B cell immunotherapy

Abstract

The invention generally features a method of treating a neurodegenerative disease (e.g., amyotrophic lateral sclerosis) or traumatic brain injury in a subject (e.g., a human) in need thereof, the method including administering to the subject a therapeutically effective amount of isolated B cells (e.g., autologous, allogeneic, or xenogeneic B cells).

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 by reference in their entireties herein. [Background technology]

[0002] 2. 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 areas 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 degenerative brain diseases, 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 investigation. However, the treatments proposed are often expensive and involve significant risks and complications. Thus, 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 PRESENT APPLICATION Provided herein are compositions comprising B cells (e.g., isolated B cells, purified B cells, or modified B cells, or a combination thereof) and uses thereof 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 a preferred embodiment, 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 includes administering a therapeutically effective amount of B cells to the subject, where 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 having 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 is an amount that 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; 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 1-7 days after administration, for 7-28 days after administration, for 1-28 weeks after administration, for 1-2 months after administration, for 2-6 months after administration, for 2-9 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 having 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 the plasma or cerebrospinal fluid (CSF) of the subject receiving treatment. Exemplary molecular markers of the 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 α-synuclein, and / or reduced 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, 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 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 (e.g., from falling, gunshot wound, sports accident, construction accident, traffic accident, or injury penetrating the skull or brain of the subject). In some embodiments, the subject with TBI and / or SCI has one or more of several physical, cognitive, social, emotional, 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 having a TBI who exhibits one or more symptoms of a TBI, the method including the steps of: administering a therapeutically effective amount of B cells to a subject, the therapeutically effective amount being 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, the therapeutically effective amount being 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., forgetfulness, inability to speak or understand language, mental 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 repetition of 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 1-7 days after administration, 7-28 days after administration, 1-28 weeks after administration, 1-2 months after administration, 2-6 months after administration, 2-9 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, levels of molecular markers of TBI and / or SCI may be determined from a sample for the subject undergoing treatment, for example, from a sample that is plasma or cerebrospinal fluid (CSF) of the subject undergoing 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, xenogeneic 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 a cell.

[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 reg In some embodiments, the 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 cells do not express CD73. reg The cells comprise at least 80% (e.g., at least 85%, 90%, 95%, or 98%) CD19+ B cells. 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 to be administered locally or systemically. In some embodiments, the B cells are formulated to be administered intravenously, intraarterially, subcutaneously, intrathecally, or intraparenchymally. In some embodiments, the B cells are formulated to be administered by intravenous infusion or intravenous bolus. In some embodiments, the B cells are formulated to be administered through an intracranial pressure (ICP) monitoring catheter.

[0039] In some embodiments, 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.

[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 0.5×10 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 pharma- ceutically acceptable excipients, where 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 pharma- ceutically 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 pharma- ceutically acceptable excipients, where 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 cell is a xenogeneic B cell.

[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 an altered B cell.

[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 performed 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, the 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 situations, including skin wounds and ulcers, muscle and heart 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 increased pro-regenerative capabilities. Factors that are generated from B cells under these unique conditions, including antibodies, cytokines, and growth factors, as well as microRNAs and other small molecules, can also be purified and applied directly to the 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., due to cerebral contusion), inflammatory disorders, or various immune diseases. Unlike any other existing cell-based therapy, B cells are easily 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 B cell therapy (allogeneic or autologous, or xenogeneic) has high applicability in clinical practice. 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 to monitor intracranial pressure, either intraparenchymal or intraventricular, providing a convenient route for administering B cells into the damaged brain.

