In some embodiments, the modified B cells are allogeneic B cells. In some embodiments, the allogeneic B cells are haploidentical allogeneic B cells, HLA-matched allogeneic B cells, or genetically modified B cells (e.g., B cells genetically modified, such as by CRISPR, to reduce the immunogenicity of the B cells).

[0050] In some embodiments, the modified B cells are autologous B cells.

[0051] In some embodiments, the modified B cells are xenogeneic B cells.

[0052] In another aspect, the invention features a method of making an altered B cell, the method comprising: i) isolating mature naive B cells from a subject; and ii) stimulating the B cells ex vivo with Toll-like receptor (TLR) agonists and / or immunomodulatory cytokines. thereby generating modified B cells.

[0053] In some embodiments, step i) further comprises isolating CD19+ mature naive B cells. In some embodiments, the isolation of CD19+ mature naive B cells is carried out by immunoprecipitation using 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 cells.

[0054] In some embodiments, the TLR agonist is an endogenous ligand selected from the following: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, uric acid crystals, mRNA, β-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, α A-crystallin, and CpG chromatin-IgG complexes.

[0055] In some embodiments, the TLR agonist is an exogenous ligand selected from the following: Pam3CSK4, triacylated lipopeptides, glycosylphosphatidylinositol (GPI)-anchored proteins, lipoarabinomannan, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acid, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinolines (e.g., imiquimod, resiquimod, loxoribine, bropirimine), antiviral compounds, unmethylated CpG oligodeoxynucleotides, and profilin.

[0056] In some embodiments, the immunomodulatory cytokine is a proinflammatory cytokine (e.g., a proinflammatory cytokine selected from IL-1β, IL-2, IL-4, IL-6, TNFα, or IFNγ).

[0057] In some embodiments, the modified B cells are reg In some embodiments, B reg The cells express the immunomodulatory cytokine IL-10. reg The cells further express one or more additional immunomodulatory cytokines selected from the following: IL-2, IL-4, IL-6, IL-35, TNF-α, TGFβ, PD-L1 FasL, and TIM1. reg The cells express one or more cell surface markers selected from 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. regThe cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. reg The cells express CD25 and CD71. reg The cells do not express CD73.

[0058] In some embodiments, the modified B cells are neuroprotective, anti-inflammatory, and / or immunomodulatory.

[0059] In yet another aspect, the present invention features B cells that can be used directly as anti-inflammatory and pro-regenerative cell-based therapeutics in a variety of disease conditions, including skin wounds and ulcers, muscle and cardiac injuries, brain and spinal cord injuries, and injuries to various internal organs. Genetically modified autologous, allogeneic, or xenogeneic cells, or cells primed with factors from the injury microenvironment, are useful because they have enhanced pro-regenerative capabilities. Factors generated by 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 injured tissue to promote healing.

[0060] Therefore, B cells can be used as a therapeutic strategy for patients with neurodegenerative diseases, TBI or SCI (e.g., resulting from cerebral contusion), inflammatory disorders, or various immune disorders. Unlike any other existing cell-based therapy, B cells are readily available from peripheral blood or other blood bank products, which is a significant advantage for developing fast-acting, off-the-shelf therapeutics. Indeed, fast-acting, minimally manipulated B cell therapies (allogeneic, autologous, or xenogeneic) have high potential for clinical application. This is particularly true not only for the treatment of neurodegenerative diseases such as ALS and PD, but also in cases of severe brain injury, where surgery is often performed to remove hematomas or penetrating bone fragments and catheters are placed, either intraparenchymally or intraventricularly, to monitor intracranial pressure, providing a convenient route for administering B cells into the injured brain.

[0061] Unlike other cell types used therapeutically, such as stem cells, B lymphocytes are mature, terminally differentiated cells with a limited natural lifespan of 5-6 weeks in vivo. In neurodegenerative settings and in the disrupted microenvironment of brain trauma, transplanted cells are expected to disappear more quickly after application. This is beneficial because longer survival of transplanted cells could raise significant safety concerns, especially considering that the central nervous system microenvironment contains several B cell-trophic factors.

[0062] Our results represent the first proof-of-principle observation that peripherally isolated mature B cells are a safe, fast-acting, and effective cell-based therapeutic strategy for several disorders disclosed herein, including ALS, PD, TBI, and SCI, for which there are no current therapeutic options to improve neurological outcomes.

[0063] 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 specific examples, while indicating preferred embodiments of the present 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.

[0064] [The present invention 1001] 1. A method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of isolated B cells. [The present invention 1002] 1001. The method of claim 1001, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS). [The present invention 1003] The method of any one of claims 1 to 10, wherein allogeneic B cells are administered. [The present invention 1004] 1001. The method of claim 1001, wherein autologous B cells are administered. [The present invention 1005] 1001. The method of claim 1001, wherein xenogeneic B cells are administered. [The present invention 1006] The method of any one of claims 1 to 10, further comprising administering a second therapeutic composition. [The present invention 1007] The method of claim 1005, wherein the second therapeutic composition is edaravone or riluzole. [The present invention 1008] The method of claim 1006, wherein the second therapeutic composition is an immunomodulatory composition. [The present invention 1009] 1001. The method of claim 1001, wherein the B cells are mature naive B cells. [The present invention 1010] The method of claim 1001, wherein the B cells are stimulated ex vivo. [The present invention 1011] 1001. The method of claim 1001, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist. [The present invention 1012] B cells are Breg 1001. The method of claim 1001, wherein the cell is a cell. [The present invention 1013] B reg The method of claim 1011, wherein the cells express the immunomodulatory cytokine IL-10. [The present invention 1014] B reg 1011. The method of claim 10, wherein the cells comprise at least 80% CD19+ B cells. [The present invention 1015] B reg 1011. The method of claim 10, wherein the cells comprise less than 10% CD138+ plasma B cells. [The present invention 1016] The method of claim 1001, wherein the B cells are formulated for local administration. [The present invention 1017] The method of claim 1001, wherein the B cells are formulated to be administered systemically. [The present invention 1018] 1001. The method of claim 1001, wherein the B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration. [The present invention 1019] 1001. The method of claim 1001, 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. [The present invention 1020] A therapeutically effective amount of at least 0.5 x 10 per administration 7 1001. The method of claim 1001, comprising administering to said patient a sample of said B cells. [The present invention 1021] A therapeutically effective amount is at least 1 x 10 per administration 8 1001. The method of claim 1001, comprising administering to said patient a sample of said B cells. [The present invention 1022] The therapeutically effective amount is at least 2 x 10 per administration 8 1001. The method of claim 1001, comprising administering to said patient a sample of said B cells. [The present invention 1023] A therapeutically effective amount is at least 1 x 10 per administration 9 1001. The method of claim 1001, comprising administering to said patient a sample of said B cells. [The present invention 1024] 1. A method of treating a subject having a traumatic brain injury (TBI), comprising administering to the subject a therapeutically effective amount of isolated B cells. [The present invention 1025] The method of claim 1022, wherein the TBI results from a head injury or a cerebral contusion. [The present invention 1026] The method of claim 1022, wherein the subject has one or more of a physical disorder, a cognitive disorder, a social disorder, an emotional disorder, and / or a behavioral disorder. [The present invention 1027] The method of claim 1022, wherein allogeneic B cells are administered. [The present invention 1028] The method of claim 1022, wherein autologous B cells are administered. [The present invention 1029] The method of claim 1022, wherein xenogeneic B cells are administered. [The present invention 1030] The method of claim 1022, further comprising administering a second therapeutic composition. [The present invention 1031] The method of claim 1022, wherein the second therapeutic composition is an antibiotic or a corticosteroid. [The present invention 1032] The method of claim 1022, wherein the B cells are mature naive B cells. [The present invention 1033] The method of claim 1022, wherein the B cells are stimulated ex vivo. [The present invention 1034] The method of claim 1022, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist. [This invention 1035] B cells are B reg The method of claim 1022, wherein the cell is a cell. [The present invention 1036] B regThe method of claim 1032, wherein the cells express the immunomodulatory cytokine IL-10. [This invention 1037] B reg The method of claim 1032, wherein the cells comprise at least 80% CD19+ B cells. [The present invention 1038] The method of claim 1032, wherein the B cells comprise less than 10% CD138+ plasma B cells. [This invention 1039] The method of claim 1022, wherein the B cells are formulated for local administration. [The present invention 1040] The method of claim 1022, wherein the B cells are formulated to be administered systemically. [The present invention 1041] The method of claim 1022, wherein the B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration. [The present invention 1042] The method of claim 1022, wherein the B cells are formulated to be administered through an intracranial pressure (ICP) monitoring catheter. [This invention 1043] The method of claim 1022, 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 yearly. [This invention 1044] A therapeutically effective amount is at least 0.5 x 10 per administration 7 1022. The method of claim 1022, comprising administering to said patient a sample of said B cells. [This invention 1045] A therapeutically effective amount is at least 1 x 10 per administration 8 1022. The method of claim 1022, comprising administering to said patient a sample of said B cells. [The present invention 1046] The therapeutically effective amount is at least 2 x 10 per administration 8 1022. The method of claim 1022, comprising administering to said patient a sample of said B cells. [This invention 1047] A therapeutically effective amount is at least 1 x 10 per administration 9 1022. The method of claim 1022, comprising administering to said patient a sample of said B cells. [This invention 1048] Any of the aforementioned methods of the present invention, wherein the subject is a human. Some aspects of the techniques and methodologies described herein are defined by any of the following numbered items:

[0065] 1. A method for treating a neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of isolated B cells. 2. The method according to item 1, wherein the neurodegenerative disease is selected from the following: amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia, Huntington's disease, infantile neuroaxonal dystrophy, progressive supranuclear palsy, dementia with Lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease. 3. The method of item 1, wherein allogeneic B cells are administered. 4. The method of item 1, wherein autologous B cells are administered. 5. The method of item 1, further comprising administering a second therapeutic composition. 6. The method of item 5, wherein the second therapeutic composition is edaravone or riluzole. 7. The method of item 6, wherein the second therapeutic composition is an immunomodulatory composition. 8. The method according to any one of items 1 to 7, wherein the B cells are mature naive B cells. 9. The method according to any one of items 1 to 8, wherein the B cells are stimulated ex vivo. 10. The method of any one of items 1 to 9, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist. 11. The method of claim 10, wherein the TLR agonist is an endogenous ligand selected from the following: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, uric acid crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and CpG chromatin-IgG complexes. 12. The method of claim 10, wherein the TLR agonist is an exogenous ligand selected from the following: Pam3CSK4, triacylated lipopeptide, glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, outer surface lipoprotein, lipopolysaccharide, cytomegalovirus envelope protein, glycoinositol phospholipid, glycolipid, GPI anchor, herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, mannuronic acid polymer, bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinoline, antiviral compound, unmethylated CpG oligodeoxynucleotide, and profilin. 13. B cells are B reg 13. The method according to any one of items 1 to 12, wherein the cell is a cell. 14. B reg 14. The method of claim 13, wherein the cells express the immunomodulatory cytokine IL-10. 15. B reg 15. The method of claim 14, wherein the cells further express one or more additional immunomodulatory cytokines selected from the following: IL-2, IL-4, IL-6, IL-35, TNF-α, TGFβ, PD-L1 FasL, and TIM1. 16. B reg16. The method of any one of items 13 to 15, wherein the cells express one or more cell surface markers selected from 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. 17. B reg 17. The method of claim 16, wherein the cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. 18. B reg 17. The method of item 16, wherein the cells express CD25 and CD71. 19. B reg 19. The method of any one of items 13 to 18, wherein the cells do not express CD73. 20. B reg 16. The method of any one of items 13 to 15, wherein the cells comprise at least 80% CD19+ B cells. 21. B reg 21. The method of any one of items 13 to 15 or item 20, wherein the cells comprise less than 10% CD138+ plasma B cells. 22. The method of any one of items 1 to 21, wherein the B cells are neuroprotective. 23. The method of any one of items 1 to 22, wherein the B cells are anti-inflammatory. 24. The method of any one of items 1 to 23, wherein the B cells are immunoregulatory. 25. The method of any one of items 1 to 24, wherein the B cells are formulated for local administration. 26. The method of any one of items 1 to 24, wherein the B cells are formulated to be administered systemically. 27. The method of any one of items 1 to 24, wherein the B cells are formulated for intravenous, intraarterial, subcutaneous, intrathecal, or intraparenchymal administration. 28. The method of item 27, wherein the B cells are formulated to be administered by intravenous infusion or intravenous bolus. 29. The method of any one of items 1 to 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 yearly. 30. The method of any one of items 1 to 29, wherein the B cells are administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. 31. The therapeutically effective amount is at least 1 x 10 per administration. 8 31. The method according to any one of items 1 to 30, comprising B cells. 32. The therapeutically effective dose is at least 2 x 10 per administration. 8 31. The method according to any one of items 1 to 30, comprising B cells. 33. The therapeutically effective amount is at least 1 x 10 per administration. 9 31. The method according to any one of items 1 to 30, comprising B cells. 34. A method of treating a subject having a traumatic brain injury (TBI), comprising administering to the subject a therapeutically effective amount of isolated B cells. 35. The method of item 34, wherein the TBI results from a head injury or a cerebral contusion. 36. The method of item 34, wherein the subject has one or more of several physical, cognitive, social, emotional, and / or behavioral disorders. 37. The method of item 34, wherein allogeneic B cells are administered. 38. The method of item 34, wherein autologous B cells are administered. 39. The method of item 34, further comprising administering a second therapeutic composition. 40. The method of item 39, wherein the second therapeutic composition is an antibiotic or a corticosteroid. 41. The method according to any one of items 34 to 40, wherein the B cells are mature naive B cells. 42. The method according to any one of items 34 to 41, wherein the B cells are stimulated ex vivo. 43. The method of any one of items 34 to 42, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist. 44. The method of claim 43, wherein the TLR agonist is an endogenous ligand selected from the following: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, uric acid crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and CpG chromatin-IgG complexes. 45. The method of item 43, wherein the TLR agonist is an exogenous ligand selected from the following: Pam3CSK4, triacylated lipopeptide, glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, outer surface lipoprotein, lipopolysaccharide, cytomegalovirus envelope protein, glycoinositol phospholipid, glycolipid, GPI anchor, herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, mannuronic acid polymer, bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinoline, antiviral compound, unmethylated CpG oligodeoxynucleotide, and profilin. 46. B cells are B reg 46. The method according to any one of items 34 to 45, wherein the cell is a cell. 47. B reg 47. The method of claim 46, wherein the cells express the immunomodulatory cytokine IL-10. 48. B reg 48. The method of paragraph 47, wherein the cells further express one or more additional immunomodulatory cytokines selected from the following: IL-2, IL-4, IL-6, IL-35, TNF-α, TGFβ, PD-L1 FasL, and TIM1. 49. B reg49. The method of any one of paragraphs 46-48, wherein the cells express one or more cell surface markers selected from 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. 50. B reg 50. The method of claim 49, wherein the cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. 51. B reg 50. The method of claim 49, wherein the cells express CD25 and CD71. 52. B reg 52. The method of any one of items 46 to 51, wherein the cells do not express CD73. 53. B reg 49. The method of any one of items 46 to 48, wherein the cells comprise at least 80% CD19+ B cells. 54. The method of any one of items 46 to 48 or item 53, wherein the B cells comprise less than 10% CD138+ plasma B cells. 55. The method of any one of items 34 to 54, wherein the B cells are neuroprotective. 56. The method according to any one of items 34 to 55, wherein the B cells are anti-inflammatory. 57. The method of any one of items 34 to 56, wherein the B cells are immunoregulatory. 58. The method of any one of items 34 to 57, wherein the B cells are formulated for local administration. 59. The method of any one of items 34 to 57, wherein the B cells are formulated to be administered systemically. 60. The method of any one of items 34 to 57, wherein the B cells are formulated for intravenous, intraarterial, subcutaneous, intrathecal, or intraparenchymal administration. 61. The method of item 60, wherein the B cells are formulated to be administered by intravenous infusion or intravenous bolus. 62. The method of any one of items 34 to 57, wherein the B cells are formulated to be administered through an intracranial pressure (ICP) monitoring catheter. 63. The method of any one of items 34 to 62, 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. 64. The method of any one of items 34 to 63, wherein the B cells are administered at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. 65. The therapeutically effective dose is at least 1 x 10 per administration. 8 64. The method according to any one of items 34 to 63, comprising B cells. 66. The therapeutically effective dose is at least 2 x 10 per administration. 8 64. The method according to any one of items 34 to 63, comprising B cells. 67. The therapeutically effective dose is at least 1 x 10 per administration. 9 64. The method according to any one of items 34 to 63, comprising 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 according to item 68, wherein the TLR agonist is an endogenous ligand selected from the following: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, uric acid crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and CpG chromatin-IgG complexes. 70. The pharmaceutical composition of item 68, wherein the TLR agonist is an exogenous ligand selected from the following: Pam3CSK4, triacylated lipopeptide, glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, outer surface lipoprotein, lipopolysaccharide, cytomegalovirus envelope protein, glycoinositol phospholipid, glycolipid, GPI anchor, herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, mannuronic acid polymer, bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinoline, antiviral compound, unmethylated CpG oligodeoxynucleotide, and profilin. 71. The pharmaceutical composition according to item 68, wherein the immunomodulatory cytokine is a proinflammatory cytokine. 72. The pharmaceutical composition according to item 71, wherein the proinflammatory cytokine is selected from the following: IL-1β, IL-2, IL-4, IL-6, TNFα, or IFNγ. 73. The pharmaceutical composition of any one of items 68 to 72, wherein the modified B cells are primary cells. 74. Modified B cells are B reg 74. The pharmaceutical composition according to any one of items 68 to 73, which is a cell. 75. B reg 75. The pharmaceutical composition of item 74, wherein the cells express the immunomodulatory cytokine IL-10. 76. B reg 76. The pharmaceutical composition of item 75, wherein the cells further express one or more additional immunomodulatory cytokines selected from the following: IL-2, IL-4, IL-6, IL-35, TNF-α, TGFβ, PD-L1 FasL, and TIM1. 77. B reg77. The pharmaceutical composition of any one of items 74 to 76, wherein the cells express one or more cell surface markers selected from 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. 78. B reg 78. The pharmaceutical composition of item 77, wherein the cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. 79. B reg 78. The pharmaceutical composition of item 77, wherein the cells express CD25 and CD71. 80. B reg 80. The pharmaceutical composition of any one of items 74 to 79, wherein the cells do not express CD73. 81. The pharmaceutical composition of any one of items 68 to 80, wherein the modified B cells are neuroprotective. 82. The pharmaceutical composition of any one of items 68 to 81, wherein the modified B cells are anti-inflammatory. 83. The pharmaceutical composition of any one of items 68 to 82, wherein the modified B cells are immunomodulatory. 84. The pharmaceutical composition according to any one of items 68 to 83, wherein the pharmaceutically acceptable excipient is an aqueous solution. 85. A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to any one of items 68 to 84. 86. The method of item 85, wherein the disease or disorder is abnormal wound healing. 87. The method of item 85, wherein the disease or disorder is a neurodegenerative disease. 88. The method according to item 87, wherein the neurodegenerative disease is selected from the following: amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia, Huntington's disease, infantile neuroaxonal dystrophy, progressive supranuclear palsy, dementia with Lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease. 89. The method of item 85, wherein the disease or disorder is traumatic brain injury (TBI). 90. The method according to item 85, wherein the disease or abnormality is selected from the following: cystic fibrosis, cardiovascular disease, keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis. 91. The method of any one of items 85 to 90, wherein the modified B cells are allogeneic B cells. 92. The method of any one of items 85 to 91, wherein the modified B cells are autologous B cells. 93. A method of producing modified B cells, comprising: i) isolating mature naive B cells from a subject; and ii) stimulating the B cells ex vivo with Toll-like receptor (TLR) agonists and / or immunomodulatory cytokines. thereby producing modified B cells. 94. The method of item 93, wherein step i) further comprises isolating CD19+ mature naive B cells. 95. The method of item 94, wherein the isolation of CD19+ mature naive B cells is carried out by immunoprecipitation using a CD19 antibody or an antigen-binding fragment thereof. 96. The method of item 95, wherein the CD19 antibody or antigen-binding fragment thereof remains bound to the modified B cells. 97. The method of claim 93, wherein the TLR agonist is an endogenous ligand selected from the following: heat shock proteins, necrotic cells or fragments thereof, oxygen radicals, uric acid crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and CpG chromatin-IgG complexes. 98. The method of Item 93, wherein the TLR agonist is an exogenous ligand selected from the following: Pam3CSK4, triacylated lipopeptide, glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, outer surface lipoprotein, lipopolysaccharide, cytomegalovirus envelope protein, glycoinositol phospholipid, glycolipid, GPI anchor, herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, mannuronic acid polymer, bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinoline, antiviral compound, unmethylated CpG oligodeoxynucleotide, and profilin. 99. The method of item 93, wherein the immunomodulatory cytokine is a proinflammatory cytokine. 100. The method of item 99, wherein the proinflammatory cytokine is selected from the following: IL-1β, IL-2, IL-4, IL-6, TNFα, or IFNγ. 101. Modified B cells are B reg 101. The method according to any one of items 93 to 100, wherein the cell is a cell. 102. B reg 102. The method of claim 101, wherein the cells express the immunomodulatory cytokine IL-10. 103. B reg 103. The method of claim 102, wherein the cells further express one or more additional immunomodulatory cytokines selected from the following: IL-2, IL-4, IL-6, IL-35, TNF-α, TGFβ, PD-L1 FasL, and TIM1. 104. B reg 104. The method of any one of paragraphs 101-103, wherein the cells express one or more cell surface markers selected from 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. 105. B reg106. The method of claim 105, wherein the cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD. 106. B reg 106. The method of claim 105, wherein the cells express CD25 and CD71. 107. B reg 107. The method of any one of items 101 to 106, wherein the cells do not express CD73. 108. The method of any one of items 93 to 107, wherein the modified B cells are neuroprotective. 109. The method of any one of items 93 to 108, wherein the modified B cells are anti-inflammatory. 110. The method of any one of items 93 to 109, wherein the modified B cells are immunomodulatory. [Brief explanation of the drawings]