[0061] Unlike other cell types used in therapy, such as stem cells, B lymphocytes are mature, terminally differentiated cells with a limited natural life span of 5-6 weeks in vivo. In neurodegenerative situations and in a microenvironment disturbed by cerebral contusion, the transplanted cells are expected to disappear in a shorter time after application. This is beneficial because the longer survival of transplanted cells may raise significant safety concerns, especially considering that the microenvironment of the central nervous system 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, rapid, and effective cell-based therapeutic strategy for several disorders disclosed herein, including ALS, PD, TBI, and SCI, where 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] 11. 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] The method of any one of claims 1 to 10, wherein autologous B cells are administered. [The present invention 1005] The method of any one of claims 1 to 10, wherein allogeneic 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 10, 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] 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] 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 B reg The method of claim 1001, which 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 The method of claim 1011, wherein the cells comprise at least 80% CD19+ B cells. [The present invention 1015] B reg The method of claim 1011, 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 any one of claims 1 to 10, wherein the B cells are formulated to be administered systemically. [The present invention 1018] The method of claim 1001, wherein the B cells are formulated for intravenous, intra-arterial, subcutaneous, intrathecal, or intraparenchymal administration. [The present invention 1019] The method of the present invention 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 is at least 0.5 x 10 per administration 7 The method of any one of claims 1 to 10, comprising administering to said patient a B cell. [The present invention 1021] The therapeutically effective amount is at least 1 x 10 8 The method of any one of claims 1 to 10, comprising administering to said patient a B cell. [The present invention 1022] The therapeutically effective amount is at least 2 x 10 8 The method of any one of claims 1 to 10, comprising administering to said patient a B cell. [The present invention 1023] The therapeutically effective amount is at least 1 x 10 9 The method of any one of claims 1 to 10, comprising administering to said patient a B cell. [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. [The present invention 1035] B cells are B reg The method of claim 1022, which is a cell. [The present invention 1036] B reg The method of claim 1032, wherein the cells express the immunomodulatory cytokine IL-10. [The present 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. [The present 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. [The present invention 1043] The method of claim 1022, 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 1044] A therapeutically effective amount is at least 0.5 x 10 per administration 7 The method of claim 1022, comprising administering to said patient a B cell. [The present invention 1045] The therapeutically effective amount is at least 1 x 10 8 The method of claim 1022, comprising administering to said patient a B cell. [The present invention 1046] The therapeutically effective amount is at least 2 x 10 8 The method of claim 1022, comprising administering to said patient a B cell. [The present invention 1047] The therapeutically effective amount is at least 1 x 109 The method of claim 1022, comprising administering to said patient a B cell. [The present invention 1048] Any of the aforementioned methods of the present invention, wherein the subject is a human. Certain 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 claim 1, further comprising administering a second therapeutic composition. 6. The method of claim 5, wherein the second therapeutic composition is edaravone or riluzole. 7. The method of claim 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 according to 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, 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. 12. The method of claim 10, wherein 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, imidazoquinoline, antiviral compounds, unmethylated CpG oligodeoxynucleotides, 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 The method of claim 13, wherein the cells express the immunomodulatory cytokine IL-10. 15. B reg 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 regThe method of any one of items 13-15, 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 The method of claim 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 13-15 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 according to any one of items 1 to 28, 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. 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 an individual B cell. 32. The therapeutically effective amount is at least 2 x 10 per administration. 8 31. The method according to any one of items 1 to 30, comprising an individual B cell. 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 an individual B cell. 34. A method of treating a subject having traumatic brain injury (TBI), comprising administering to the subject a therapeutically effective amount of isolated B cells. 35. The method of item 34, wherein the TBI results from a head injury or cerebral contusion. 36. The method of claim 34, wherein the subject has one or more of a physical, cognitive, social, emotional, and / or behavioral disorder. 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 claim 34, further comprising administering a second therapeutic composition. 40. The method of claim 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 according to any one of items 34 to 42, wherein the B cells are stimulated with a Toll-like receptor (TLR) agonist. 44. The method according to item 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, 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. 45. The method according to item 43, wherein 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, imidazoquinoline, antiviral compounds, unmethylated CpG oligodeoxynucleotides, 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 claim 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 regThe 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 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 according to any one of items 46 to 48, wherein the cells comprise at least 80% CD19+ B cells. 54. The method according to 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 according to 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 amount is at least 1 x 10 per administration. 8 64. The method according to any one of items 34 to 63, comprising the 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 the B cells. 67. The therapeutically effective amount is at least 1 x 10 per administration. 9 64. The method according to any one of items 34 to 63, comprising the B cells. 68. A pharmaceutical composition comprising modified B cells and one or more pharma- ceutically 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, 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. 70. The pharmaceutical composition according to item 68, wherein 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, imidazoquinoline, antiviral compounds, non-methylated CpG oligodeoxynucleotides, and profilin. 71. The pharmaceutical composition described in 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. The modified B cells are 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 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 74-76, 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 according to 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 pharma- ceutically 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 described in 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 according to 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 claim 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 according to 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 an altered B cell. 94. The method according to item 93, wherein step i) further comprises isolating CD19+ mature naive B cells. 95. The method according to 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 according to item 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, 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. 98. The method according to item 93, wherein 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, imidazoquinoline, antiviral compounds, unmethylated CpG oligodeoxynucleotides, and profilin. 99. The method of item 93, wherein the immunomodulatory cytokine is a proinflammatory cytokine. 100. The method of claim 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 The method of claim 101, wherein the cells express the immunomodulatory cytokine IL-10. 103. B reg 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 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 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 cell does 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 description of the drawings]

[0066] [Figure 1A] We show 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 main stages of wound healing in a wild-type mouse wound model. [Figure 1B]We show that application of B cells induces complex changes in the molecular microenvironment of the wound. Figure 1B shows a heat map summarizing the expression dynamics over time for proteins whose expression was significantly altered in response to application of B cells. A total of 213 proteins, including proteins that were significantly altered in association with treatment with B cells (n = 111; p < 0.05, unpaired t-test) and a collection of such proteins with high fold changes for each time point regardless of the significance level (top 20 up- or down-regulated proteins) (n = 112), were classified according to the process involved in wound healing. The heat map shows the fold change in expression after treatment with B cells at 0, 1, 4, and 10 days 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. [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 following treatment with B cells. 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 expression of proteins that normally either decrease or increase during injury and healing, significantly reducing the inflammatory peak observed in normal healing, preventing the decrease of anti-apoptotic factors (arrows) and oxidative stress protectants, and increasing proliferation (Figure 2A-B), reducing the decline of antioxidant stress protectants and cell proliferation, and maintaining low levels of cell migration (Figure 2C-D), maintaining constant levels of proteins related to remodeling and secondary skin structures (Figure 2E-F), decreasing the levels of proteolysis and autophagy observed early in injury in controls, and increasing the levels of proteins related to 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. [Diagram 3]Experimental paradigm for in vivo evaluation of B cell application in acute wound healing. A total of four full-thickness wounds were made 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 subcutaneously injected under the intact skin as an internal control. After a defined survival period, wounded or uninjured skin tissues were 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 almost absent in either location, they can be easily detected in large numbers after experimental application (red arrows). [Figure 4] Flow cytometric 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 a mixture of neutrophils, monocytes and macrophages, dendritic cells, and T cells, and fibroblasts (CD140a+ / B220-). These cell categories were assessed for markers of activation and cytokine production. [Diagram 5]Dynamics of activation markers and key cytokines in B cells recovered from wounds after defined exposure periods to the wound microenvironment are shown. B cells were exposed to the wound niche in vivo or injected under uninjured skin (control at equivalent location). Control B cells kept 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 excising and dissociating the tissue and further characterized by flow cytometry for both surface markers and intracellular cytokines. B cells exposed to the wound microenvironment transiently upregulated multiple immunomodulatory cytokines, peaking 2 days after application. Several immunomodulatory cytokines, including TGFβ and IL-6, remain elevated at day 4, and IL-10 remains elevated until day 10. N = 3-6 animals / group. [Figure 6] Heatmap as aggregation of the 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. 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 strongest 4 days after injury and B cell application and persisted for up to 10 days. N = 3-6 animals / group. [Figure 8] Heatmap showing the pattern of increased production of anti-inflammatory cytokines (IL-10 and TGFβ) in the presence of B cells in infiltrating non-B cell leukocytes in the wound microenvironment as an aggregation of the average values ​​for each marker. [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, wound fibroblasts produced less of the proinflammatory cytokine TNFα at both 4 and 10 days after injury when B cells were applied. [Figure 10] Heatmap of the mean values ​​for each marker in wound and subcutaneous 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, while the levels of these anti-inflammatory cytokines were reduced in wounds treated with saline. 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] We show that functional TLR signaling and IL-10 production are essential components of the regenerative function of foreign B cells in wound healing. Full-thickness excisional wounds (shown here at healing day 6) 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 up to days 2-3, while MyD88- / - and IL-10- / - B cells showed no benefit on wound closure, similar to saline application. [Figure 12]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 of 3809 proteins (rows) consistently expressed in all animals at four different time points (columns) after injury in both B cell and saline treated wounds using complete linkage method. Pseudocolor scale shows normalized, log transformed fold change in expression values ​​for each protein. Dendrogram shows 15 protein clusters derived from this analysis, with the color of each cell in (A) corresponding to the average expression value of the cluster at the respective time point. Proteins are clustered by their expression pattern over time. (B) Heatmap of 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 explore the 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] We present an experimental paradigm to evaluate the effect of B cell application on functional (behavioral) and histological recovery after contusion TBI. 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 before injury, a single injection of 2 x 106 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, as well as memory performance, anxiety, and depression-like behaviors 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. (A) Rotarod assessment showed a significant protective effect of B cells administered at the time of CCI. Notably, during repeated trials, the latency to fall was increased in mice treated with B cells similar to sham-injured animals, suggesting a component of motor learning. No such improvement was observed in controls treated with T cells or saline. 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 showed a significant effect of injury compared to sham-injured controls, but no statistically significant differences were observed between treatment conditions in injured mice. Data are mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. CCI + B cells, n = 12 mice; CCI + T cells, n = 12 mice; CCI + saline, n = 12 mice; Sham injury + B cells, n = 10 mice; Sham injury + saline, n = 10 mice. [Figure 16]Effects of a single acute application of B cells on learning and memory. (A)-(D) Morris water maze assessment. Learning curves showed significant improvement in CCI mice treated with B cells compared to saline-treated CCI animals (p < 0.05). No significant differences were observed between B cell-treated lesioned animals and either sham-lesioned condition after the third trial (p > 0.98) (A). Visible platform trials showed no difference between lesioned and non-lesioned treatment conditions (B). (C) Probe trials showed that CCI-lesioned animals treated with B cells spent time above chance level in the target quadrant, not significantly different from sham-lesioned mice. In contrast, control CCI-lesioned mice treated with either T cells or saline spent only chance time exploring the target quadrant, and were significantly different from sham-lesioned animals (p < 0.05). Dashed lines indicate chance level. (D) Representative swim path traces during the probe trial show spatial exploration patterns in CCI-B cell mice, as well as both sham-lesioned groups, while CCI mice in the T cell and saline groups adopted 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 the lesioned groups treated with T cell or saline, and performed similarly to the sham-lesioned 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-lesioned + B cell, n = 10 mice; sham-lesioned + saline, n = 10 mice. [Figure 17]Figure 1 shows the effect of treatment with B cells on anxiety and depression-like behavior after CCI. (A) Elevated plus maze assay of anxiety-like behavior. No significant overall differences were observed between treatment groups, except for slight differences in 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 for depression-like behavior. No injury or treatment 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 treatment with B cells on histological outcomes after CCI is shown. (A) Representative coronal sections through the injury site 35 days after injury. In animals treated with B cells, a portion 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 mice treated with B cells 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. The results show a consistent reduction in lesion size in injured brains treated with B cells. (D) The total volume of compensated hippocampus in the injured hemisphere was significantly greater in animals treated with B cells compared to either of the 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]Effect of treatment with B cells on gliosis and microglial activation. (A)-(D) Confocal images showing immunolabeling for GFAP and CD68 in the whole-body view of the medial aspect of the injury 35 days after CCI and treatment with either saline (A), B cells (B), or T cells (C), or in sham-injured controls treated with saline (D). (E) Quantitative analysis of the area occupied by GFAP immunostaining showed a significant reduction in reactive astrogliosis in injured animals treated with B cells compared to either saline-treated CCI controls or CCI controls treated with T cells. (F) Quantitative analysis of CD68 immunostaining showed a significant reduction in the presence of CD68 in animals treated with either T or B cells after CCI compared to 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] Showing B cell survival and persistence in the brain. (A) Representative example of intravital imaging of WT C57Bl6 / J mice 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 until approximately 14 days after application, with the number of viable cells decreasing significantly after 7 days. [p / s] = photons per second. [Figure 21]B cell localization 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 B220+ B cells clustered 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, although the intensity of vital staining was reduced 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 can be observed throughout the region, but no co-staining for B220 and Ki67 was observed. (E) Enlarged version of the boxed area in (D). (F) In sham-injured animals, the needle track through the cortex, bordered 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 are 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]Normalized body weight (percentage of the value on day 76 for each individual animal) over time, measured twice weekly, in 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 progressive weight gain was observed over the study period, regardless of treatment. Results also showed a delayed decline for transgenic SOD1 animals receiving B cell administration (arrow). N=32 per treatment condition. [Figure 24] Figure 1 shows an analysis of peak body weight in SOD1-G93A animals. A. Survival plots show the time points at which animals reached their peak body weight. B. Treatment with B cells significantly delayed the onset of complete paralysis as shown by the time to reach peak body weight. Statistics: A: Gehan-Breslow-Wilcoxon test; B: unpaired t-test. N = 32 animals per group. [Diagram 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. Animals that reached a neuroscore of 4 (complete paralysis) were considered to have died of ALS. Treatment with naive B cells significantly extended survival time compared to saline control treatment (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. 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 and 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 differences within this group. D: A significant benefit of treatment with B cells was evident when the percentage of degenerated and pyknotic motor neurons was specifically analyzed. Statistics: (left): 2-way ANOVA; (right): unpaired t-test. N = 19-24 animals per group. Note that collection of tissue samples was not possible in all animals tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 those commonly understood by those skilled in the art to which the present invention belongs. In carrying out 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. Numerical ranges include the numbers that define the range.