[0066] [Figure 1A] Figure 1A shows that application of B cells induces complex changes in the molecular microenvironment of the wound. Figure 1A is a schematic representation of the average duration of the major stages of wound healing in a wild-type mouse wound model. [Figure 1B]The application of B cells induces complex changes in the wound's molecular microenvironment. Figure 1B shows a heat map summarizing the expression dynamics over time for proteins whose expression significantly changed in response to B cell application. A total of 213 proteins, including proteins significantly altered in association with B cell treatment (n = 111; p < 0.05, unpaired t-test) and those with high fold changes for each time point (top 20 up- or down-regulated proteins) (n = 112), regardless of significance level, were categorized according to their role in wound healing. The heat map shows the fold change in expression after B cell treatment at days 0, 1, 4, and 10 after injury. Red = upregulated; green = downregulated. Of particular note was the downregulation of multiple proteins associated with inflammation and inflammatory cells 4 days after injury, and the substantial upregulation of proteins associated with cell proliferation, protection from apoptosis (cell death) and from oxidative stress, and tissue remodeling (formation of hair follicles and muscle) 4 to 10 days after injury. [Figure 2-1]Figures 2A-H show the average expression of proteins by functional family over time in saline-treated wounds (control, normal wound healing) or in wounds after treatment with B cells. This analysis demonstrates the overall effect of B cells as homeostatic agents rather than inducers or inhibitors of protein expression. Application of B cells was associated with maintaining constant levels of protein expression that normally either decreases or increases during injury and healing, significantly reducing the inflammation peak observed in normal healing, preventing the decrease in anti-apoptotic factors (arrows) and oxidative stress protectants, and increasing proliferation (Figure 2A-B), reducing the decline in antioxidant stress protectants and cell proliferation, and maintaining low levels of cell migration (Figure 2C-D), maintaining constant levels of proteins associated with remodeling and secondary skin structure (Figure 2E-F), reducing the levels of proteolysis and autophagy observed early in injury in controls, and increasing the levels of proteins associated with angiogenesis and nerve regeneration in the later stages of healing (Figure 2G-H). [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 2-4] See description of Figure 2-1. [Figure 3]This figure shows the experimental paradigm for the in vivo evaluation of B cell application in acute wound healing. A total of four full-thickness wounds were created in the dorsal skin of wild-type C57B16 mice, and mature naive B cells purified from isogenic animals were applied directly to the wound surface. Control animals received saline application. To create a similar microenvironment without injury, B cells or saline control were also injected subcutaneously under the intact skin as an internal control. After the specified survival period, wounded or uninjured skin tissue was harvested, dissociated, and processed for flow cytometry analysis. The scatter plot on the right shows the typical distribution of cell suspensions from each treatment category. Wound samples showed a characteristic influx of leukocytes (hollow white arrows), which are almost absent in uninjured tissue. Although B cells are typically few in number at either location, they can be readily detected in large numbers after experimental application (red arrows). [Figure 4] Flow cytometry gating strategy and analysis of cell suspensions from B cell-treated and control wounds are shown. Live cells were gated into three major categories: B cells (CD19+ / B220+ lymphocytes), non-B cell leukocytes (CD140a- / B220- leukocytes), including neutrophils, monocytes and macrophages, dendritic cells, and a mixture of T cells, and fibroblasts (CD140a+ / B220-). These cell categories were evaluated for markers of activation and cytokine production. [Figure 5]This figure shows the dynamics of activation markers and key cytokines in B cells recovered from wounds after defined exposure periods to the wound microenvironment. B cells were exposed to the wound niche in vivo or injected under uninjured skin (control at equivalent locations). Control B cells maintained on ice immediately after isolation for the same duration are shown for comparison. After periods including 18 hours, 2 days, 4 days, and 10 days, the wounds were treated with brefeldin A for 4 hours to induce intracellular retention of cytokines. B cells were then recovered by tissue excision and dissociation and further characterized by flow cytometry for both surface markers and intracellular cytokines. B cells exposed to the wound microenvironment transiently upregulated multiple immunoregulatory cytokines, peaking 2 days after application. Several immunoregulatory cytokines, including TGFβ and IL-6, remained elevated at 4 days, and IL-10 remained elevated up to 10 days. N = 3-6 animals / group. [Figure 6] Heatmap as aggregation of mean values for each marker in B cells exposed to the wound microenvironment, B cells exposed to subcutaneous control, or B cells maintained on ice (no exposure). [Figure 7] Dynamics of activation markers and key cytokines in infiltrating non-B cell leukocyte aggregates present in wounds are shown. Overall, when B cells were present in the wound, infiltrating leukocytes produced more of the anti-inflammatory cytokines IL-10, TGFβ, and IL-35, and less of the pro-inflammatory cytokines TNFα and IL-2. This effect was most pronounced 4 days after injury and B cell application and persisted for up to 10 days. N = 3-6 animals per group. [Figure 8] Heatmap showing the pattern of increased production of anti-inflammatory cytokines (IL-10 and TGFβ) in the presence of B cells, as an aggregation of the mean values for each marker in infiltrating non-B cell leukocytes in the wound microenvironment. [Figure 9]We show the dynamics of activation markers and key cytokines in the CD140a+ fibroblast population in wound and subcutaneous tissue. When wounds were exposed to B cells, wound-resident fibroblasts produced significantly more IL-10 and TGFβ at 10 days after injury. Furthermore, when B cells were applied, wound fibroblasts produced less of the pro-inflammatory cytokine TNFα at both 4 and 10 days after injury. [Figure 10] Heatmaps show aggregated mean values for each marker in wound and subcutaneous tissue fibroblasts treated with either B cells or saline. Fibroblasts are the most important source of anti-inflammatory and pro-regenerative factors in wound healing and produce high levels of IL-10 and TGFβ, regardless of treatment. Nevertheless, fibroblasts from wounds treated with B cells continued to produce higher levels of both IL-10 and TGFβ at 4 and 10 days after injury, whereas saline-treated wounds showed reduced levels of these anti-inflammatory cytokines. Interestingly, a significant effect of B cell application was observed in reducing pro-inflammatory cytokines, including IL-6 and TNFα, in wound fibroblasts. [Figure 11] These results demonstrate that functional TLR signaling and IL-10 production are essential components for the regenerative function of foreign B cells in wound healing. Full-thickness excisional wounds (shown here at day 6 of healing) were treated on day 0 with B cells lacking the common TLR signaling adaptor myeloid differentiation factor 88 (MyD88), B cells lacking IL-10, or WT B cells as a control. Saline was also included in each test animal as an internal control. WT B cells consistently promoted wound closure in WT animals by days 2–3, whereas MyD88– / – B cells and IL-10– / – B cells, similar to saline application, showed no benefit on wound closure. [Figure 12]Figure 1 shows an unsupervised hierarchical cluster analysis of identified proteins expressed in skin wound samples. Only identified proteins found to be consistently present across all samples were included in the analysis. (A) Hierarchical clustering using complete linkage of 3809 proteins (rows) consistently expressed in all animals at four different time points after injury (columns) in both B cell-treated and saline-treated wounds. The pseudocolor scale indicates the normalized, log-transformed fold-change in expression value for each protein. A dendrogram shows 15 protein clusters derived from this analysis, with the color of each cell in (A) corresponding to the cluster's mean expression value at each time point. Proteins are clustered by their expression pattern over time. (B) Heatmap of the hierarchical clusters from (A) showing all 3809 proteins. (C) Gene Ontology analysis of the 15 hierarchical clusters. The mouse GOslim gene list from QuickGO (accessible at https: / / www.ebi.ac.uk / QuickGO) was used to examine 15 hierarchical clusters. Bar graphs show the top biological function category for each cluster. [Figure 13] The distribution of proteins that were significantly altered in response to treatment with B cells at each time point during wound healing evaluated is shown. [Figure 14] An experimental paradigm for assessing the effects of B cell application on functional (behavioral) and histological recovery after contusion TBI is presented. Adult male C57BL / 6J mice were anesthetized, and a 5 mm circular craniotomy was performed over the left parietal-temporal cortex, and the bone flap was removed. Immediately prior to injury, a single injection of 2 x 10 B cells was delivered intraparenchymally into the ipsilateral hemisphere. Mice then underwent CCI or were sham-injured. After recovery, motor function, motor and spatial learning, and memory performance, anxiety, and depressive-like behavior were assessed using multiple assays. At the end of behavioral testing (day 35), animals were euthanized, and brains were harvested to assess total injury volume. [Figure 15] Effects of acute treatment with B cells on vestibular-related motor function and striatal-related learning are shown. (A) Rotarod assessment demonstrated a significant protective effect of B cells administered at the time of CCI. Notably, during repeated trials, the latency to fall increased in B cell-treated mice, similar to that in sham-injured animals, suggesting a component of motor learning. No such improvement was observed in T cell- or saline-treated controls. After the second trial, no significant differences were observed between CCI-injured mice receiving B cell treatment and sham-injured animals treated with B cells. (B) Assessment of recovery of vestibular-related motor skills after injury using the wire grip assay demonstrated a significant effect of injury compared with sham-injured controls, but no statistically significant differences were observed between treatment conditions in injured mice. Data are means ± 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; sham injury + B cells, n = 10 mice; sham injury + saline, n = 10 mice. [Figure 16]The effects of a single acute application of B cells on learning and memory are shown. (A)-(D) Morris water maze assessment. Learning curves showed significant improvement in CCI mice treated with B cells compared with saline-treated CCI animals (p < 0.05). After the third trial, no significant differences were observed between B cell-treated injured animals and either sham-injured condition (p > 0.98) (A). The visible platform trial showed no difference between the injured and uninjured treatment conditions (B). (C) The probe trial showed that CCI-injured animals treated with B cells spent above chance levels in the target quadrant, with no significant difference compared with sham-injured mice. In contrast, control CCI-injured mice treated with either T cells or saline spent only chance levels exploring the target quadrant, which was significantly different from sham-injured animals (p < 0.05). The dashed line indicates chance levels. (D) Representative swim path traces during the probe trial show a spatial exploration pattern in CCI-B cell mice, similar to both sham-injured groups, while CCI mice in the T cell and saline groups employed a non-spatial strategy. (E) Y-maze assessment of short-term learning and memory. CCI mice treated with B cells had significantly higher alternation scores than T cell- or saline-treated groups and performed similarly to the sham-injured group. All data are shown as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. CCI + B cell, n = 12 mice; CCI + T cell, n = 12 mice; CCI + saline, n = 12 mice; sham-injured + B cell, n = 10 mice; sham-injured + saline, n = 10 mice. [Figure 17]The effects of B cell treatment on anxiety- and depression-like behavior after CCI are shown. (A) Elevated plus maze assay of anxiety-like behavior. No significant overall differences were observed between treatment groups, except for slight differences in the time spent in the closed arms between CCI-injured mice receiving B cells at the time of injury and animals receiving the same number of T cells at the time of injury (*p < 0.05). (B) Forced swim assay of depression-like behavior. No injury- or treatment-related effects were observed in this assay. All data are presented as mean ± SEM. *p < 0.05. CCI + B cells, n = 12 mice; CCI + T cells, n = 12 mice; CCI + saline, n = 12 mice; sham injury + B cells, n = 10 mice; sham injury + saline, n = 10 mice. [Figure 18] The effect of B cell treatment on histological outcomes after CCI is shown. (A) Representative coronal sections through the injury site 35 days after injury. In B cell-treated animals, portions of the hippocampus often compensated for the injured hemisphere (arrow). The section shown is located approximately -2.2 mm from bregma. (B) The total volume of brain injury in B cell-treated mice was significantly reduced by 40-60% compared to saline and T cell controls 35 days after TBI. (C) Lesion area in transverse brain sections along the anterior-posterior axis of the brain. Results show a consistent reduction in lesion size in injured brains treated with B cells. (D) The total compensated hippocampal volume in the injured hemisphere was significantly greater in B cell-treated animals compared to both CCI controls. All data are presented 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; sham injury + B cells, n = 10 mice; sham injury + saline, n = 10 mice. [Figure 19]The effect of B cell treatment on gliosis and microglial activation is shown. (A)-(D) Confocal images showing immunolabeling for GFAP and CD68 in the entire medial aspect of the injury 35 days after CCI and treatment with either saline (A), B cells (B), or T cells (C), or in saline-treated sham-injured controls (D). (E) Quantitative analysis of the area occupied by GFAP immunostaining showed a significant decrease in reactive astrogliosis in injured animals treated with B cells compared with either saline-treated or T cell-treated CCI controls. (F) Quantitative analysis of CD68 immunostaining showed a significant decrease in the presence of CD68 in animals treated with either T or B cells after CCI compared with saline-treated CCI controls. n = 4 imaging fields per animal. All data are presented as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. [Figure 20] Demonstrating B cell survival and persistence in the brain. (A) Representative example of in vivo imaging of a WT C57B16 / J mouse at multiple time points after CCI and intraparenchymal application of 5 x 106 Bluc cells. (B) Light emission from the head (n = 6 mice) at the location of the CCI wound site shows that cells survive in situ for approximately 14 days after application, with the number of viable cells decreasing significantly after 7 days. [p / s] = photons per second. [Figure 21]Figure 1 shows the localization of B cells at the injury site after CCI. (A) Immediately after intraparenchymal injection and CCI, pre-labeled B cells are visible at the injury site. The black rectangle indicates the area imaged in (B). (B) Confocal microscopy image of a coronal section through the injury site showing clustered B220+ B cells at the injection site (arrow). Cell proliferation, as indicated by Ki67 immunolabeling, was not observed immediately after injury. (C) Four days after B cell injection and CCI, labeled B cells are still observable at the injury site; however, the intensity of vital staining has decreased by this time point compared to immediately after injection. The black rectangle indicates the area imaged in (D). (D) Confocal image of a coronal section through the injection site 4 days after injury and B cell administration. B220+ B cells can still be found clustered and in large numbers at the injury site. Vigorous cell proliferation was observed throughout the region, but no co-staining for B220 or Ki67 was observed. (E) Enlarged version of the boxed area in (D). (F) In sham-injured animals, the needle track through the cortex, outlined by astroglial scar formation, is still visible 35 days after treatment. No B220+ B cells can be observed at the original injection site at this time point. (G) High-magnification confocal image of the boxed area in (F). In all confocal images, cell nuclei were counterstained with DAPI. n = 4 animals per time point. [Figure 22] An outline of the experimental design is shown below: Body weight and Neuroscore assessments were performed twice weekly at the same time of day by an experimenter blinded to treatment conditions. [Figure 23]Figure 1 shows normalized body weight (percentage of the value on day 76 for each individual animal) measured twice weekly over time in the B cell-treated and saline-treated groups. The graph shows a composite index of normalized body weight and viability, with dead animals receiving a weight score of 0. We observed the expected difference in body weight between non-carrier control animals and SOD1 transgenic animals in each treatment group. In non-carrier control animals, a gradual weight gain was observed over the study period, regardless of treatment. Results also showed a delayed decline in transgenic SOD1 animals receiving B cells (arrows). N = 32 per treatment condition. [Figure 24] Figure 1 shows an analysis of peak body weight in SOD1-G93A animals. A. Survival plot shows the time points at which animals reached their peak body weight. B. Treatment with B cells significantly delayed the onset of complete paralysis, as indicated by the time to reach peak body weight. Statistics: A: Gehan-Breslow-Wilcoxon test; B: Unpaired t-test. N = 32 animals per group. [Figure 25] Neuroscore values are shown over time. In the combined graph of neurological score and survival, animals that died already reached a neuroscore of 4, but were still assigned a value of 4 for the remainder of the study. The increase in neuroscore values typically observed in transgenic SOD1 animals was slower in animals treated with B cells, especially in the early stages of disease progression (orange boxes). Statistics: 2-way ANOVA with Tukey post-hoc correction for multiple comparisons. [Figure 26]Survival analysis of transgenic SOD1-G93A animals is shown. Animals that reached a neuroscore of 4 (complete paralysis) were considered to have died of ALS. Compared to saline control treatment, treatment with naive B cells significantly extended survival (N = 32 animals per group). Statistics: (Left): Gehan-Breslow-Wilcoxon test; (Right): 1-way ANOVA. N = 32 animals per group. [Figure 27] End-point assessment of lumbar motor neurons is shown. 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 pathological, abnormal neurons (arrows) showing morphological features of injury / degeneration were counted by an experimenter blinded to treatment. C: The total number of motor neurons was significantly reduced in transgenic animals, but there were no treatment-related differences within this group. D: A significant benefit of treatment with B cells was revealed when the percentage of degenerated, pyknotic motor neurons was specifically analyzed. Statistics: (Left): 2-way ANOVA; (Right): Unpaired t-test. N = 19–24 animals per group. Note that tissue sampling was not possible in all animals tested. DETAILED DESCRIPTION OF THE INVENTION