[0068] The headings provided herein are not intended to limit the various aspects or embodiments of the invention which may be had by reference to the specification as a whole, and therefore 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 disorder 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, many similarities are found between these diseases that relate to each other. 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 circuitry, ranging from molecular to systemic. Neurodegeneration may 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 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 α-synuclein, and / or reduced 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 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 disturbance of the normal function of the 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 injury penetrating the skull or brain of the subject).TBI is diagnosed according to clinical guidelines known to those skilled in the art. TBI can be further characterized by molecular markers of disease progression, such as protein biomarkers 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), post-injury autoimmune response (autoantibodies targeting brain antigens), etc. (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). 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 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 injury that penetrates the spinal cord of the subject).SCI is diagnosed according to clinical guidelines 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 abnormality that has an inflammatory or immune component in the etiology, pathogenesis, progression, or symptomology of the disease.For example, an inflammatory or immune disorder may include dysregulation of inflammatory or immune pathways and / or 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 the change (e.g., reduction) in symptoms associated with neurodegenerative disorder, 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 disorder, 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 can 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 can be characterized by a decrease in inflammatory markers (e.g., a decrease in proinflammatory 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, etc., and / or may decrease the activity of cells associated with an immune response, e.g., by decreasing proinflammatory markers, e.g., cytokines, etc.

[0077] As used herein, the term "B cell" or "B lymphocyte" used interchangeably herein refers to a small subtype of lymphocyte, a type of white blood cell. Unlike the other two classes of lymphocytes, T cell and natural killer cell, B cells express B cell receptors (BCR) on their cell membrane. BCR allows B cells to bind with a specific antigen against which the B cell initiates 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 cell" 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 of skill 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, 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 cytokines" refers to cytokines secreted from immune cells that promote inflammation. Immune cells that produce and secrete proinflammatory cytokines include T cells (e.g., Th cells), macrophages, B cells, and mast cells. Proinflammatory cytokines include: interleukin-1 (IL-1, e.g., IL-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, e.g., TNFα), interferon gamma (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 receptor (TLR), 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 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 complex. 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, imidazoquinolines, 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 change the natural course of the individual receiving the treatment, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, the prevention of the occurrence or recurrence of a disease or disorder (such as those described herein), alleviation of symptoms of such a disease, reduction of any direct or indirect pathological consequences resulting from the disease, and alteration of 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 "administer" refers to a method of administering a drug to a subject.The compositions utilized in the methods described herein can be, for example, administered intravitreally (e.g., by intravitreal injection), administered by eye drops, administered intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraparenchymally, intraprostatically, intrathoracically, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, The composition may be administered subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by direct local perfusion of the target cells, by catheter, by perfusion, as a cream, or as a lipid composition. The compositions utilized in the methods described herein may also be administered systemically or locally. For local administration, the dosage is administered as a lotion, cream, ointment, or gel. The method of administration may vary depending on various factors, such as the composition 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., human), a rodent (e.g., rat or mouse), or another type of mammal (e.g., livestock, 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 having, having had, or having symptoms of an immunological disorder, a neurodegenerative disorder, a TBI, an SCI, an immunological disorder, or a mammal having 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 "isolate" or "isolation" refers to both the physical identification and the separation of a cell or cell population from a cell culture or biological sample. Isolation can be carried out by applying suitable cell biology techniques, either based on the investigation of cell cultures and the characterization (and physical separation, if possible and desirable) of cells that meet a criterion, or based on automated sorting of cells (e.g., by FACS, etc.), for example, by characteristics such as the presence / absence of antigens and / or cell size. In some embodiments, the term "isolate" or "isolation" can include a further step of physical separation and / or quantification of cells, especially by performing flow cytometry. Physical separation also includes enrichment of cells or cell populations of a particular characteristic. 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 that essentially consists of or includes cells as defined herein. A cell population may essentially consist 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 certain cellular components or products (e.g., RNA or protein), activity of certain biochemical pathways, proliferation capacity and / or proliferation kinetics, differentiation potential and / or response to differentiation signals, or behavior during in vitro culture. Thus, such demonstrable characteristics may define a cell population, or a fraction thereof. A cell population may be "substantially homogeneous" when a substantial 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, for example, 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), where 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%-99%, or 70%-90% B cells (or a subpopulation of B cells, for example, 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 collection 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 obtained 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 includes pre-pro 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 of skill 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 use the 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 a primary antibody that recognizes molecules on the surface of the cells to be separated from the 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 precursor B cells. Such populations are 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 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 that 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 in 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 that are bound to them.

[0096] To achieve positive or negative selection, the heterogeneous cell population is incubated with the primary antibody for a sufficient time to achieve binding of the antibody to the antigen on the cell surface. If the primary antibody is labeled, separation can be performed at this stage. If a secondary antibody is utilized, the secondary (anti-primary) antibody is then incubated with the cells bound to the primary antibody for a sufficient time 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 microbeads labeled with the secondary antibody then form a complex, which is left behind when a magnet is applied, while the other unlabeled cells are removed with the cell culture medium. The positively labeled cells are then eluted and ready for further processing. Negative selection is 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 cell lysis, or from peripheral blood mononuclear cells (PBMCs) after density gradient centrifugation. Both positive selection and depletion strategies can be followed 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 separate 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 off platelets, the product (CD19 up to 4 x 10 per vial of CliniMACS Reagent) is enriched for B cells. 10 A total of 5 x 10 cells 9 The target fraction (CD19+ cells) was treated with CD19 microbeads separated by an LS column for enrichment of CD19+ cells. The target fraction was washed and then the infusion medium was supplemented with 25% HSA (final concentration 1%) Plasma-Lyte A.

[0100] The method generally includes: Day 1 a. The donor apheresis product is received and sampled for sterility, cell count, viability, and flow cytometry. b. The product is stored in the cold overnight. Day 2 The product is removed from the refrigerator, mixed thoroughly, and allowed to equilibrate to ambient temperature for 30 minutes. Samples are removed for sterility, cell count, viability, 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 following standard CliniMacs procedures except incubation is performed at 4 degrees. d. After incubation with the beads, one antibody wash with chilled (4 degrees) media is performed 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 counts, 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 from stem cell populations can also involve a negative selection process, in which red blood cell lysis is first performed in bone marrow by placing the bone marrow in a hypotonic buffer and centrifuging the red blood cells from the buffer. The red blood cell debris remains in the supernatant, which is removed from the tube. The cells derived from bone marrow are then resuspended in a buffer that has the appropriate conditions for antibody binding. Alternatively, density gradient centrifugation can be performed on bone marrow. The buffy coat layer containing the cells derived from bone marrow is removed from the gradient after centrifugation. The cells are washed and resuspended in 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 with 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 as described herein. Using such ex vivo stimulation, IL-10 producing B cells can be produced. 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 preferably 10 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 having 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 in the form of an aqueous solution.

[0108] The B cells described herein may be administered in any physiologically compatible carrier, such as, for example, 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 may be administered in PlasmaLyte infusion buffer. PlasmaLyte is a family of regulated crystalloid solutions with several different formulations and is available worldwide depending on local clinical practices and preferences. It closely mimics human plasma in its electrolyte content, osmolality, and pH. PlasmaLyte solution also has additional buffering capacity and can be used to inject anions, such as bicarbonate, CO 2 , and acetate, gluconate, and especially lactate, which are converted to water. The advantage of PlasmaLyte is that it includes 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 (C 6 H 11 NaO 7 ); 368 mg of sodium acetate trihydrate (C 2 H 3 NaO 2 3H 2 HO); 37 mg potassium chloride (KCl); and 30 mg magnesium chloride (MgCl 2 6H 2 O). It does not contain antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is about 7.4 (e.g., 6.5 to 8.0).

[0109] Other pharma- ceutically acceptable carriers and diluents include physiological saline, buffered aqueous solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid media, such as Dulbecco's Modified Eagle Medium (DMEM), sterile saline, sterile phosphate-buffered saline, Leibovitz's medium (L15, Invitrogen, Carlsbad, Calif.), sterile aqueous dextrose solution, 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, which includes, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or as well as vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of 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. The solution is preferably 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, and the like. Solutions of the present invention can be prepared using a pharma- ceutically 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, the solution containing the pharmaceutical composition described herein may be appropriately buffered, and if necessary, the liquid diluent is first made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.In this regard, the sterile aqueous medium available in the present disclosure is known to those skilled in the art.In any case, the person in charge of administration determines the appropriate dose for each subject.In addition, for human administration, the preparation may meet the sterility, pyrogen regulation, general safety and purity standards required in the FDA agency's biological product standards.

[0112] Pharmaceutical compositions can also include excipients that promote cell membrane stability. For example, highly soluble osmoregulatory proteins, such as highly soluble osmoregulatory proteins with high molecular weight, 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 that include cells. EXAMPLES

[0113] The present invention will be described in more 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 regarded as an integral part of this specification and as an explanation of the present invention.All references cited are specifically incorporated herein by reference in their entirety as described therein.The following examples are provided for illustrative purposes, but are not provided 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, with a significant decrease in proteins associated with inflammatory responses and an increase in proteins associated with tissue proliferation and remodeling. By recovering applied exogenous B cells from the wound niche at various time points after application and examining the cell populations using multicolor flow cytometry, we determined that mature naive B cells applied to the wound transitioned toward 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, with a peak at 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, with a decrease in the expression of proinflammatory cytokines, including IL-2, IL-4, IL-6, and IFN-γ. Thus, naive B cells located at the site of injury detect local inflammatory signals and damage-associated molecular patterns (DAMPs) by 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 capacity.

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

[0117] animal Wound healing studies were performed in 7-9 week old male wild type C57Bl6 / J mice (Jackson Laboratories). Male WT C57Bl6 / J 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 12:12 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 of Massachusetts General Hospital. All efforts were made to reduce the number of animals used and to minimize animal suffering.

[0118] Cell isolation Mouse spleens were harvested 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 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 Full-thickness excision wounds through the dorsal skin were 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 and a 5 mm biopsy punch was penetrated 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 ​​100 x 100 mm. A silicone splint (Sigma-Aldrich) with an internal diameter of 7 mm was attached to the periphery of the wound using Vetbond tissue adhesive (3M). The wound with the attached splint was then covered with Tegaderm™ transparent dressing (3M). The cell suspension in PBS 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 with equal doses of B cells or saline under the dorsal skin. 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, the subjects were re-inoculated with 3% isoflurane in O. 2Mice were lightly anesthetized using 50 mM NaCl, 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 4 hours of incubation, mice were euthanized and tissue biopsies were taken, including the wound and subcutaneous injection sites. Tissue biopsies were enzymatically dissociated for 30 min at 37°C with gentle shaking 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), 0.025 mg / ml, >10 U / mg dispase I (Millipore Sigma). Tissues were then mechanically minced into smaller pieces, followed by further enzymatic dissociation in the same solution for an additional 30 min at 37°C with gentle shaking. Digested tissue from 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 6 Wounds were treated with either a solution of purified B cells or saline control at 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 1 M urea in 50 mM HEPES, pH 8.5. Vortexing, sonication, and manual disruption were used to increase solubility. The solubilized proteins were digested in a two-step process, starting with an overnight digestion at room temperature with 3 μg of Lys-C (Wako), followed by a 6-h digestion at 37°C with 3 μg of trypsin (sequencing grade, Promega). 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. Peptides were incubated with the reagent for 1 h at room temperature. The labeling reaction was terminated by adding 6 μL of 5% hydroxylamine. The labeled samples were then acidified by adding 50 μL of 1% TFA, and the peptide mixtures were pooled as ten-plex TMT samples. The pooled samples were desalted by C18 SPE on a Sep-Pak cartridge as described above.