[0067] Detailed Description The present invention will now be described in detail, using the following definitions and examples for reference purposes only. All patents and publications referenced herein are expressly incorporated by reference. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In practicing or testing the present invention, any methods and materials similar or equivalent to those described herein can be used, but preferred methods and materials are described. Ranges using numerical values include the numbers defining the range.

[0068] The headings provided herein are not limitations of the various aspects or embodiments of the present invention which may be had by reference to the specification as a whole, and accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0069] definition As used herein, the term "neurodegenerative disease" refers to a neurological disease, disorder, or abnormality characterized by the progressive loss of neuronal structure or function, including neuronal death, for example, in the central nervous system (CNS). At the subcellular level, these diseases share many similarities. Furthermore, there are many similarities between different neurodegenerative disorders, including abnormal protein assembly and induced cell death. Neurodegeneration can be found at many different levels of neural circuits, ranging from molecular to systemic. Neurodegeneration can be characterized by molecular markers of disease progression, such as, for example, T-tau (total tau), P-tau (hyperphosphorylated tau), Aβ42 (amyloid beta 42), the Aβ42 / Aβ40 ratio, YKL-40 (chitinase 3-like protein 1), VLP-1 (visinin-like protein 1), NFL (neurofilament light chain), pNFH (phosphorylated neurofilament heavy chain subunit), Ng (neurogranin), and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43 (TAR DNA-binding protein 43), decreased α-synuclein, and / or decreased levels of 3,4-dihydroxyphenylacetic acid (see, e.g., Robey and Panegyres. Cerebrospinal fluid biomarkers in neurodegenerative disorders. Future Neurol. 14(1). (2019), 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 neuroaxonal dystrophy, progressive supranuclear palsy, dementia with Lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

[0070] 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.CNS injury can be caused by external mechanical force, as described herein.CNS injury includes traumatic brain injury (TBI) and / or spinal cord injury (SCI).

[0071] As used herein, the term " traumatic brain injury (TBI) " refers to the disruption of the normal function of brain caused by external mechanical force.For example, TBI can be caused by head trauma or brain contusion (for example, can be caused by falling, gunshot wound, sports accident, construction accident, traffic accident, or the injury that penetrates the skull or brain of the subject).TBI is diagnosed according to the clinical guideline known to those skilled in the art. TBI can be further characterized by molecular markers of disease progression, such as protein biomarkers for neuronal cell body injury (UCH-L1, NSE), astroglial injury (GFAP, S100B), neuronal cell death (αII-spectrin breakdown products), axonal injury (NF proteins), white matter injury (MBP), post-injury neurodegeneration (total tau and phosphorylated tau), and post-injury autoimmune responses (autoantibodies targeting brain antigens) (see, e.g., Wang et al. An update on diagnostic and prognostic biomarkers for traumatic brain injury. Expert Rev Mol Diagn. 18(2): 165-180 (2018)), which is incorporated by reference in its entirety. TBI can occur simultaneously with SCI and can result from the same injury or the same accident.

[0072] As used herein, the term " spinal cord injury (SCI) " refers to the injury to spinal cord that is caused by external mechanical force.For example, SCI can be caused by spinal cord trauma or spinal cord contusion (for example, can be caused by falling, gunshot wound, sports accident, construction accident, traffic accident, or the injury that penetrates the spinal cord of the subject).SCI is diagnosed according to the clinical guideline known to those skilled in the art.SCI can occur at the same time as TBI, and can be caused by the same injury or the same accident.

[0073] As used herein, the term "inflammatory disease" or "immune disease" refers to a disease, disorder, or condition that has an inflammatory or immune component to its etiology, onset, progression, or symptomatology. For example, an inflammatory or immune disorder can involve dysregulation of an inflammatory or immune pathway and / or an abnormal inflammatory or immune response to a stimulus. Exemplary inflammatory or immune disorders include cystic fibrosis, cardiovascular disease, keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis.

[0074] As used herein, the term "neuroprotective" refers to the property of preventing, inhibiting, or reducing neuronal cell death.For example, a composition or method that is neuroprotective can be characterized by a change (e.g., a reduction) in the symptoms associated with neurodegenerative disorders, TBI, or SCI.Alternatively, a composition or method that is neuroprotective can also be characterized by the effect of the composition or method on molecular markers of disease, such as the molecular markers described herein for neurodegenerative disorders, TBI, or SCI.

[0075] As used herein, the term "anti-inflammatory" refers to the property of preventing, inhibiting, or reducing inflammation. For example, a composition or method that is anti-inflammatory may be characterized by a change (e.g., a reduction) in symptoms associated with an inflammatory disorder. Alternatively, a composition or method that is anti-inflammatory may be characterized by a decrease in inflammatory markers (e.g., a decrease in pro-inflammatory cytokines) or an increase in anti-inflammatory markers (e.g., an increase in anti-inflammatory cytokines).

[0076] As used herein, the term "immunomodulatory" refers to the property of initiating or altering (e.g., increasing or decreasing) the activity of cells associated with an immune response. An immunomodulatory composition or method may increase the activity of cells associated with an immune response, e.g., by increasing proinflammatory markers, e.g., cytokines, and / or may decrease the activity of cells associated with an immune response, e.g., by decreasing proinflammatory markers, e.g., cytokines.

[0077] As used herein, the terms "B cells" or "B lymphocytes," used interchangeably herein, refer to a small subtype of lymphocyte, a type of white blood cell. Unlike the other two classes of lymphocytes, T cells and natural killer cells, B cells express B cell receptors (BCRs) on their cell membranes. The BCR enables B cells to bind to specific antigens against which the B cells initiate an antibody response. B cells function as 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 cells" refers to B cells that have completed the process of B cell maturation, for example, in the bone marrow of a mammal. Mature B cells leave the bone marrow and migrate to secondary lymphoid tissues, where they can interact with foreign antigens and / or T helper cells. The stages of B cell maturation are well characterized in the scientific literature and known to those skilled in the art.

[0078] As used herein, the term "naive B cells" refers to B cells that have not been exposed to an antigen.

[0079] As used herein, the term "Breg cells" or "regulatory B cells" refers to a type of B cell that is involved in immune regulation and suppression of immune responses. The Breg cells of the present disclosure are mature naive B cells that express 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 CD138. When introduced into an injured environment, Breg cells can produce immunomodulatory cytokines, including, but not limited to, IL-2, IL-4, IL-6, IL-10, IL-35, TNF-α, TGF-β, and interferon-γ. In particular, Breg cells are characterized by the production of IL-10.

[0080] As used herein, the term "cytokine" refers to a small protein involved in cell signaling. Cytokines can be produced and secreted by immune cells, such as T cells, B cells, macrophages, and mast cells, and can include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. As used herein, the term "proinflammatory cytokine" refers to a cytokine secreted by immune cells that promotes 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-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, e.g., TNFα), interferon-γ (IFNγ), and granulocyte-macrophage colony-stimulating factor (GMCSF).

[0081] As used herein, the term "Toll-like receptor (TLR) agonist" refers to a ligand that binds to and activates Toll-like receptors (TLRs), causing downstream TLR cell signaling. TLR agonists are known to those skilled in the art and include endogenous and exogenous ligands. Exemplary endogenous ligands that are TLR agonists include heat shock proteins, necrotic cells or their fragments, oxygen radicals, uric acid crystals, mRNA, β-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1, pulmonary surfactant protein A, low-density lipoprotein (LDL), pancreatic elastase, polysaccharide fragments of heparan sulfate, soluble hyaluronan, α A-crystallin, and CpG chromatin-IgG complexes. Exemplary exogenous ligands that are TLR agonists include: Pam3CSK4, triacylated lipopeptides, glycosylphosphatidylinositol (GPI)-anchored proteins, lipoarabinomannan, outer surface lipoproteins, lipopolysaccharides, cytomegalovirus envelope proteins, glycoinositol phospholipids, glycolipids, GPI anchors, herpes simplex virus 1 or fragments thereof, lipoteichoic acid, mannuronic acid polymers, bacterial outer membrane porins, zymosan, double-stranded RNA, single-stranded RNA, poly(I), poly(C), taxol, flagellin, modulin, imidazoquinoline, antiviral compounds, unmethylated CpG oligodeoxynucleotides, and profilin.

[0082] As used herein, the term "treatment" (and variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual receiving treatment, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to: preventing the occurrence or recurrence of a disease or disorder (such as those described herein), alleviating the symptoms of such a disease, reducing any direct or indirect pathological consequences resulting from the disease, and altering the immune response. In addition, treatment refers to clinical intervention related to any of the diseases or disorders described herein.

[0083] As used herein, the term "administering" refers to the method of administering medication to a subject.The composition utilized in the method described herein can be, for example, administered intravitreally (for example, by intravitreal injection), by eye drops, intramuscularly, intravenously, intradermally, transdermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraparenchymally, intraprostatically, intrathoracically, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, The compositions may be administered subconjunctivally, intravesically, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, by inhalation, injection, implantation, infusion, continuous infusion, by direct local perfusion of target cells, by catheter, by irrigation, as a cream, or as a lipid composition. The compositions used in the methods described herein may also be administered systemically or locally. For local administration, the medication may be administered as a lotion, cream, ointment, or gel. The method of administration may vary depending on various factors (e.g., the composition being administered and the severity of the immune dysregulation disorder, disease, or disorder being treated).

[0084] The subject treated according to the present invention is a mammal. The mammal can be, for example, a primate (e.g., a human), a rodent (e.g., a rat or a mouse), or another species of mammal (e.g., a livestock animal or other domesticated animal). In each of the above methods, the mammal can have any of the diseases or disorders disclosed herein. In a preferred embodiment, the subject is a human.

[0085] A mammal "in need" of treatment may include, but is not limited to, a mammal with, or having previously had, a neurodegenerative disorder, a TBI, an SCI, an immunological disorder, or a mammal with symptoms of an immunological disorder, or an inflammatory disorder or disease. Exemplary disorders are disclosed herein.

[0086] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result or to achieve a particular stated purpose. An "effective amount" can be determined experimentally and by known techniques related to the stated purpose.

[0087] The term "isolating" or "isolation" refers to both the physical identification and separation of a cell or cell population from a cell culture or biological sample. Isolation can be performed by applying suitable cell biological techniques, either based on examining the cell culture and characterizing (and, if possible and desirable, physically separating) cells that meet a criteria, or based on automated sorting of cells (e.g., by FACS) by characteristics such as, for example, the presence / absence of an antigen and / or cell size. In some embodiments, the term "isolating" or "isolation" can include the further step of physical separation and / or quantification of cells, particularly by performing flow cytometry. Physical separation also includes enrichment of cells or cell populations of particular characteristics. An "isolated" cell or an "isolated" cell population is a cell or cell population that has been identified and / or separated as described above.

[0088] The term "cell population" or "population of cells" generally refers to a group of cells. Unless otherwise specified, the term refers to a cell group consisting essentially of or including cells as defined herein. A cell population may consist essentially of cells with a common phenotype, or may include at least a fraction of cells with a common phenotype. Cells are said to have a common phenotype if they are substantially similar or identical in one or more demonstrable characteristics, including, but not limited to, morphological appearance, expression levels of specific cellular components or products (e.g., RNA or protein), activity of specific biochemical pathways, proliferation capacity and / or growth kinetics, differentiation potential and / or response to differentiation signals, or behavior during in vitro culture. Thus, a cell population, or a fraction thereof, can be defined by such demonstrable characteristics. A cell population can be "substantially homogeneous" if a substantially majority of the cells have a common phenotype. A "substantially homogeneous" cell population can include 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, such as B cells (e.g., B reg Furthermore, a cell population may share a common phenotype, such as a phenotype specific to B cells (e.g., B cells), if any other cells present in the population do not change or have a substantial effect on the overall properties of the cell population, and thus the cell population can be defined as a cell line. reg Thus, an isolated cell population (or, for example, isolated B cells) typically comprises at least 60%, or 60% to 99%, or 70% to 90% of B cells (or a subpopulation of B cells, such as B cells). reg cells, etc.