[0123] Fractionation of samples by basic pH reversed-phase liquid chromatography (bRPLC). Fractionation of samples was performed by bRPLC39, 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 dried under vacuum, reconstituted with a solution of 5% formic acid and 5% ACN, and then analyzed by LC-MS2 / MS3 for identification and quantification.

[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 in a microcapillary column (100 μm i.d.; 360 μm o.d.) that was stretched and packed in-house. The column was first packed with ∼0.5 cm of Magic C4 resin (5 μm, 100 Å, Michrom Bioresources), followed by ∼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 11% to 30% ACN in 0.125% formic acid at a flow rate of 300 nL / min over 165 min with the column 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 resulted in 6 x 10 NMR spectra across the m / z range of 500–1,200 in the Orbitrap. 4 For the MS1 survey scan, the automatic gain control (AGC) was set to 5 x 10 5The chromatographic speed was set to 100 s, the maximum injection time was set to 100 ms, and the radio frequency (RF) setting of the S-lens 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 started 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 600-1200, since triply and quadruply charged ions had to be detected in the m / z range of 500-1200. The ion intensity threshold was 5 x 10 5 The AGC was set to 0.5 ms. When acquiring MS2 spectra, ions were isolated using the quadrupole by applying a window of 0.5 m / z 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 maximizing the 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 at 2.5 m / z, and fragmentation was performed by HCD at a normalized collision energy of 50%. Fragment ions in the MS3 spectrum were detected in the Orbitrap at a resolution of 60,000 and m / z of ≥110. The AGC target was ions 5 x 10 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 an in-house developed software suite (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 made 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 the entire protein sequence 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, as well as carbamidomethylation of cysteines (+57.02146 Da). Oxidation of methionines (+15.99492 Da) was included as a variable modification. Data were filtered to 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 score combined from the following peptide and spectral properties: XCorr, ΔCn, tryptic uncleavage, peptide mass accuracy, and peptide length. The probability that a peptide-spectrum match was correct was calculated using a posteriori error histograms, the probabilities of all peptides that were assigned to one particular protein were combined by multiplication, and the dataset was refiltered for an FDR of <1% of protein assignments for the entire dataset of all proteins identified across all of the analyzed samples. Peptides that matched to more than one protein were assigned to the protein containing the greatest number of matched redundant peptide sequences according to the rule of parsimony.

[0129] For quantitative analysis, the intensities of TMT reporter ions were extracted from the 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 specificity of isolation for the precursor ion was ≥ 0.75. Protein intensities were calculated by summing the TMT reporter ions for all peptides belonging to one protein.

[0130] Flow cytometry To assess cell viability after recovery from tissue digests, cell suspensions were washed and resuspended in PBS and stained with Zombie UV fixable viability kit (Biolegend, Inc.) for 30 min in the dark at 4° C. with gentle shaking. Stained cells were then washed and resuspended for 10 min in the dark at 4° C. in PBS containing: 1% FBS, 0.01% sodium azide (RICCA Chemical, Arlington, TX), and 5% FcR blocking reagent (Miltenyi Biotec, Inc.). Blocked cells were incubated with the following fluorophore-conjugated primary surface antibodies for 30 minutes at 4° 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), PE-CF594-conjugated rat anti-mouse CD140a (clone APA5) (both from BD Biosciences, San Jose, Calif.). Surface stained cells were washed and resuspended in Fixation Buffer (Biolegend, Inc.) for 30 min 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 min 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 from Biolegend, NY). Inc.), fluorescein-conjugated rat anti-mouse IFN-β (clone RMMB-1), 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 at 0 (intact), 1, 4, 10, and 16 days after injury were fixed in 4% buffered paraformaldehyde for 24-48 hours at 4°C, then cryoprotected in 1M 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° 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 for 5 min each in TBS.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 h 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 min at room temperature. Sections were washed three times for 7 min in TBS and embedded with Fluoromount (Novus Biologicals). Antibody controls included incubation of tissue sections with isotype antibodies and omission of primary antibodies when secondary antibodies were used for visualization. No nonspecific signals were detected in 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 wound healing time course endpoint were taken using a 10 mm biopsy punch and fixed in 4% paraformaldehyde in PBS for 24–48 h at 4°C, after which the samples were dehydrated by 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 / pixel using an Aperio CS2 scanner (Leica Biosystems). 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 using linear mixed-effects modeling in SPSS 23 (IBM Corporation) separately for each cell population examined. For variance stabilization, the proportion of gated cells for each sample and each marker examined was logit (log of odds) transformed before analysis. A three-way (or four-way, where applicable) full factorial design was used, including survival time (18, 45, or 93 hours), environment (wound, subcutaneous, or B cells only on ice), marker, and, where applicable, condition (treatment with B cells or saline) as fixed factors. Technical replicates (day of operation) and biological replicates (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 main stages of wound healing in a wild-type mouse wound model is shown in FIG. 1A. A heat map summarizing the expression dynamics over time for proteins whose expression was significantly changed in response to B cell application is shown in FIG. 1B. A total of 213 proteins, including proteins that were significantly changed in association with B cell treatment (n = 111; p < 0.05, unpaired t-test) and a collection of such proteins with high fold changes for each time point regardless of the significance level (top 20 up- or down-regulated proteins) (n = 112), were classified according to their process in wound healing. The heat map shows the fold change in expression after treatment with B cells at 0, 1, 4, and 10 days 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] Expression of proteins by functional family The average expression of proteins per 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 expression of proteins that normally either decrease or increase during injury and healing, significantly reducing the inflammatory peak observed in normal healing, preventing the decrease of anti-apoptotic factors (arrows) and oxidative stress protectants, and increasing proliferation (Figure 2A-B), reducing the decline of antioxidant stress protectants and cell proliferation, and maintaining low levels of cell migration (Figure 2C-D), maintaining constant levels of proteins related to remodeling and secondary skin structures (Figure 2E-F), decreasing the levels of proteolysis and autophagy observed early in injury in controls, and increasing the levels of proteins related to 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 FIG. 12. Only identified proteins that were found to be consistently present across all samples were included in the analysis. Hierarchical clustering of 3809 proteins (rows) that were consistently expressed in all animals at four different time points (columns) after injury in both B cell and saline treated wounds using the complete linkage method is shown in FIG. 12A. The pseudocolor scale shows 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 (FIG. 12A) corresponding to the average expression value of the cluster at the respective time point. Proteins are clustered by their expression pattern over time. In FIG. 12B, a heatmap of the hierarchical clusters from (FIG. 12A) is shown, showing all 3809 proteins. Gene ontology analysis of the 15 hierarchical clusters is shown in FIG. 12C. The mouse GOslim gene list from QuickGO (accessible at https: / / www.ebi.ac.uk / QuickGO) was used to explore the 15 hierarchical clusters. Bar graphs show the top biological function category for each cluster.

[0138] At each time point during wound healing evaluated, 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 FIG. 3. A total of four full-thickness wounds were made 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 subcutaneously injected under the intact skin as an internal control. After the defined survival period, wounded or uninjured skin tissues were 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 show a characteristic influx of leukocytes (hollow white arrows), which are almost absent in uninjured tissue. Although B cells are typically almost absent in either location, they can be easily detected in large numbers after experimental application (red solid arrows).

[0140] Flow cytometric analysis of cell suspensions from B cell-treated and control wounds. To assess the changes in both B cells and cells of the wound environment between the various 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 is shown in Figure 4. Live cells were gated into three main categories: B cells (CD19+ / B220+ lymphocytes), non-B cell leukocytes (CD140a- / B220- leukocytes), including a mixture of neutrophils, monocytes and macrophages, dendritic cells, and 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 (controls at comparable locations). Control B cells kept 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 excising and dissociating the tissue and further characterized by flow cytometry for both surface markers and intracellular cytokines. B cells exposed to the wound microenvironment transiently upregulated multiple immunomodulatory cytokines, peaking 2 days after application. Several immunomodulatory cytokines, including TGFβ and IL-6, remain elevated at day 4, and IL-10 remains elevated until day 10. N = 3-6 animals / group.

[0142] Heatmap Analysis Heat maps as aggregations of the 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) 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 strongest 4 days after injury and B cell application and persisted for up to 10 days. N = 3–6 animals / group.

[0143] Heat maps 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 average values ​​for each marker are shown in Figure 8.

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

[0145] Additional heat maps as aggregates of the average values ​​for each marker in wound and subcutaneous tissue fibroblasts treated with either B cells or saline are shown in FIG. 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 the levels of these anti-inflammatory cytokines were reduced in wounds treated with saline. 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 of the regenerative function of foreign B cells in wound healing. Full-thickness excision 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 up to days 2-3, while MyD88- / - and IL-10- / - B cells showed no benefit on wound closure, similar to the application of saline.

[0147] Example 2: Treatment with B cells improves outcomes after TBI This example shows 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 possible 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 the same number of T cells or saline, and sham-injured mice (craniotomy only) received B cells or saline. The sham-injured groups showed similar performance in motor and learning tests. Compared with the saline or T cell-treated CCI groups, the injured mice that received B cells showed significant improvement in performance after injury in the rotarod, Y-maze, and Morris water maze (MWM). Furthermore, the injury volume in mice treated with B cells was significantly reduced by 40% compared to saline and T cell controls 35 days after TBI, and astrogliosis and microglial activation were reduced. In vivo tracking of exogenous B cells showed that the cells have a limited life span of approximately 14 days in situ 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 site of injury.

[0149] Thus, in the following study, the potential of mature naive B cells for protection from cognitive and histopathological damage in a mouse CCI TBI model was investigated.Compared to administration of splenic T cells or saline, a single dose of B cells delivered by intraparenchymal injection at the time of injury was associated with significant improvements in hippocampal-dependent and striatal-dependent behavioral tasks.The observed behavioral improvements were associated with a significant reduction in injury volume in animals treated with B cells, which was accompanied by compensation of hippocampal structures.In vivo tracking of exogenously applied B cells after intraparenchymal injection showed that the cells have limited viability in situ for approximately 2 weeks, indicating that the cells 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 Institutional Animal Care and Use Committee of Massachusetts General Hospital. Studies were performed in adult male C57Bl6 / J 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 isolation of B and T cells. Animals were socially housed (4-5 per cage) and maintained under standard laboratory care conditions at temperatures ranging from 20-23°C, with a 12-h light:dark cycle and with 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 splenocyte suspensions were processed for negative selection of B or T cells by immunomagnetic separation and retention of non-target cells using a commercial 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 that had less than 1% contamination with other leukocytes. 16 Purified lymphocytes were cultured at 4 x 10 5 The cells 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 vaporized with 70% N containing 4.5% isoflurane (Baxter, Deerfield, IL) for 90 seconds using a Fluotec3 vaporizer (Colonial Medical, Windham, NH). 2 O and 30% O 2Mice were anesthetized with a mixture of 100% ethanol and mounted in a stereotaxic frame. Anesthesia was maintained with 4.5% isoflurane. After a midline incision in the scalp, 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. Ipsilateral intraparenchymal injections were delivered to a depth of 3 mm through the left parietal cortex, approximately −1 mm from bregma on the anterior-posterior axis and +2 mm from bregma on the mediolateral axis. A total volume of 5 μl 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 selected were previously optimized in skin injury models exhibiting similar injury volumes. 16 To ensure accurate and consistent cell injection while the brain structures were intact, cell application was performed immediately prior to CCI. Immediately afterwards, mice received CCI using a pneumatic cylinder fitted with a 3 mm blunt-tip impounder at a velocity of 6 m / s, a depth of 0.6 mm, and an impact duration of 100 ms. Sham-injured mice received 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 min. Mice were tested in the battery of assays according to the schedule described in Figure 14. Vestibular-related motor abilities were assessed by wire grip assay 1, 3, and 7 days after injury. Rotarod testing was performed 7, 9, 10, 13, and 14 days after injury. 17 days after injury, animals were subjected to anxiety assessment using an elevated plus maze assay. Morris water maze (MWM) testing was performed 20, 21, 22, 23, and 24 days after injury, and a probe test was performed 27 days after injury. Mice were subjected to a forced swim test to assay depressive-like behavior 29 days after injury, and a Y-maze test, an assay for hippocampal-dependent working memory, was performed 30 days after injury.