[0089] B cell harvesting and isolation Any source of B cell, also known as B lymphocyte, can be used for collecting purpose.As known to those skilled in the art, such B cell can be derived from bone marrow, spleen, lymph node, blood or other allogeneic tissue that is the source of B cell.The preferred source of B cell is bone marrow and blood.Preferably, autologous B cell, or allogeneic B cell, or xenogeneic B cell is collected.

[0090] Using aseptic techniques, in one embodiment, bone marrow is obtained, preferably from the posterior superior ilium. After isolation and relative purification, the resulting B cells can be used immediately, stored for later use, or cultured for a period of time before use. The B cell population in bone marrow includes prepro-B cells, pro-B cells, pre-B cells, immature B cells, and some mature B cells.

[0091] In this application, the term B cells encompasses prepro-B 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 skilled in the art.

[0092] Methods for obtaining B cells, or for example, precursor B cells, from a heterogeneous cell population are known. Many of these techniques utilize a primary antibody that recognizes a molecule on the surface of the desired B cells or precursor B cells and uses that antibody to positively select for and separate these cells from unwanted cells. This technique is known as positive selection.

[0093] Another commonly used technique uses primary antibodies that recognize molecules on the surface of cells to be separated from desired B cells or precursor B cells. In this manner, molecules on the unwanted cells bind to these antisera and the cells are removed from the heterogeneous cell population. This technique is known as negative selection.

[0094] A combination of positive and negative selection techniques can be utilized to obtain a relatively pure population of B cells or progenitor B cells. Such a population is referred to as isolated B cells. As used herein, relatively pure means at least 60% pure, 65% pure, 70% pure, 75% pure, 80% pure, 85% pure, 88% pure, or higher degrees of purity, such as at least 90% pure, at least 95% pure, at least 97% pure, or at least 98%-99% pure.

[0095] Numerous techniques for separating antibodies bound to cells are available to those skilled in the art. Antibodies can be linked to various molecules that provide labels or tags to facilitate separation. In one embodiment, primary antibodies can be linked to magnetic beads that allow separation in a magnetic field. In another embodiment, primary antibodies can be linked to fluorescent molecules that allow separation in a fluorescence-activated cell sorter. Fluorescent and magnetic labels are commonly used on primary and / or secondary antibodies to achieve separation. Secondary antibodies that bind to primary antibodies can be labeled with fluorescent molecules that allow cell separation in a fluorescence-activated cell sorter. Alternatively, metal microbeads can be linked to primary or secondary antibodies. In this case, a magnet can be used to isolate these antibodies and the cells bound to them.

[0096] To achieve positive or negative selection, a heterogeneous cell population is incubated with a primary antibody for a time sufficient to achieve antibody binding to antigens on the cell surface. If the primary antibody is labeled, separation can occur at this stage. If a secondary antibody is used, the secondary (anti-primary) antibody is then incubated with the cells bound to the primary antibody for a time sufficient to achieve binding of the secondary antibody to the primary antibody. If the secondary antibody has a fluorescent label, the cells are then sent to a fluorescence-activated cell sorter to isolate the labeled antiserum bound to the desired cells. If the secondary antibody has a magnetic label, the selected cells with the primary antibody and the secondary antibody-labeled microbeads then form a complex, which is left behind when a magnet is applied, while other unlabeled cells are removed along with the cell culture medium. The positively labeled cells are then eluted and ready for further processing. Negative selection involves the collection of unlabeled cells that have passed through a magnetic field.

[0097] Miltenyi Biotec has developed a number of products for the direct magnetic separation of B cells and different B cell subsets. B cells can be isolated either 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. Both positive selection and depletion strategies can be pursued for the direct isolation of B cells and for the isolation of B cells by standard methods.

[0098] Thus, in a practical embodiment, patients and potential donors are tested for HLA (A, B, and DR-B1), for example, by the American Red Cross. Potential donors found to be haploidentical to the recipient are available as allogeneic donors. The donor then undergoes apheresis to isolate and harvest B cells. A B cell product is then prepared for infusion.

[0099] Upon receipt of donor allogeneic mononuclear cells - MNC (A), the apheresis product is enriched for B cells using Miltenyi Biotec's CliniMACS® CD19 selection. After washing away platelets, the product (CD19 CliniMACS reagent) is enriched for B cells up to 4 x 10 per vial. 10 A total number of cells, up to a maximum of 5 x 10 9 The target fraction (CD19+ cells) was treated with CD19 microbeads separated on an LS column to enrich for CD19+ cells. The target fraction was washed, and then the infusion medium was supplemented with 25% HSA (final concentration 1%) and Plasma-Lyte A.

[0100] The method generally includes: Day 1 a. The donor apheresis product is received and sampled for sterility determination, cell count, viability determination, and flow cytometry. b. The product is stored in the cold overnight. Day 2 a. The product is removed from the refrigerator, mixed thoroughly, and allowed to equilibrate to ambient temperature for 30 minutes. Samples are removed for sterility determination, cell count, viability determination, and flow cytometry (DuraClone panel with CD20). b. Platelet washout is performed according to standard CliniMacs procedures. c Beads are added and incubated for 30 minutes on a shaker according to standard CliniMacs procedures except that the incubation is carried out at 4°C. d. After incubation with the beads, one antibody wash is performed with chilled (4 degrees) media and the product is loaded onto a CliniMacs LS column according to the standard CliniMacs procedure: e. Separation is performed using the CliniMacs Enrichment 1.1 program. f. The CD19 enriched target fraction is sampled for cell counting, flow cytometry, stability determination, and sterility determination. g. CD19-depleted (non-target fraction) is sampled for cell counting and flow cytometry.

[0101] Isolation of B cells from heterogeneous cell populations and stem cell populations can also involve a negative selection process in which red blood cell lysis is first performed in the bone marrow by placing the bone marrow in a hypotonic buffer and then centrifuging the red blood cells from the buffer. Red blood cell debris remains in the supernatant, which is removed from the test tube. The bone marrow-derived cells are then resuspended in a buffer with appropriate conditions for antibody binding. Alternatively, density gradient centrifugation can be performed on the bone marrow. The buffy coat layer containing the bone marrow-derived cells is removed from the gradient after centrifugation. The cells are washed and resuspended in an antibody binding buffer, and then incubated with primary antibodies directed against stem cells, T cells, granulocytes, and monocytes / macrophages (called lineage depletion), followed by positive selection using antibodies against B cells.

[0102] Different subpopulations of B cells can be distinguished based on the differential expression of various surface markers and collected accordingly.

[0103] Ex vivo B cell stimulation Once isolated, the B cells can be treated or stimulated by exposing them to one or more of the TLR agonists or immunomodulatory cytokines described herein. IL-10-producing B cells can be stimulated using such ex vivo stimulation. reg The production of cells is useful in the methods and treatment strategies described herein.

[0104] Administration The number of cells administered can be related to the area or volume of the affected area to be treated and can be related to the delivery method.

[0105] One non-limiting range of numbers of B cells for administration is 10 4 ~10 14 B cells, which varies depending on the volume of tissue or organ being treated. Other ranges include 10 5 ~10 12 B cells, and 10 6 ~10 10 B cells (e.g., B reg The pharmaceutical composition containing the cells is 4 ~10 14 B cells, 10 5 ~10 12 B cells, or 10 6 ~10 10 The antibody may comprise B cells.

[0106] Individual injection volumes can include the non-limiting ranges of 1 μl to 1000 μl, 1 μl to 500 μl, 10 μl to 250 μl, or 20 μl to 150 μl. Total injection volumes per animal range from 10 μl to 10 ml, which varies depending on the species, method of delivery, and volume of tissue or organ being treated.

[0107] Pharmaceutical Compositions The B cells described herein can be incorporated into a vehicle for administration to a patient, such as a human patient with a disease or disorder described herein. Pharmaceutical compositions comprising B cells can be prepared using techniques known in the art. For example, such compositions can be prepared using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); incorporated herein by reference) and in a desired form, such as an aqueous solution.

[0108] The B cells described herein can be administered in any physiologically compatible carrier, such as buffered saline or a solution containing one or more electrolytes (e.g., one or more of sodium chloride, magnesium chloride, potassium chloride, sodium gluconate, or sodium acetate trihydrate). For example, the B cells can be administered in PlasmaLyte infusion buffer. PlasmaLyte is a family of balanced crystalloid solutions with several different formulations available worldwide, depending on local clinical practice and preferences. It closely mimics human plasma in its electrolyte content, osmolality, and pH. PlasmaLyte solutions also have additional buffering capacity and contain anions, such as acetate, gluconate, and especially lactate, which are converted to bicarbonate, CO2, and water. Advantages of PlasmaLyte include the correction of volume and electrolyte deficiencies while addressing acidosis. In a preferred embodiment, the infusion buffer is PlasmaLyte A. PlasmaLyte A is a sterile, pyrogen-free, isotonic solution for infusion (e.g., intravenous) administration. Each 100 mL of PlasmaLyte A contains: 526 mg sodium chloride (NaCl); 502 mg sodium gluconate (CH 11 NaO7); 368 mg sodium acetate trihydrate (C2H3NaO2 3H2O); 37 mg potassium chloride (KCl); and 30 mg magnesium chloride (MgCl2 6H2O). This does not contain an antimicrobial agent. The pH is adjusted using sodium hydroxide. The pH is approximately 7.4 (e.g., 6.5 to 8.0).

[0109] Other pharmaceutically acceptable carriers and diluents include saline, aqueous buffer 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 Dulbecco's Modified Eagle's Medium (DMEM), sterile saline, sterile phosphate-buffered saline, Leibovitz's medium (L15, Invitrogen, Carlsbad, Calif.), sterile aqueous dextrose, and any other physiologically acceptable liquid.

[0110] Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by using a coating agent, such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it may be preferable to include an isotonic agent, such as sugar or sodium chloride. The solution is preferably sterile and fluid to the extent that easy puncture is present. Preferably, the solution is stable under the conditions of manufacture and storage, and is protected against the contaminating action of microorganisms, such as bacteria and fungi, by the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Solutions of the present invention can be prepared using a pharmaceutically acceptable carrier or diluent, and optionally other ingredients listed above, followed by filter sterilization, and then incorporating the B cells described herein.

[0111] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered, and if necessary, the liquid diluent is 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, sterile aqueous media that can be used in connection with the present disclosure are known to those skilled in the art. In any case, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for human administration, preparations may meet the sterility, pyrogen control, general safety, and purity standards required by the FDA's biological product standards.

[0112] Pharmaceutical compositions can also contain excipients that promote cell membrane stability. For example, highly soluble osmoregulatory proteins, such as those with high molecular weights, can be added to the injection medium. Serum proteins, such as human serum albumin (HSA), can be included in the pharmaceutical compositions described herein as a medium supplement to maintain cell membrane stability. HSA includes recombinant albumin. Alternatively, human serum can be used to stabilize pharmaceutical compositions containing cells. [Example]

[0113] The present invention will be described in further detail in the following examples, which are not intended to limit the scope of the claimed invention in any way. The accompanying drawings are intended to be considered as an integral part of this specification and as an explanation of the invention. All cited references are specifically incorporated herein by reference in their entirety. The following examples are provided for illustrative purposes, but are not intended to limit the claimed invention.

[0114] Example 1: Foreign B cells regulate immune infiltration and responses This example demonstrates the large-scale analysis of the molecular effects of B cells on wound healing using isobaric labeling multiplexed proteomics.

[0115] Our data showed that B cell application had a significant homeostatic effect on the wound microenvironment, accompanied by a significant decrease in proteins associated with inflammatory responses and an increase in proteins associated with tissue proliferation and remodeling. By recovering applied foreign B cells from the wound niche at various time points after application and examining cell populations using polychromatic flow cytometry, we determined that mature naive B cells applied to the wound transitioned to a regulatory phenotype characterized by expression of CD138 and the immunoregulatory cytokines IL-10, IL-35, and TGF-β. This Breg-like phenotype appeared transiently, peaking 2 days after application. Furthermore, the phenotype of monocytes and macrophages in the wound environment was significantly altered as a result of B cell application, accompanied by a decrease in the expression of proinflammatory cytokines, including IL-2, IL-4, IL-6, and IFN-γ. Thus, naive B cells deployed at the site of injury detect local inflammatory signals and damage-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-β, which act on nearby immune cells and fibroblasts and bias their phenotype toward an anti-inflammatory and pro-regenerative one. 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 capabilities.

[0116] material and method The following materials and methods were utilized in this study.

[0117] animal Wound healing studies were performed in 7- to 9-week-old male wild-type C57Bl6 / J mice (Jackson Laboratories). Male WT C57Bl6 / J mice were used as isogenic donors for B cell isolation. Animals were maintained under standard laboratory conditions at temperatures ranging from 20 to 23°C, with a 12-h:12-h light:dark cycle and free access to food and acidified water. All animal handling was performed in accordance with the Public Health Service Policy on Humane Care of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. All efforts were made to reduce the number of animals used and minimize animal suffering.

[0118] Cell isolation Mouse spleens were collected in ice-cold EasySep™ buffer (STEMCELL Technologies), which contained 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS). Spleens were mechanically dissociated by passing them through a 40 μm cell strainer, and splenocyte suspensions were processed for negative selection of B or T cells by immunomagnetic separation using a commercially available cell isolation kit (STEMCELL Technologies) according to the manufacturer's instructions.

[0119] Wound model, and tissue sampling A full-thickness excision wound through the dorsal skin was created as previously described (Wang et al. (2013) Nat Protoc. 8(2):302-9.). Briefly, mice were anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg), and the dorsal skin was shaved and depilated. Analgesia was administered preoperatively with 0.08 mg / kg buprenorphine injected subcutaneously. The dorsal skin was spread open, and a 5 mm biopsy punch was inserted into the folded skin to create two symmetrical wounds on either side of the back. Each wound was approximately 20 mm 2 The wounds had an initial area of 1000 x 1000 mm. A silicone splint (Sigma-Aldrich) with an inner diameter of 7 mm was attached around the wound using Vetbond tissue adhesive (3M). The splinted wound was then covered with Tegaderm™ transparent dressing (3M). PBS containing the cell suspension or an equal volume of PBS solution alone (saline control) was applied directly to the wound using a manual pipette. Each mouse also received two local subcutaneous injections under the dorsal skin with equal doses of B cells or saline. Each of the treated wound and subcutaneous sites contained 15–20 x 10 6 Each mouse received 20 μl of PBS containing 10 B cells.

[0120] After a defined period of 18 hours, 2 days, or 4 days, mice were lightly anesthetized with 3% isoflurane in O2, and 10-20 μl of a working solution of Brefeldin A (GolgiPlug™, BD Pharmingen) in PBS was applied to each treated wound and subcutaneous site to promote intracellular cytokine accumulation. After a 4-hour incubation, mice were euthanized, and tissue biopsies were taken, including the wound and subcutaneous injection site. Tissue biopsies were enzymatically dissociated in RPMI medium containing 5% FBS, 0.5% L-glutamine, 0.5% penicillin-streptomycin, 1.5 mg / ml, 0.25 U / mg collagenase D (Roche), 1.5 mg / ml, >400 U / mg bovine testicular hyaluronidase (Millipore Sigma), 0.4 mg / ml, 400 U / mg DNAase I (Roche), and 0.025 mg / ml, >10 U / mg dispase I (Millipore Sigma) for 30 minutes at 37°C with gentle shaking. The tissue was then mechanically minced into smaller fragments, followed by further enzymatic dissociation in the same solution for an additional 30 minutes at 37°C with gentle shaking. The digested tissue from the individual wound and subcutaneous samples was then pooled for each mouse and passed through a 100 μm cell strainer followed by a 40 μm cell strainer to obtain a single cell suspension.

[0121] Proteomics Tissue Sampling. Full-thickness excision wounds were created in the dorsal skin of mice as described above. 2 x 10 cells suspended in 20 μl of saline were used. 6 Wounds were treated with either a solution of purified B cells or saline control. After defined periods of 0 (approximately 10 min), 1, 4, and 10 days, mice (n = 3-5 per condition) were euthanized, and the wound area, including the wound edges and subcutaneous layer, was excised and snap-frozen in liquid nitrogen and then stored at -80°C until thawed.

[0122] Protein digestion and tandem mass tag (TMT) labeling. Sample processing was performed as previously described (Lapek et al. (2017) Nat Biotechnol. 35(10):983-989). Protein concentration of cell lysates was determined using a BCA assay (Thermo Scientific). Proteins were then reduced with DTT and alkylated with iodoacetamide as previously described. Reduced and alkylated proteins were precipitated by methanol-chloroform precipitation. Precipitated proteins were reconstituted in 300 μL of 50 mM HEPES, pH 8.5, containing 1 M urea. Vortexing, sonication, and manual disruption were used to increase solubility. The solubilized proteins were digested in a two-step process, starting with overnight digestion with 3 μg of Lys-C (Wako) at room temperature, followed by a 6-hour digestion with 3 μg of trypsin (sequencing grade, Promega) at 37°C. The digest was acidified with trifluoroacetic acid (TFA). The digested peptides were desalted using C18 solid-phase extraction (SPE) (Sep-Pak, Waters). The concentration of the desalted peptide solution was measured using a BCA assay, and the peptides were vacuum-dried in 50 μg aliquots and stored at -80°C until they were labeled with the TMT reagent. The TMT reagent (Thermo Scientific) was suspended in anhydrous acetonitrile (ACN) at a concentration of 20 μg / μL. Dried peptides (50 μg) were resuspended in 200 mM HEPES, pH 8.5, containing 30% ACN, and 5 μL of the appropriate TMT reagent was added to the sample. The peptides were incubated with the reagent for 1 hour at room temperature. The labeling reaction was terminated by adding 6 μL of 5% hydroxylamine. The labeled sample was then acidified by adding 50 μL of 1% TFA, and the peptide mixture was pooled as a ten-plex TMT sample. The pooled sample was desalted by C18 SPE on a Sep-Pak cartridge as described above.

[0123] Fractionation of samples by basic pH reversed-phase liquid chromatography (bRPLC). Sample fractionation was performed by bRPLC, and fractions were pooled for analysis by mass spectrometry. Briefly, samples were resuspended in a solution containing 5% formic acid and 5% ACN and separated through a 4.6 mm x 250 mm ZORBAX Extend C18 column (5 μm, 80 Å, Agilent Technologies) on an Agilent 1260 HPLC system equipped with a fraction collector, degasser, and variable wavelength detector. Separation was performed by applying a gradient of 10 mM ammonium bicarbonate containing ACN, increasing from 22% to 35%, at a flow rate of 0.5 mL / min for 60 min. All 96 fractions were combined as previously described (Edwards et al. (2016) Methods Mol Biol. 1394:1-13). The combined fractions were vacuum dried, reconstituted with a solution of 5% formic acid and 5% ACN, and then analyzed by LC-MS2 / MS3 for identification and quantification.

[0124] Liquid chromatography coupled to mass spectrometry. All LC-MS2 / MS3 experiments were performed on an Orbitrap Fusion (Thermo Fisher Scientific) coupled to an Easy-nLC 1000 (Thermo Fisher Scientific) with a cooled autosampler. Peptides were separated on a microcapillary column (inner diameter 100 μm; outer diameter 360 μm) that was stretched and packed in-house. The column was first packed with approximately 0.5 cm of Magic C4 resin (5 μm, 100 Å, Michrom Bioresources), followed by approximately 0.5 cm of Maccel C18 AQ resin (3 μm, 200 Å; Nest Group), and then finally packed with GP-C18 (1.8 μm, 120 Å; Sepax Technologies) to a length of 30 cm. Peptides were eluted with a linear gradient of 0.125% formic acid containing ACN from 11% to 30% at a flow rate of 300 nL / min over 165 min while the column was heated to 60° C. Electrospray ionization was achieved by applying 1,800 V via a PEEK T-junction at the inlet of the microcapillary column.

[0125] The Orbitrap Fusion was operated in data-dependent mode, which allowed for 6 x 10 NMR spectra across the m / z range of 500 to 1,200 in the Orbitrap. 4 For the MS1 survey scan, the automatic gain control (AGC) was set to 5 x 10 5The ion intensity was set to 0.05, the maximum injection time was set to 100 ms, and the S-lens radio frequency (RF) setting was 60. The most abundant ions detected in the survey scan were subjected to MS2 and MS3 experiments using the "top speed" setting, which allows the maximum number of spectra to be acquired in a 5-second experimental cycle before the next cycle is initiated with another survey full MS scan. For MS2 analysis, a decision tree option was available, with precursors selected based on charge state and m / z range. Doubly charged ions were selected from the m / z range of 600 to 1,200, since triply and quadruply charged ions had to be detected in the m / z range of 500 to 1,200. The ion intensity threshold was 5 x 10 5 The AGC target was set to 1 x 10. When acquiring MS2 spectra, ions were isolated using the quadrupole by applying a 0.5 m / z window and fragmented using collision-induced dissociation (CID) at a normalized collision energy of 30%. The fragment ions were detected in the ion trap at a fast scan rate. The AGC target was set to 1 x 10. 4 and the maximum injection time of ions was set to 35 ms.

[0126] MS3 analysis was performed using synchronous precursor selection (MultiNotch MS3), which allows for maximizing sensitivity for quantification of TMT reporter ions. Up to 10 MS2 precursors were simultaneously isolated and fragmented for MS3 analysis. The isolation window was set to 2.5 m / z, and fragmentation was performed by HCD at 50% normalized collision energy. Fragment ions in the MS3 spectrum were detected in the Orbitrap at a resolution of 60,000 and m / z ≥ 110. The AGC target was set to 5 x 10 ions. 4The MS2 spectrum was set to 100 ms, and the maximum ion injection time was set to 250 ms. Fragment ions in the MS2 spectrum with m / z 40 m / z below and 15 m / z above the precursor m / z were excluded from selection for MS3 analysis.

[0127] Data processing and analysis. Data were processed using a software suite developed in-house (Huttlin et al. (2010) Cell. 143(7):1174-89). RAW files were converted to mzXML format using a modified version of ReAdW.exe (http: / / www.ionsource.com / functional_reviews / readw / t2x_update_readw.htm). Spectral assignment of MS2 data was performed using the Sequest algorithm to search the Uniprot database of mouse protein sequences, including known contaminants such as trypsin.

[0128] The database included a decoy database consisting of all protein sequences in reverse order. Searches were performed using a precursor mass tolerance of 50 ppm. Static modifications included ten-plex TMT tags (+229.162932 Da) at lysine residues and peptide N-termini, and carbamidomethylation of cysteines (+57.02146 Da). Methionine oxidation (+15.99492 Da) was included as a variable modification. Data were filtered for a false discovery rate (FDR) of <1% for peptides and proteins using a target-decoy search strategy (Elias et al. (2010) Methods Mol Biol 604: 55-71). This was achieved by first applying linear discriminant analysis to filter peptide annotations (peptide-spectrum matches) using a combined score from the following peptide and spectral characteristics: XCorr, ΔCn, tryptic uncleavage, peptide mass accuracy, and peptide length. The probability of a peptide-spectrum match being accurate was calculated using a posteriori error histograms, and the probabilities of all peptides assigned to a particular protein were combined multiply. The dataset was then refiltered for an FDR of <1% for protein assignment across the entire dataset of all proteins identified across all analyzed samples. Peptides that matched more than one protein were assigned to the protein containing the greatest number of matched redundant peptide sequences according to the principle of parsimony.

[0129] For quantitative analysis, the intensities of TMT reporter ions were extracted from MS3 spectra by selecting the most intense ion within a 0.003 m / z window centered on the predicted m / z value for each reporter ion, and signal-to-noise (S / N) values were extracted from the raw files. Spectra were used for quantification if the sum of the S / N values of all reporter ions was ≥386 and the isolation specificity for the precursor ion was ≥0.75. Protein intensities were calculated by summing the TMT reporter ions for all peptides assigned to a protein.

[0130] Flow cytometry To assess cell viability after recovery from tissue digests, cell suspensions were washed and resuspended in PBS and stained with a Zombie UV fixable viability kit (Biolegend, Inc.) for 30 minutes at 4°C in the dark with gentle shaking. Stained cells were then washed and resuspended in PBS containing 1% FBS, 0.01% sodium azide (RICCA Chemical, Arlington, TX), and 5% FcR blocking reagent (Miltenyi Biotec, Inc.) for 10 minutes at 4°C in the dark. Blocked cells were incubated with the following fluorophore-conjugated primary surface antibodies for 30 minutes at 4°C in the dark: Brilliant Violet 785-conjugated rat anti-mouse CD19 (clone 6D5), Alexa Fluor® 700-conjugated rat anti-mouse / human CD45R / B220 (clone RA3-6B2), APC / Cy7-conjugated rat anti-mouse CD138 (clone 281-2) (all from Biolegend Inc.), Brilliant Ultraviolet 395-conjugated hamster anti-mouse CD69 (clone H1.2F3), and PE-CF594-conjugated rat anti-mouse CD140a (clone APA5) (both from BD Biosciences, San Jose, CA). Surface stained cells were washed and resuspended in fixation buffer (Biolegend, Inc.) for 30 minutes at 4°C, followed by resuspension in clearing wash buffer (1X) (Biolegend, Inc.).The permeabilized cells were then incubated with the following fluorophore-conjugated primary intracellular antibodies for 30 minutes at 4°C in the dark: brilliant violet 421-conjugated mouse anti-mouse TGF-β1 (clone TW7-16B4), brilliant violet 510-conjugated rat anti-mouse IFN-γ (clone XMG1.2), brilliant violet 605-conjugated rat anti-mouse IL-4 (clone 11B11), brilliant violet 711-conjugated rat anti-mouse TNF-α (clone MP6-XT22), PerCP / Cy5.5-conjugated rat anti-mouse IL-2 (clone JES6-5H4), PE / Cy7-conjugated rat anti-mouse IL-10 (clone JES5-16E3), and APC-conjugated rat anti-mouse IL-6 (clone MP5-20F3) (all Biolegend). The antibodies used were: fluorescein-conjugated rat anti-mouse IFN-β (clone RMMB-1), and PE-conjugated rat anti-mouse IL-27 / IL-35 EBI3 subunit (clone 355022) (both from R&D Systems, Minneapolis, MN). Cells were analyzed on an LSRFortessa X-20 flow cytometer (BD Biosciences, San Jose, CA) equipped with BD FACSDIVA™ software and lasers at 355 nm, 405 nm, 488 nm, 561 nm, and 640 nm. At least 100,000 events were collected from each sample for analysis. Data were analyzed using FlowJo software, version 10.3 (TreeStar, Inc., Ashland, OR).

[0131] immunohistochemistry Wound biopsies taken on days 0 (intact), 1, 4, 10, and 16 after injury were fixed in 4% buffered paraformaldehyde for 24–48 hours at 4°C, then cryoprotected in 1 M sucrose solution for an additional 24–48 hours at 4°C, and embedded in tissue freezing medium (Electron Microscopy Sciences). Transverse sections through the wound were cut at 10 μm thickness using a cryostat (Leica Biosystems) and thaw-mounted onto SuperFrost Plus Gold slides (Fisher Scientific). For immunohistochemical detection of antigens, sections were washed with three changes of Tris-buffered saline (TBS), pH 7.4, then cleared and blocked by incubation with TBS containing 5% bovine serum albumin, 5% FBS, and 0.3% Triton X-100 for 1 h at room temperature. Sections were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: APC-conjugated rat anti-mouse CD45R / B220 (clone RA3-6B2; BioLegend, Inc.), PE-conjugated rat anti-mouse CD31 (clone MEC 13.3; BD Biosciences), Alexa Fluor® 488-conjugated mouse anti-tubulin β3 (clone TUJ1; BioLegend, Inc.), Alexa Fluor® 488-conjugated rat anti-mouse F4 / 80 (clone BM8; BioLegend, Inc.), PE-conjugated rat anti-mouse CD11b (clone M1 / 70; BioLegend, Inc.), rabbit polyclonal anti-Ki67 (Abcam), and rabbit monoclonal anti-active caspase 3 (clone C92-605; BD Pharmigen). Unbound primary antibody was removed by rinsing three times in TBS for 5 min each.When unconjugated primary antibodies were used, antigenic sites were visualized by incubating sections with Alexa Fluor 488®-conjugated F(ab')2 goat anti-rabbit IgG (Thermo Fisher Scientific) diluted 1:200 in blocking solution for 2 hours at room temperature. Sections were counterstained by incubation with 2 μg / ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) in PBS for 3 minutes at room temperature. Sections were washed three times for 7 minutes in TBS and embedded with Fluoromount (Novus Biologicals). Antibody controls included incubation of tissue sections with isotype antibodies and omission of the primary antibody when a secondary antibody was used for visualization. No nonspecific signals were detected in the control samples. Stained tissue sections were imaged using a Zeiss LSM 710 laser scanning microscope (Carl Zeiss) equipped with 20x, 40x, and 63x objectives. Confocal images were acquired 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.

[0132] histology Wound biopsies at the time course of wound healing were taken using a 10 mm biopsy punch and fixed in 4% paraformaldehyde in PBS at 4°C for 24–48 hours. The specimens were then dehydrated through graded ethanol and xylene washes and embedded in paraffin. Transverse sections through the wound were cut at 5 μm thickness and mounted on microscope slides. Serial sections were stained with hematoxylin and eosin and with Masson's trichrome stain to visualize collagen fibers. Stained slides were digitized at a resolution of 0.25 μm per pixel using an Aperio CS2 scanner (Leica Biosystems). The digitized slides were used for scoring of tissue regeneration by an experimenter blinded to the treatment conditions.

[0133] statistics The time-dependent effect of B cell application on the expression of cellular markers of interest was assessed separately for each tested cell population using linear mixed-effects modeling in SPSS 23 (IBM Corporation). To stabilize variance, the proportions of gated cells for each sample and each tested marker were logit (log of odds) transformed before analysis. A three-way (or four-way, if applicable) full factorial design was used, with survival time (18, 45, or 93 hours), environment (wound, subcutaneous, or B cells only on ice), marker, and, if applicable, condition (B cell treatment or saline) included as fixed factors. Technical replicate (day of administration) and biological replicate (mouse / sample ID) were included as random effects. Post-hoc contrasts between levels of fixed factors were adjusted for multiple comparisons using the Dunn-Sidak method. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0134] result B cell application induces complex changes in the molecular microenvironment of the wound Proteomic analysis of whole wound lysates identified up to 9125 proteins across all samples and treatments. For analysis across treatment conditions and time points, only proteins present in all samples (n = 30 animals) were considered. This resulted in a total of 3809 proteins (Figure 12).

[0135] A schematic of the average duration of the major stages of wound healing in a wild-type mouse wound model is shown in Figure 1A. A heat map summarizing the expression dynamics over time for proteins whose expression significantly changed in response to B cell application is shown in Figure 1B. A total of 213 proteins were categorized according to their role in wound healing, including proteins significantly changed in association with B cell treatment (n = 111; p < 0.05, unpaired t-test) and those with high fold changes for each time point, regardless of significance level (top 20 up- or down-regulated proteins) (n = 112). The heat map shows the fold change in expression after B cell treatment at days 0, 1, 4, and 10 after injury. Red = up-regulated; green = down-regulated. Of particular note was the downregulation of multiple proteins associated with inflammation and inflammatory cells 4 days after injury, and the substantial upregulation of proteins associated with cell proliferation, protection from apoptosis (cell death) and from oxidative stress, and tissue remodeling (formation of hair follicles and muscle) 4 to 10 days after injury.

[0136] Protein expression by functional family The average expression of proteins by functional family over time in saline-treated wounds (control, normal wound healing) or in wounds after treatment with B cells is shown in Figures 2A-2H. This analysis indicates the overall effect of B cells as homeostatic agents rather than inducers or inhibitors of protein expression. Application of B cells was associated with maintaining constant levels of protein expression that normally either decreases or increases during injury and healing, significantly reducing the inflammation peak observed in normal healing, preventing the decrease in anti-apoptotic factors (arrows) and oxidative stress protectants, and increasing proliferation (Figure 2A-B), reducing the decline in antioxidant stress protectants and cell proliferation, and maintaining low levels of cell migration (Figure 2C-D), maintaining constant levels of proteins associated with remodeling and secondary skin structure (Figure 2E-F), reducing the levels of proteolysis and autophagy observed early in injury in controls, and increasing the levels of proteins associated with angiogenesis and nerve regeneration in the later stages of healing (Figure 2G-H).

[0137] An unsupervised hierarchical cluster analysis of the identified proteins expressed in skin wound samples is shown in Figure 12. Only identified proteins found to be consistently present across all samples were included in the analysis. Hierarchical clustering using complete linkage of 3809 proteins (rows) consistently expressed in all animals at four different time points after injury (columns) in both B cell-treated and saline-treated wounds is shown in Figure 12A. The pseudocolor scale indicates the normalized, log-transformed fold-change expression value for each protein. A dendrogram shows 15 protein clusters derived from this analysis, with the color of each cell in (Figure 12A) corresponding to the mean expression value of the cluster at each time point. Proteins are clustered by their expression pattern over time. A heatmap of the hierarchical clusters from (Figure 12A) is shown in Figure 12B, representing all 3809 proteins. Gene ontology analysis of the 15 hierarchical clusters is shown in Figure 12C. The mouse GOslim gene list from QuickGO (accessible at https: / / www.ebi.ac.uk / QuickGO) was used to examine 15 hierarchical clusters. Bar graphs show the top biological function category for each cluster.

[0138] At each time point during wound healing that was assessed, the distribution of proteins that were significantly changed in response to treatment with B cells was determined and is shown in FIG.

[0139] In vivo evaluation of B cell recruitment in acute wound healing The experimental paradigm for the in vivo evaluation of B cell application in acute wound healing is shown in Figure 3. A total of four full-thickness wounds were created in the dorsal skin of wild-type C57B16 mice, and mature naive B cells purified from isogenic animals were applied directly to the wound surface. Control animals received saline application. To create a similar microenvironment without injury, B cells or saline control were also injected subcutaneously under the intact skin as an internal control. After the specified survival period, wounded or uninjured skin tissue was harvested, dissociated, and processed for flow cytometry analysis. The scatter plot on the right shows the typical distribution of cell suspensions from each treatment category. Wound samples showed a characteristic influx of leukocytes (open white arrows), which are almost absent in uninjured tissue. Although B cells are typically few in number at either location, they could be readily detected in large numbers after experimental application (solid red arrows).

[0140] Flow cytometric analysis of cell suspensions from B cell-treated and control wounds. To assess changes in both B cells and cells of the wound environment across different conditions and time points, samples were analyzed using flow cytometry. Our gating strategy and analysis of cell suspensions from B cell-treated and control wounds by flow cytometry are shown in Figure 4. Live cells were gated into three major categories: B cells (CD19+ / B220+ lymphocytes), non-B cell leukocytes (CD140a- / B220- leukocytes), including neutrophils, monocytes and macrophages, dendritic cells, and a mixture of T cells, and fibroblasts (CD140a+ / B220-). These cell categories were assessed for markers of activation and cytokine production.

[0141] Dynamics of activation markers and key cytokines in B cells recovered from the wound The dynamics of activation markers and key cytokines in B cells recovered from wounds after defined exposure periods to the wound microenvironment are shown in Figure 5. B cells were exposed to the wound niche in vivo or injected under uninjured skin (control at equivalent locations). Control B cells maintained on ice immediately after isolation for the same duration are shown for comparison. After periods including 18 hours, 2 days, 4 days, and 10 days, the wounds were treated with brefeldin A for 4 hours to induce intracellular retention of cytokines. B cells were then recovered by tissue excision and dissociation and further characterized by flow cytometry for both surface markers and intracellular cytokines. B cells exposed to the wound microenvironment transiently upregulated multiple immunoregulatory cytokines, peaking 2 days after application. Several immunoregulatory cytokines, including TGFβ and IL-6, remained elevated at day 4, and IL-10 remained elevated until day 10. N = 3-6 animals / group.

[0142] Heatmap analysis Heatmaps of the aggregated mean values for each marker for B cells exposed to the wound microenvironment, B cells exposed to subcutaneous control, or B cells maintained on ice (unexposed) are found in Figure 6. The dynamics of activation markers and key cytokines in aggregates of infiltrating non-B cell leukocytes present in the wound are shown in Figure 7. Overall, when B cells were present in the wound, infiltrating leukocytes produced more of the anti-inflammatory cytokines IL-10, TGFβ, and IL-35, and less of the pro-inflammatory cytokines TNFα and IL-2. This effect was most pronounced 4 days after injury and B cell application and persisted for up to 10 days. N = 3–6 animals per group.

[0143] A heat map showing the pattern of increased production of anti-inflammatory cytokines (IL-10 and TGFb) in the presence of B cells in infiltrating non-B cell leukocytes in the wound microenvironment as an aggregation of the mean values for each marker is shown in Figure 8.

[0144] The dynamics of activation markers and key cytokines in the CD140a+ fibroblast population in the wound and subcutaneous tissue are shown in Figure 9. When the wound was exposed to B cells, wound-resident fibroblasts produced significantly more IL-10 and TGFβ at 10 days after injury. Furthermore, when B cells were applied, wound fibroblasts produced less of the pro-inflammatory cytokine TNFα at both 4 and 10 days after injury.

[0145] Additional heat maps of the mean values for each marker in wound and subcutaneous tissue fibroblasts treated with either B cells or saline are shown in Figure 10. Fibroblasts are the most important source of anti-inflammatory and pro-regenerative factors in wound healing and produce high levels of IL-10 and TGFβ, regardless of treatment. Nevertheless, fibroblasts from wounds treated with B cells continued to produce higher levels of both IL-10 and TGFβ at 4 and 10 days after injury, while saline-treated wounds showed reduced levels of these anti-inflammatory cytokines. Interestingly, a significant effect of B cell application was observed in reducing pro-inflammatory cytokines, including IL-6 and TNFα, in wound fibroblasts.

[0146] TLR signaling and IL-10 production are essential components of regenerative foreign B cell function in wound healing As shown in Figure 11, functional TLR signaling and IL-10 production are essential components for the regenerative function of foreign B cells in wound healing. Full-thickness excisional wounds (shown here at day 6 of healing) were treated on day 0 with B cells lacking the common TLR signaling adaptor myeloid differentiation factor 88 (MyD88), B cells lacking IL-10, or WT B cells as a control. Saline was also included in each test animal as an internal control. WT B cells consistently promoted wound closure in WT animals by days 2–3, whereas MyD88− / − B cells and IL-10− / − B cells, similar to saline application, showed no benefit on wound closure.

[0147] Example 2: Treatment with B cells improves outcomes after TBI This example demonstrates that exogenously applied B cells significantly improved performance after injury in a mouse TBI model.

[0148] Cerebral contusion leads to neurological dysfunction mediated in part by the inflammatory response to injury. B lymphocytes are dynamic regulators of the immune system, but they have not been systematically studied in TBI. Using a controlled cortical impact (CCI) model in mice, we evaluated the potential beneficial role of exogenously applied B cells on histopathological and functional outcomes after TBI. Mice were inoculated with 2 x 10 6Mature naive syngenic splenic B cells were intraparenchymally injected and then subjected to CCI. Control CCI mice received either the same number of T cells or saline, and sham-injured mice (craniotomy only) received either B cells or saline. The sham-injured groups performed similarly in motor and learning tests. Compared with the saline- or T-cell-treated CCI groups, injured mice receiving B cells showed significantly improved post-injury performance in the rotarod, Y-maze, and Morris water maze (MWM). Furthermore, the injury volume in B-cell-treated mice was significantly reduced by 40% 35 days after TBI compared with saline and T-cell controls, and astrogliosis and microglial activation were reduced. In vivo tracking of exogenous B cells showed that they have a limited in situ lifespan of approximately 14 days and do not appear to proliferate. The data suggest proof-of-principle that local administration of B lymphocytes is a therapeutic option for the treatment of cerebral contusion, especially when clinical management involves procedures that allow access to the injury site.

[0149] Therefore, the potential of mature naive B cells for protection from cognitive and histopathological impairment in a mouse CCI TBI model was investigated in the following study. Compared with administration of splenic T cells or saline, a single dose of B cells delivered by intraparenchymal infusion at the time of injury was associated with significant improvements in hippocampal- and striatal-dependent behavioral tasks. The observed behavioral improvements were associated with a significant reduction in lesion volume in B cell-treated animals, which was accompanied by compensation of the hippocampal structure. In vivo tracking of exogenously applied B cells after intraparenchymal infusion showed that the cells had limited in situ viability of approximately two weeks, indicating that they may be a safe and viable option for the treatment of acute and subacute contusion TBI.

[0150] material and method The following materials and methods were utilized in this study.

[0151] Animals: All animal handling was performed in accordance with the NIH Guide for Care and Use of Laboratory Animals and the Public Health Service's Code for Humane Care of Laboratory Animals. All protocols were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. Studies were performed in male C57Bl6 / J adult mice (Jackson Laboratories, Bar Harbor, ME) aged 12–14 weeks and weighing 25–32 g. Male C57Bl6 / J and FVB-Tg(CAG-luc-GFP) L2G85Chco / J mice (all from Jackson Laboratories, Bar Harbor, ME) were used as isogenic donors for B and T cell isolation. Animals were socially housed (4–5 per cage) and maintained under standard laboratory conditions at temperatures ranging from 20–23°C, with a 12-hour light:dark cycle and free access to food and acidified water. Animals were age-matched and randomly assigned to experimental conditions. Animals from different treatment arms were housed together to avoid bias.

[0152] Lymphocyte isolation: Cell isolation was performed using negative immunomagnetic selection as previously described. 16Briefly, mouse spleens were harvested in ice-cold buffer containing 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS). Spleens were mechanically dissociated by passing through a 40 μm cell strainer, and the splenocyte suspension was processed for negative selection of B or T cells by immunomagnetic separation and retention of non-target cells using a commercially available cell isolation kit (STEMCELL Technologies, Inc., Vancouver, Canada) according to the manufacturer's instructions. The B cell isolation procedure was verified by flow cytometry analysis and typically yielded >98% pure, mature, naive CD45R B cells, although some residual red blood cells may be present. + / CD19 + A population of B lymphocytes was obtained with less than 1% contamination by other leukocytes. 16 Purified lymphocytes were cultured at 4 x 10 5 were resuspended in sterile PBS at a concentration of 100 cells / μl.

[0153] Controlled cortical impact (CCI): All surgical procedures, including injury and application of cells or saline, were performed by an experimenter blinded to the treatment conditions and who did not participate in the preparation of the treatment dose for injection. Mice were anesthetized with a mixture of 70% N2O and 30% O2 containing 4.5% isoflurane (Baxter, Deerfield, IL) for 90 seconds using a Fluotec3 vaporizer (Colonial Medical, Windham, NH) and mounted in a stereotaxic frame. Anesthesia was maintained with 4.5% isoflurane. After a midline scalp incision, a craniotomy was performed over the left parietal-temporal cortex using a portable drill and a 5-mm trephine, and the bone flap was discarded. An ipsilateral intraparenchymal injection was delivered approximately -1 mm from bregma on the anterior-posterior axis and approximately +2 mm from bregma on the medial-lateral axis, to a depth of 3 mm through the left parietal cortex. A total volume of 5 μl of saline containing either 2 million B cells, 2 million T cells, or no cells was injected using a 10 μl Hamilton syringe fitted with a 26s gauge blunt needle (Hamilton Company, Franklin, MA). The cell doses chosen were those previously optimized in skin injury models exhibiting similar injury volumes. 16 To ensure accurate and consistent cell injection while the brain structure was intact, cell application was performed immediately before CCI. Immediately thereafter, mice underwent CCI using a pneumatic cylinder equipped with a 3 mm blunt-tipped impounder at a velocity of 6 m / s, a depth of 0.6 mm, and an impact duration of 100 ms. Sham-injured mice underwent anesthesia, craniotomy, and intraparenchymal injection of the same number of B cells or saline, but did not receive a CCI injury. The craniotomy was left open, and the skin was sutured over the skull using 6-0 nylon sutures (Fisher Scientific, Waltham, MA).

[0154] Behavioral testing schedule: Behavioral testing was performed during the light phase of the circadian cycle by an experimenter blinded to the treatment conditions. Before each test, mice were allowed to acclimate to the testing space for at least 30 minutes. Mice were tested in the battery of assays according to the schedule described in Figure 14. Vestibular-related motor abilities were assessed using a wire grip assay 1, 3, and 7 days after injury. The rotarod test was performed 7, 9, 10, 13, and 14 days after injury. At 17 days after injury, animals underwent anxiety assessment using an elevated plus maze assay. Morris water maze (MWM) testing was performed 20, 21, 22, 23, and 24 days after injury, with a probe test conducted 27 days later. At 29 days after injury, mice underwent a forced swim test to assess depressive-like behavior, and at 30 days, they underwent a Y-maze test, an assay for hippocampal-dependent working memory.

[0155] Wire grip test: Vestibular-related motor function was assessed using the wire grip test (Bermpohl et al. (2007) J Cereb Blood Flow Metab 27, 1806-1818). Mice were placed on a 45 cm long metal wire suspended 45 cm above the ground and allowed to cross the wire for 60 seconds. The latency to fall during the 60 seconds was measured, and the wire grip score was quantified using a 5-point scale. Tests were performed in triplicate, and the mean value was calculated for each mouse on each test day.

[0156] Rotarod: Mice were placed on an automated rotarod apparatus (Harvard Apparatus, Holliston, MA) that accelerated from 4 revolutions / min to 40 revolutions / min over 60 seconds. The maximum duration of a trial was 300 seconds or until the mouse fell off the rotarod. Each mouse was assessed five times per day, with a 5-minute rest interval. The mean latency to fall and mean speed of revolutions / min across the five trials were recorded for each day of testing.

[0157] MWM: The MWM was performed as previously described with slight modifications (Mannix et al. (2013) Ann Neurol 74, 65-75). Spatial learning was assessed at approximately the same time of day. Each mouse received seven invisible platform trials (one to two trials per day) with a randomized set of start locations in one of the four quadrants. A trial consisted of the average latency from each of the four start locations. If the mouse failed to find the platform within 90 seconds, it was placed on the platform for approximately 10 seconds. A probe trial was performed 24 hours after the last invisible platform trial by having the mouse swim in a tank without a platform for 30 seconds and recording the time spent in the target quadrant.

[0158] Porsolt forced swim test: Mice were placed in a 30 cm (height) x 20 cm (diameter) cylindrical clear glass tank filled with water (25°C) to a height of 20 cm. A white Styrofoam box was installed to blindfold them on three sides. Mice were placed in the water for 6 min, and swimming behavior was recorded. The total time active (swimming, paddling / climbing the beaker wall) vs. the time inactive (floating without resistance) was quantified for the last 4 min of the test.

[0159] Y-maze spontaneous alternation test: The Y-maze test was conducted in a white, opaque acrylic apparatus consisting of three 40-cm-long arms connected at a 120-degree angle and with 15-cm-high walls. Each arm was marked with a contrasting visual indicator (black square, circle, or star on a white background). Mice were placed in the center of the apparatus and allowed to explore the maze for 10 minutes. Mouse behavior was recorded using a webcam mounted directly above and Photo Booth software (ANY-maze). Normal exploratory behavior in rodents involves a tendency to preferentially enter arms other than the one previously entered (spontaneous alternation). The alternation score was calculated by dividing the number of three consecutive choices, including one for each arm, by the total number of arm entries (i.e., alternation opportunities). The apparatus was disinfected with 70% ethanol between trials.

[0160] Elevated plus maze: The apparatus consisted of two 130 cm x 8 cm platforms, elevated 60 cm above the ground, with an 8 cm x 8 cm square area at their intersection. The closed arms of the platform had 10 cm walls, while the open arms had no walls. Each mouse was placed in the center area of the maze and videotaped for 5 minutes. The apparatus was disinfected with 70% ethanol between trials. Video recordings were analyzed for mean speed and percent time in the closed and open arms using ANY-Maze (Stoelting Co., Wood Dale, IL) software.

[0161] IVIS imaging: Splenic B cells were isolated from mice homozygous for the CAG-luc-eGFP L2G85 transgene, which expresses firefly luciferase and enhanced green fluorescent protein under the CAG promoter (Jackson Laboratories, Bar Harbor, ME). Approximately 5 million luciferase-expressing B cells in 5 μl PBS were infused into the left hemisphere of recipient WT C57B16 / J mice as described above. Mice were imaged using an IVIS Lumina II system (Caliper Life Sciences, Waltham, MA) on the day of surgery and at regular intervals thereafter for a total of 4 weeks. For each imaging session, anesthesia was induced with 3% isoflurane in oxygen and maintained with 2–3% isoflurane at 1 L / min throughout the imaging session. To visualize luciferase activity, 100 μl of a 30 mg / ml D-luciferin solution (Regis Technologies, Inc., Morton Grove, IL) was injected subcutaneously near the injury site at least 6 minutes before imaging. Mice were imaged for 10 minutes, and the same parameters were maintained for each repeated imaging.

[0162] Tissue Sampling: Thirty-five days after CCI and treatment, mice were deeply anesthetized with ketamine (100 mg / kg) and xylazine (10 mg / kg), transcardially perfused with 10–15 ml of heparinized PBS to remove blood, and decapitated. Brains were rapidly removed on ice, frozen in vapor phase liquid nitrogen, and stored at −80°C. For cryosectioning, brains were embedded in M-1 embedding matrix (Thermo Fisher Scientific, Waltham, MA) and sectioned coronally at 16 μm thickness using a cryostat. Sections were taken at 500 μm intervals along the anterior-posterior axis and thaw-mounted onto SuperFrost Plus Gold slides (Fisher Scientific, Waltham, MA).

[0163] Immunohistochemistry: Tissue processing for immunohistochemical analysis was performed as previously described (Sirbulescu et al. (2017) Wound Repair Regen 25, 774-791). Briefly, sections were washed with PBS, then cleared and blocked by incubation for 1 hour at room temperature with PBS containing 5% bovine serum albumin, 5% fetal bovine serum, and 0.3% Triton X-100. Sections were then incubated overnight at 4°C with the following primary antibodies diluted in blocking solution: Alexa Fluor® 594-conjugated rat anti-mouse CD45R / B220 (clone RA3-6B2; BioLegend, Inc., San Diego, CA), Alexa Fluor® 488-conjugated mouse anti-mouse CD45.1 (clone A20; BioLegend, Inc., San Diego, CA), Alexa Fluor® 488-conjugated mouse anti-glial fibrillary acidic protein (GFAP) (clone 2E1.E9; BioLegend, Inc., San Diego, CA), Alexa Fluor® 647-conjugated rat anti-mouse CD68 (clone FA-11; BioLegend, Inc., San Diego, CA), and rabbit polyclonal anti-Ki67 (Abcam, Cambridge, MA). Unbound primary antibodies were removed by rinsing three times in PBS. When unconjugated primary antibodies were used, antigenic sites were visualized by incubating sections with Alexa Fluor 488®-conjugated F(ab')2 goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA) diluted 1:200 in blocking solution for 2 hours at room temperature. Sections were counterstained by incubation with 2 μg / ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich).Antibody controls included incubation of tissue sections with isotype antibodies and omission of the primary antibody when a secondary antibody was used for visualization. No nonspecific signals were detected in the control samples. 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).

[0164] Measurement of lesion volume: Sections were stained with hematoxylin, and high-resolution full-body photographs of the slides were acquired. Morphometric image analysis in ImageJ (NIH, Bethesda, MD) 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 loss, expressed in mm 3 It is expressed as:

[0165] Image Analysis: For unbiased quantification of GFAP and CD68 immunolabeling, confocal images (n = 4 fields of 1400 x 1400 μm per animal) collected from the periphery of the injury site, including both the medial and lateral aspects of the injury area, were analyzed using standard functions in MATLAB (The MathWorks, Inc., Natick, MA). For each image, the region of interest was semi-automatically defined by morphological closing, morphological opening, and interactive filling of a binary mask generated by applying a minimum intensity threshold of 10 across all imaged channels to a maximum intensity projection process. Background levels were determined by image smoothing using a 3 x 3 pixel moving average, followed by morphological opening using a 10-pixel radius structuring element for each channel separately. Foreground objects were identified as sets of pixels with intensities above the corresponding background level (>25 for GFAP immunolabeling and >50 for CD68 immunolabeling). For each marker analyzed, the relative labeled area was calculated as the percentage of pixels within the region of interest that were marked as foreground, while the mean labeling intensity was calculated as the average intensity of all foreground pixels found within the region of interest. All image analysis was performed by an experimenter blinded to treatment conditions.

[0166] Statistical Analysis: Prior to statistical testing, all data sets were assessed for normal distribution using the D'Agostino-Pearson normality test and were found to pass (p > 0.2). The statistical significance of differences between experimental groups for repeated behavioral tests, including the wire grip assay, rotarod, MWM invisible and visible platform, and elevated plus maze, was assessed using a two-way (treatment x number) repeated measures analysis of variance (with trial / timepoint as the repeated measures factor) with matched subjects, followed by post hoc multiple comparisons using Tukey's or Sidak's test. Single-timepoint measurements, including the MWM probe, Y-maze, forced swim, and histological comparisons, were assessed using a one-way analysis of variance followed by Tukey's multiple comparison test. All reported descriptive statistics are estimated marginal means ± standard error of the sample mean (SEM). All statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). P < 0.05 was considered statistically significant.

[0167] result Application of B lymphocytes at the time of injury improves cognitive recovery after CCI A total of 63 mice completed the study, and one died. Intraparenchymal treatment with B lymphocytes had a significant protective effect in the rotarod assay (Figure 15A). While all injury groups performed worse than sham-injured animals, B cell-treated injured mice performed better than T cell- or saline-treated groups (p < 0.01 for each group), and their performance was no different from that of sham-injured B cell-treated mice. Interestingly, successive trials showed sustained improvement in performance in the CCI + B cell group, as well as in both sham-injured and control groups, suggesting that procedural learning occurred in these groups. There were no significant differences between the B cell-treated CCI group and the sham-injured mice, except for trial 3, in which B cell-treated sham-injured mice performed better (p < 0.01). In contrast, CCI mice treated intraparenchymally with either saline or 2 million T cells at the time of injury showed minimal day-to-day improvement in performance and significantly shorter latencies to fall compared with either CCI-injured animals treated with B cells or sham-injured animals (p < 0.0001). There were no statistically significant differences between the CCI-injured groups receiving T cells and those receiving saline.

[0168] In the wire grip test, all injured groups performed worse than those with sham injury, but no group differences were observed among the B cell-, saline-, and T cell-treated CCI groups (Figure 15B).

[0169] In the MWM (Figures 16A-16D), sham-injured mice performed significantly better than all CCI groups on the invisible platform trial, indicating a robust effect of CCI (p < 0.001 for group), with no differences observed among sham-injured groups. Among the injury groups, there was a highly significant effect of treatment (p < 0.0001) for B cell-treated vs. saline-treated mice on trial 7 (p = 0.016), with a significant treatment x trial interaction (p = 0.02) (Figure 16A). The rate of performance improvement over the seven invisible platform trials, assessed as the difference between trial 1 and trial 7 in mean time to reach the platform, was significantly increased in injured animals receiving B cells compared with saline controls (p < 0.01). No significant differences were observed among any of the groups on the visible platform trial (Figure 16B). In the probe trial, injured mice treated with B cells performed similarly to the sham-injured group (Fig. 16C). In contrast, CCI-injured mice receiving either T cells or saline performed worse than sham-injured mice (p < 0.05). The swimming patterns of injured mice treated with B cells, similar to those of the sham-injured group, showed evidence of a spatial exploratory strategy, whereas the exploratory strategies of injured mice treated with T cells and saline were non-spatial (Fig. 16D).

[0170] In the Y-maze test (Figure 16E), CCI-injured mice receiving intraparenchymal administration of B cells had an alternation score of 76.65 ± 2.46, which was significantly higher than either T-cell-treated (46.86 ± 2.41) or saline-treated (38.92 ± 2.56; p < 0.0001) injured animals and similar to sham-injured B-cell-treated mice. There were no significant differences between either injury group and treatment group with respect to the total number of maze arm entries (p > 0.13) or total distance traveled (p > 0.07).

[0171] In the elevated plus maze (Figure 17A), there were no significant differences between any of the treatment groups with respect to time spent in the open arms (p > 0.84) or time spent in the central area of the starting point (p > 0.94). There was a significant treatment x location interaction (p < 0.05). Within the lesion group, mice receiving B cells spent significantly more time in the closed arms of the maze compared with animals receiving the same number of T cells (p < 0.05).

[0172] In the forced swim test, the sham-lesioned and lesioned groups did not differ from each other, suggesting that the lesion paradigm employed in this study does not affect the depressive-like behavior measured in this assay (Fig. 17B).

[0173] Application of B lymphocytes at the time of injury is associated with reduced injury volume and reduced glial scar formation 35 days after CCI To assess whether the behavioral improvements observed with B cell treatment had neuropathological correlates, the brains of all animals undergoing behavioral assessment were collected for histological examination 35 days after injury (Figure 18). Analysis of brain tissue damage in the CCI group showed cavitation of the injury area in all CCI-injured mice, while the sham-injured group showed no brain tissue loss (Figure 18A). Quantitative analysis of lesion volume across all groups (Figure 18B) demonstrated a significant effect of CCI, as expected (p < 0.0001). Brain tissue loss was significantly reduced in CCI-injured mice treated with B cells compared with the saline-injured group (p < 0.001) or the T cell-injured group (p < 0.0001). There was no significant difference in lesion volume between the T cell-injured and saline-injured CCI-injured groups. Analysis of the distribution of lesion area in serial sections of the whole brain (Fig. 18C) showed that the most striking differences between treatment conditions were observed in the posterior two-thirds of the lesion, a level that primarily includes the hippocampus. Indeed, targeted volumetric measurements showed that B cell-treated CCI mice had a significantly greater proportion of compensated hippocampal tissue in the injured hemisphere compared with either saline- or T cell-treated CCI controls (p < 0.0001; Fig. 18D).

[0174] To assess the effect of B cell treatment on long-term reactive responses in the injured brain, including astrogliosis and microglial activation, tissue sections taken 35 days after injury were immunolabeled for GFAP and the activation-associated marker CD68. Unbiased analysis of confocal images collected from a location adjacent to the cavitation lesion formed at the site of the initial CCI injury demonstrated a significant effect of B cell treatment delivered at the time of injury compared with saline- and T cell-treated controls (Figure 19). Glial scar formation, indicated by strong GFAP immunolabeling and significant astrocyte hypertrophy, was significantly reduced in B cell-treated animals, as indicated by decreased immunolabeling for CD68, a microglial activation marker (Figures 19E and 19F).

[0175] B lymphocytes do not proliferate in situ and have a limited life span of approximately two weeks after administration To determine whether exogenously delivered B cells persist in vivo after intraparenchymal injection, we used purified B cells constitutively expressing firefly luciferase under the CAG promoter. Luciferase has a short biological half-life, so it cannot be detected unless it is continuously produced. Because the signal rapidly disappears upon death of luciferase-expressing cells, this assay can be used to determine the long-term viability of experimentally introduced B cells (Zinn et al. (2008) ILAR J 49, 103-115). Because only viable exogenous B cells generate a light signal, this method allows for noninvasive tracking of cell viability and persistence over time until the number of cells is reduced to a few hundred. Results showed that in all animals tested, infused B cells were clearly detectable immediately after application (Figure 20A). Quantitative analysis of total photon flux in the head showed that the enzyme activity active in the injected cells initially increased slowly, peaking between days 3 and 7, and then rapidly decreased after day 7, reaching undetectable levels by day 17 after injection (Fig. 20B). These results support the notion that exogenous B cells have a limited lifespan of approximately 2 weeks in situ.

[0176] To investigate whether the introduced B cells could proliferate in situ, thereby potentially establishing a longer-lived population in the brain parenchyma, coronal brain sections through the injection site were taken and immunolabeled for B cell and proliferation markers. Ex vivo labeling of B cells with toluidine blue prior to injection demonstrated that after delivery, cells were distributed exclusively within a radius of approximately 1 mm around the injection site (Figure 21A). Confocal imaging confirmed that injected B cells remained largely localized to the application site up to 4 days after delivery, with some dispersion throughout the injury (Figure 21D). Immunolabeling for the proliferation marker Ki67 revealed that B cells did not express this marker at the 0 and 4 day timepoints, while adjacent non-B cells showed high levels of Ki67 immunopositivity 4 days after injury in all animals examined (n = 4 animals per timepoint; Figures 21D, 21E). This finding was consistent with the fact that no B cells were observed 35 days after application in tissue sections immunolabeled for CD45R (B220) and CD45.1 in either injured or sham-injured animals (data not shown). In CCI-injured animals, the original injection site was destroyed by cavitation at day 35, whereas in sham-injured controls, it was readily detectable due to gliosis surrounding the needle-induced injury (Figure 21F). By day 35, no B220-positive B cells were found at the injection site of B-cell-treated sham-injured animals (Figures 21F, 21G).

[0177] conclusion This study describes, to our knowledge, the first use of direct application of B lymphocytes in a preclinical TBI model to modulate structural and functional outcomes after injury. We found that a single intraparenchymal delivery of purified (>95%) mature naive B cells during CCI significantly reduced learning and memory deficits and decreased brain tissue loss after injury. These results suggest a previously unknown protective effect of endogenous B cells in this model of brain contusion.

[0178] The CCI injury model produces highly reproducible injuries and has a very low mortality rate (Xiong et al., (2013) Nat Rev Neurosci 14, 128-142). Although the impact is delivered to the cortical surface with the dura intact, the neuropathological consequences of the injury are typically widespread and include cortical, hippocampal, and thalamic degeneration (ibid.). These pathologies are associated with long-term cognitive impairment and changes in emotional behavior (ibid.). In this CCI injury paradigm, the impact force is applied directly to the cortex, so this region reliably and consistently degenerates in all animals regardless of treatment condition. However, differences between treatment conditions were observed in compensation in subcortical structures, particularly the hippocampus. The observed behavioral benefits of B cell treatment also correlated with the functions supported by these structures, suggesting a link between the localization of the introduced lymphocytes, tissue compensation around the injection site, and functional neuroprotection. In support of this hypothesis, in this model, B lymphocytes were injected approximately 1 mm posterior to the bregma and remained mostly localized between the caudate-putamen and the hippocampus (Lein et al. (2007) Nature 445, 168-17).

[0179] In this study, confocal imaging of tissue sections taken from the CCI site showed that B cells clustered around the initial injection site and were somewhat distributed throughout the injured tissue up to 4 days after injury and application. These results indicate that intraparenchymal injected B cells appear to remain at the injury site for their entire in situ lifespan. Although the molecular mechanisms underlying the observed neuroprotective effects of B cells are not fully understood, it is possible that diffusible factors emanating from the cells may be involved, which overcome the strict localization of the applied cells and reach distal regions. This hypothesis is supported by the observation that animals treated with B cells during CCI had significantly reduced injury volume and significantly reduced potentially deleterious reactive phenomena, including astrogliosis and microglial activation, at the injury site compared with controls.

[0180] Thus, B cells can be used as a therapeutic strategy for patients with cerebral contusion. Unlike any other existing cell-based therapy, B cells are readily available from peripheral blood or other blood bank products, which is a key advantage for developing fast-acting, off-the-shelf therapeutics. Indeed, fast-acting, minimally manipulated autologous B cell therapy may have high clinical applicability. This is particularly true in cases of severe brain injury, where surgery is often performed to remove hematomas or impinging bone fragments and catheters are placed either intraparenchymally or intraventricularly to monitor intracranial pressure (Stocchetti et al. (2017) Lancet Neurol 16, 452-464; Galgano et al. (2017) Cell Transplant 26, 1118-1130), providing a convenient route for administering B cells into the injured brain.

[0181] Unlike other cell types used in therapy, such as stem cells, B lymphocytes are mature, terminally differentiated cells with a limited natural lifespan of 5-6 weeks in vivo. When applied in a disrupted microenvironment following a cerebral contusion injury, transplanted cells are expected to disappear more quickly. This is beneficial because longer survival of transplanted cells could raise significant safety concerns, especially considering that the CNS microenvironment contains several B cell trophic factors. To monitor the presence and persistence of metabolically active transplanted cells over time, we used in vivo visualization of luciferase-expressing B cells. We observed that the cell signal rapidly decreased after day 7 and reached undetectable levels by day 17. With CCI, luciferase signal intensity initially increased slowly 3-7 days after intraparenchymal injection, which may indicate that the transplanted cells underwent a period of adaptation and / or stimulation in the local microenvironment of the CCI injury. The increased signal intensity is unlikely to be due to in situ cell proliferation, because immunohistochemical examination of the injection site either immediately after B cell administration and CCI or up to 4 days later showed that the transferred B cells did not express markers of cell proliferation. Indeed, although B cells enhanced the proliferation of nearby cells, no B cell proliferation was observed after application to the wound.

[0182] This study describes the first proof-of-principle observation that peripherally isolated mature B cells can be a safe, rapid, and effective cell-based therapeutic strategy for the acute and subacute treatment of contusion TBI, where there are no current therapeutic options to improve neurological outcomes.

[0183] Example 3: Evaluation of B cell immunotherapy in the SOD1-G93A mouse model of ALS This example demonstrates the use of SOD1, a standard mouse model of ALS. G93A 1 illustrates the safety and efficacy of intravenous (iv) administration of B cells in mice.

[0184] Male and female transgenic SOD1 G93A Mice were bred at 5 x 10 day-to-day intervals for a total of 10 weeks, starting at 10 weeks of age (day 72), along with an equal number of sex-matched non-carrier controls (n = 32 / condition). 6 The mice were treated with saline containing 100 B cells (or saline control) once a week. G93A We found that B cell treatment delayed the onset of symptoms (p < 0.0001), as indicated by the attainment of peak body weight and significantly prolonged survival (p < 0.05) in mice. Although the total number of motor neurons was unchanged by treatment, B cell treatment was associated with a significant reduction in the relative number of damaged / degenerated motor neurons in the lumbar spinal cord at endpoint (p < 0.05). In non-transgenic wild-type control littermates receiving the same treatment, B cell treatment was not associated with any observable adverse effects (behavioral or phenotypic), and the controls continued to gain weight over the duration of repeated treatment. Taken together, these results demonstrate that B cell therapy may provide a viable method for reducing neuroinflammation in ALS.

[0185] ALS is a fatal disease characterized by the progressive degeneration of the upper motor neuron in the motor cortex of the brain and the lower motor neuron in the brainstem and anterior horn of the spinal cord.To date, there is no cure for ALS, highlighting the urgent and unmet need in the art.

[0186] Test Plan To evaluate the efficacy of B cell immunotherapy in ALS, we investigated the well-established B6.SOD1 G93AWe used a transgenic mouse model following published guidelines for design and interpretation of results (Galgano (2017) Cell Transplant 26, 1118-1130; Nordstrom et al. (2014) Ann Neurol 75, 374-381). Mice transgenic with the G93A mutant form of human SOD1 exhibit a phenotype similar to ALS in humans. The mice progressively lose one or both limbs, eventually resulting in complete paralysis due to loss of motor neurons from the spinal cord. Transgenic mice are also short-lived. Neurodegenerative symptoms typically begin to appear around 12-14 weeks of age, and the mice die at approximately 20-24 weeks of age. Thus, this model demonstrates rapid and aggressive disease progression. The model is highly stable and reproducible, allowing for the evaluation of therapeutic options for this neurodegenerative disorder.

[0187] A total of 15-17 female and male animals (according to guidelines recommended in the art (ibid.)) were used for each experimental condition (treatment) throughout the study, along with an equal number of sex-matched non-littermate non-carrier controls (Table 1). Beginning at 10 weeks of age (day 72), all animals received a total of 10 weekly intravenous infusions of B cells (or saline control), delivered by retro-orbital injection (Figure 22). For feasibility, the study was conducted in two overlapping cohorts, as described in Table 1 (overview of animals used by genotype, sex, and treatment for each cohort).

[0188] [Table 1]

[0189] material and method The following materials and methods were used in this study.

[0190] Animals: B6SJL-Tg(SOD1*G93A)1Gur / J(SOD1-G93A) transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME; Stock No: 002726). This founder strain (often referred to as G1H) has been reported by Jackson Laboratory to carry a high copy number of the SOD1 transgene. All animals were individually genotyped by Jackson Laboratory before shipping, and only heterozygous animals carrying a high copy number of the SOD1 transgene (top third of the distribution) are sold. Control animals were littermates (non-carriers) lacking the SOD1 transgene.

[0191] Donor animals for B cell isolation were C57BL / 6J mice, which were also purchased from Jackson Laboratory (Stock number: 000664).

[0192] All animal handling was performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Public Health Service Code for the Humane Care of Laboratory Animals. All protocols were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee under Protocol Number 2019N000004.

[0193] B cell isolation: All cell isolation procedures were performed under sterile conditions in a clean biosafety cabinet, and the resulting cell suspension was delivered in sterile phosphate-buffered saline (PBS). Cells were isolated and purified from the spleens of C57BL6 wild-type donor animals (similar to siblings) with a genetic background similar to that of the recipient. Spleens were dissociated into splenocyte suspensions, and a commercially available kit (Miltenyi B Cell Isolation Kit, mouse; 130-095-873, Miltenyi Biotec) was used to isolate total B cells by negative immunomagnetic selection, as described in our previously published protocols (DeKosky et al. (2013) Nat Rev Neurol 9, 192-200; Stocchetti et al. (2017) Lancet Neurol 16, 452-464). As confirmed by flow cytometry analysis after isolation, the resulting cells were >98% CD19+ B cells and typically >85-90% CD19+ / B220+ / IgM+ / IgD+, containing approximately 5% CD138+ plasma cells, and <1% other cell populations (DeKosky et al. (2013) Nat Rev Neurol 9, 192-200). This constituted the naive B cell fraction (for treatment) and was infused into animals on the same day after isolation.

[0194] Treatment: Beginning at 10 weeks of age (day 72), all animals received weekly intravenous infusions of B cells (or saline control) delivered via retro-orbital injection for a total of 10 doses. Animals were anesthetized with 3% isoflurane in oxygen, and a 100 μl bolus of 5 million naive B cells in saline (treatment) or saline without cells (saline control) was injected into the retro-orbital sinus. Ophthalmic ointment was then applied to the treated eye. This administration method was chosen because it has a significantly lower risk of failure compared with tail vein injections for cell transplantation, especially when repeated administration is required.

[0195] Safety Assessment: The health status of all animals was assessed three times a week according to IACUC guidelines for potential adverse effects of the treatment (weight loss, apathy, poor health, piloerection, hunchback posture).

[0196] Efficacy assessment: Body weight and neurological scores (Neuroscore) were assessed twice weekly by an experimenter blinded to treatment conditions and used to assess disease progression, as described, for example, in Hatzipetros, T. et al. (2015) J. Vis. Exp. (104), e53257, doi:10.3791 / 53257; Mashkouri et al. (2016) Neural Regen Res 11, 1379-1384.

[0197] Neuroscore 0 (pre-symptomatic) When the mouse is suspended by the tail, the hind limbs exhibit normal splay, i.e., the hind limbs are fully spread from the lateral midline and remain in this position for more than 2 seconds. When the mouse is allowed to walk, a normal gait is observed.

[0198] Neuroscore 1 (initial symptoms) When mice are suspended by the tail, the hind limbs exhibit abnormal splay, i.e., the hind limbs are folded or partially folded toward the lateral midline, or While suspended by the tail, is there tremor in the hind limbs? or Hind limbs are contracted / flexed. When the mouse is allowed to walk, or A slightly slower gait is observed.

[0199] Neuroscore 2 (onset of paresis) When a mouse is suspended by its tail, the hind limbs are partially or The legs are completely folded and do not splay much (joint movement is still observed). When the mouse is allowed to walk, the hind limbs are used for forward movement, but the toes curl downward or point upward at least twice during a 90 cm walk. or Drag any part of the foot. If the mouse is placed on its left side, andWhen placed right side down, From either side It can return to its correct position within 10 seconds.

[0200] Neuroscore 3 (complete paralysis) When mice are suspended by the tail, complete tonic paralysis of the hind limbs is observed. or Minimal movement of the joints is observed. When the mouse is allowed to walk, forward movement is observed, but the hind limbs are not used for forward movement. not present If you place the mouse on its left side, and When placed right side down, From either side It can return to its correct position within 10 seconds.

[0201] Neuroscore 4 (humane endpoint) When a mouse is hung by its tail, it shows complete tonic paralysis of the hind limbs. When the mouse is made to walk, no forward movement is observed. When the mouse is placed on its left side, and When placed right side down, From either side and return to its normal position within 10 seconds. I can't i.e., the absence of a righting reflex.

[0202] Body weight was used as a reliable and unbiased assessment of disease progression. The onset of disease was determined retrospectively using the age at peak body weight, which is a reliable and objective criterion for the onset of muscle denervation, as previously described (Turner et al. (2014) Neurobiol Aging 35, 906-915). Histology: After euthanasia, lumbar spinal cords were harvested from all animals, preserved in fixative, and processed for histological analysis. To visualize ventral horn motor neurons, spinal cords were sectioned longitudinally in the horizontal plane at a thickness of 10 μm and stained with hematoxylin and eosin (H&E). Motor neurons were easily identifiable based on their large size and unique morphology and were counted in three to six regions of interest, each measuring at least 500 × 500 μm, randomly collected from two to three longitudinal sections per animal. We also separately quantified healthy motor neurons (large, rounded cell bodies; nuclei with a single, distinct nucleolus; presence of Nissl substance; see Figure 27A) and damaged / degenerated motor neurons (shrunken cell bodies, hyperbasophilia, strongly clumped nuclear chromatin, pyknotic nuclei; see Figure 27B). All counts were performed by an experimenter blinded to the treatment conditions.

[0203] result The following results summarize the data collected from all animal studies. The study was terminated on postnatal day 150, when the last transgenic SOD1 animal was euthanized.

[0204] safety No observable (behavioral and phenotypic) adverse effects (weight loss, apathy, poor health, piloerection, hunched posture) were found in control non-carrier animals administered naive B cells (at a dose of approximately 200 million cells / kg). Control animals continued to gain weight throughout the study period, regardless of treatment with B cells (Figure 23). Furthermore, in transgenic SOD1-G93A animals, no such adverse effects attributable to cell infusion treatment were observed in the period before the onset of symptoms (days 72-90 of treatment).

[0205] Efficacy of treatment for symptoms of ALS progression a. Peak body weight (shown in Figure 24). Peak body weight was defined as the time point at which the measured body weight of an individual animal subsequently showed a continuous decrease. This information was used to perform survival analysis, in which the time point of peak body weight represents "survival." Only transgenic SOD1-G93A animals were used in this analysis because control non-carrier mice did not show weight loss.

[0206] Peak body weight (i.e., symptom onset, associated with weight loss) was delayed by an average of approximately 28 days in the naive B cell treatment condition compared to saline-treated controls (p < 0.0001, one-way ANOVA with Sidak post-hoc correction for multiple comparisons) (see Turner et al., Neurobiol. Aging 35, 906-915 (2014)).

[0207] b. Neurological score assessment (shown in Figure 25). For this analysis, disease onset was defined as the time point at which an animal received a neuroscore of 1 on three consecutive assessments and did not subsequently experience a decrease in neuroscore. A gradual increase in neurological score over time was observed in all animals, although treatment with B cells was associated with a slower rate of progression (Figure 25).

[0208] c. Survival analysis (shown in Figure 26). As described above, animals that became completely paralyzed and were unable to return to their upright position within 10 seconds of being placed on either side were classified as having a neuroscore of 4 and were humanely euthanized. These animals were counted as having died from ALS. Analysis of death as a consequence of disease progression demonstrated a significant benefit in overall survival associated with intravenous administration of naive B cells (p = 0.0286, one-way ANOVA with Sidak post-hoc correction for multiple comparisons; Figure 26).

[0209] d. Histological analysis of spinal motor neurons (shown in Figure 27). All histological sample processing and examination were performed by experimenters blinded to the treatment conditions. As expected, a highly significant decrease 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 transgenic SOD1 animals treated with B cells and control transgenic SOD1 animals, the percentage of these dead or dying motor neurons was found to be significantly lower in animals treated with B cells compared to saline controls.

[0210] conclusion In this study using the transgenic SOD1-G93A mouse model of ALS, intravenous infusion of purified allogeneic mature naive B cells, administered to mice once weekly for 10 consecutive weeks, was associated with: (i) a statistically significant 28-day delay in the onset of symptoms, (ii) a significant increase in survival time, and (iii) a significant reduction in pathological, dead, or dying neurons in the lumbar spinal cord of treated animals compared to saline-treated controls. No observable adverse side effects related to treatment were observed in either wild-type or transgenic animals receiving B cell infusions at a dose of 200,000 cells / gram (or 20 billion B cells / kg).

[0211] Example 4. Administration of B cells to treat Parkinson's disease A composition comprising a therapeutically effective amount of B cells, such as any of the compositions described herein, can be administered to a subject with Parkinson's disease. Treatment of Parkinson's disease can include administering therapeutic B cells (e.g., B reg Cells) to an appropriate animal model for Parkinson's disease (see, e.g., Bobela W. et al. Overview of mouse models of Parkinson's disease. Curr Protoc Mouse Biol. (2014)) and monitoring therapeutic efficacy according to techniques known to those skilled in the art. Methods for monitoring response include assessment of motor function, pain, neuroinflammation, and death of substantia nigra neurons (see, e.g., Peng Q. et al. The Rodent Models of Dyskinesia and Their Behavioral Assessment. Front Neurol. (2019)).

[0212] Responsiveness to treatment can be monitored by a reduction in the rate of disease progression (eg, a reduction in the rate of progression as measured by the severity of symptoms associated with Parkinson's disease). Alternatively, responsiveness to treatment can be monitored by determining levels of molecular markers of disease progression associated with neurodegenerative diseases, such as T-tau (total tau), P-tau (hyperphosphorylated tau), Aβ42 (amyloid beta 42), the Aβ42 / Aβ40 ratio, YKL-40 (chitinase 3-like protein 1), VLP-1 (visinin-like protein 1), NFL (neurofilament light chain), pNFH (phosphorylated neurofilament heavy chain subunit), Ng (neurogranin), and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43 (TAR DNA-binding protein 43), decreased α-synuclein, and / or decreased levels of 3,4-dihydroxyphenylacetic acid (see, e.g., Robey and Panegyres. Cerebrospinal fluid biomarkers in neurodegenerative disorders. Future Neurol. 14(1). (2019)).

[0213] 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 neuroaxonal dystrophy, progressive supranuclear palsy, dementia with Lewy bodies, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.

[0214] Exemplary animal models for the study of such neurodegenerative diseases are described in the art, for example, in: Alzheimer's disease (see, e.g., Esquerda-Canals G. et al. Mouse Models of Alzheimer's Disease. J Alzheimers Dis. (2017)); Chronic traumatic encephalopathy (CTE) (see, e.g., Dapul HR, et al. Concussive injury before or after controlled cortical impact exacerbates histopathology and functional outcome in a mixed traumatic brain injury model in mice. J Neurotrauma. 30(5):382-91 (2013)); and Huntington's disease (see, e.g., Farshim PP et al. Mouse Models of Huntington's Disease. Methods Mol Biol. (2018)).

[0215] Responsiveness to treatment can be monitored by a reduction in the rate of disease progression (e.g., a reduction in the rate of progression as measured by the severity of symptoms associated with the neurodegenerative disease). Alternatively, responsiveness to treatment can be monitored by determining the level of a molecular marker of disease progression associated with the neurodegenerative disease, such as, for example, the molecular marker of disease progression provided in Example 4.

[0216] Example 6. Administration of B Cells to Treat Inflammatory or Immune Diseases The B cells described herein can be administered to treat inflammatory or immune disorders, such as, for example, cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, or rheumatoid arthritis. Treatment of these inflammatory or immune disorders can be assessed using the methods described herein by administering therapeutic B cells (e.g., Breg cells) to an appropriate animal model and monitoring therapeutic effectiveness according to methods known to those skilled in the art.

[0217] Exemplary animal models for the study of such inflammatory or immune diseases are described in the art, for example, in: Cystic fibrosis (see, e.g., Dreano, E. et al. Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9. Animal Model Exp Med. 2(4):297-311 (2019)); Cardiovascular disease (Goodchild, TT et al. Bone marrow-derived B cells preserve ventricular function after acute myocardial infarction. JACC Cardiovasc Interv. 2(10):1005-16 (2009)); keratoconus (see, e.g., Tachibana M. et al. Androgen-dependent hereditary mouse keratoconus: linkage to an MHC region. Invest Ophthalmol Vis Sci. 43(1):51-7 (2002)); Osteoarthritis (see, e.g., Kuyinu, EL et al. Animal models of osteoarthritis: classification, update, and measurement of outcomes. J Orthop Surg Res. 11:19 (2016)); Osteoporosis (see, e.g., Komori T. Animal models for osteoporosis. Eur J Pharmacol. 759:287-94 (2015)); Pulmonary arterial hypertension (see, e.g., Sztuka K. and Jasinska-Stroschein M. Animal models of pulmonary arterial hypertension: A systematic review and meta-analysis of data from 6126 animals. Pharmacol Res. 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. Histol Histopathol. 25(7):933-44 (2010)); and Rheumatoid arthritis (see, e.g., Asquith DL et al. Animal models of rheumatoid arthritis. Eur J Immunol. 39(8):2040-4 (2009)).

[0218] All publications (including patents and patent applications, including U.S. Provisional Patent Application Nos. 62 / 795,629, 62 / 837,765, and 62 / 965,032) mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0219] Other Aspects It will be apparent from the foregoing description that changes and modifications may be made to the invention described herein to adapt it to various applications and conditions, and such embodiments also fall within the scope of the appended claims.

Claims

1. A pharmaceutical composition for treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising modified B cells, wherein a therapeutically effective amount of the modified B cells is administered to the subject, and the modified B cells are mature naive B cells stimulated ex vivo.

2. 10. The pharmaceutical composition of claim 1 used in combination with a second therapeutic composition.

3. 3. The pharmaceutical composition of claim 2, wherein the second therapeutic composition is edaravone or riluzole or an immunomodulatory composition.

4. 2. The pharmaceutical composition of claim 1, wherein the modified B cells are allogeneic B cells, autologous B cells, or xenogeneic B cells.

5. The pharmaceutical composition of claim 1, wherein the modified B cells are mature naive B cells stimulated with a Toll-like receptor (TLR) agonist.

6. The pharmaceutical composition of claim 1, wherein the modified B cells are Breg cells, and the Breg cells express the immunoregulatory cytokine IL-10 or contain at least 80% CD19+ B cells.

7. the modified B cells are formulated for local administration; or the modified B cells are formulated for systemic administration; 10. The pharmaceutical composition of claim 1.

8. 10. The pharmaceutical composition of claim 1, wherein the modified B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration.

9. 10. The pharmaceutical composition of claim 1, wherein the modified 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 yearly.

10. The therapeutically effective amount is at least 0.5 x 10 per administration. 7 B cells, at least 1 x 10 per dose 8 B cells, at least 2 x 10 per dose 8 B cells, or at least 1 x 10 per dose 9 10. The pharmaceutical composition of claim 1, comprising B cells.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the subject is a human.

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