[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 in 60 s. The maximum duration of a trial was 300 s or until the mouse fell off the rotarod. Each mouse was assessed five times per day with a 5-min rest interval. The mean latency to fall and the mean speed of revolutions / min obtained over the five trials were recorded for each day of testing.

[0157] MWM: The MWM was performed with slight modifications as previously described (Mannix et al. (2013) Ann Neurol 74, 65-75). Spatial learning was assessed at approximately the same time of day. Each mouse received seven trials of the invisible platform (1–2 trials per day) with a randomized set of start locations in one of the four quadrants. One trial consisted of the average latency from each of the four start locations. If the mouse failed to find the platform within 90 s, it was placed on the platform for approximately 10 s. A probe trial was performed 24 h after the last invisible platform trial by having the mouse swim in a tank without a platform for 30 s and recording the time spent in the target quadrant.

[0158] Porsolt forced swim test: Mice were placed in a cylindrical clear glass tank, 30 cm (height) x 20 cm (diameter), filled with water (25°C) to a height of 20 cm. A white Styrofoam box was provided for blindfolding on three sides. Mice were placed in the water for 6 min and swimming behavior was recorded. Total time active (swimming, paddling / climbing the beaker wall) vs. inactive time (floating unresisted) was quantified for the last 4 min of the test.

[0159] Y-maze spontaneous alternation test: The Y-maze test was performed in a white opaque acrylic apparatus with 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 display (black square, circle, star on white background). The mouse was placed in the center of the apparatus and allowed to explore the maze for 10 minutes. The mouse's movements were recorded using a webcam mounted directly above and Photo Booth software (ANY-maze). Normal exploration behavior in rodents involves a preference for entering the arm of the maze that was not the one that was entered most recently (spontaneous alternation). The alternation score was calculated by dividing the number of three consecutive choices, including one in 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 a square area of ​​8 cm x 8 cm 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. Videotapes were analyzed by ANY-Maze (Stoelting Co., Wood Dale, IL) software for mean speed and percent time in closed and open arms.

[0161] IVIS imaging: Splenic B cells were isolated from mice homozygous for the CAG-luc-eGFP L2G85 transgene, which exhibits widespread expression of firefly luciferase and enhanced green fluorescence protein under the CAG promoter (Jackson Laboratories, Bar Harbor, ME). Approximately 5 million luciferase-expressing B cells in 5 μl PBS were injected into the left hemisphere of recipient WT C57Bl6 / J mice as described above. Mice were imaged on the day of surgery and at regular intervals thereafter for a total of 4 weeks using an IVIS Lumina II system (Caliper Life Sciences, Waltham, MA). 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 aqueous solution of D-luciferin (Regis Technologies, Inc., Morton Grove, Ill.) was injected subcutaneously near the injury site at least 6 min before imaging. Mice were imaged for 10 min, and identical 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), perfused transcardially 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: Processing of tissues 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 with PBS containing 5% bovine serum albumin, 5% fetal bovine serum, and 0.3% Triton X-100 for 1 hour at room temperature. Sections were then incubated overnight at 4° 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 primary antibodies when secondary antibodies were used for visualization. No nonspecific signals were detected in 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 injury volume: Sections were stained with hematoxylin, and high-resolution gross 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 This 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 to a maximum intensity projection process across all imaged channels. Background levels were determined by image smoothing using a 3 x 3 pixel moving average, followed by morphological opening using a structuring element of 10 pixel radius for each channel separately. Foreground objects were identified as the set of pixels with intensities above the corresponding background levels of >25 for GFAP immunolabeling and >50 for CD68 immunolabeling, respectively. For each marker analyzed, the relative area labeled was calculated as the percentage of pixels in 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 in the region of interest. All image analyses were performed by an experimenter blinded to the 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 found to pass (p > 0.2). Statistical significance of differences between experimental groups for behavioral tests performed in replicates, including wire grip assay, rotarod, MWM invisible and visible platforms, 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 MWM probe, Y-maze, forced swim, and histological comparisons, were assessed using one-way analysis of variance followed by Tukey's multiple comparison test. All descriptive statistics reported 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. In the rotarod assay, intraparenchymal treatment with B lymphocytes had a significant protective effect (Figure 15A). While all injury groups performed worse than sham-injured animals, injured mice treated with B cells performed better than injury groups treated with T cells or saline (p < 0.01 for groups), and their performance was not different from that of sham-injured mice treated with B cells. Interestingly, successive trials showed a sustained improvement in performance in the CCI + B cell group, as well as both sham-injured control groups, suggesting that procedural learning occurred in these groups. There were no significant differences between the CCI group treated with B cells and the sham-injured mice, except for trial 3, where sham-injured mice treated with B cells performed better (p < 0.01). In contrast, CCI mice that received intraparenchymal treatment with either saline or 2 million T cells at the time of injury showed minimal day-to-day improvement in performance and had 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 that received T cells and the CCI-injured groups that received saline.

[0168] In the wire grip test, all injured groups performed worse than sham-injured ones, but no group differences were observed between 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 in the invisible platform trial, indicating a robust effect of CCI (p < 0.001 for groups) with no differences observed among the sham-injured groups. Among the injured groups, there was a highly significant effect of treatment (p < 0.0001) with a significant treatment x trial interaction (p = 0.02) in B cell-treated vs saline-treated mice on trial 7 (p = 0.016) (Figure 16A). The rate of performance improvement over the seven invisible platform trials, assessed as the difference between trial 1 and trial 7 in the mean time to reach the platform, was significantly elevated in injured animals receiving B cells compared to saline controls (p < 0.01). No significant differences were observed among any of the groups in the visible platform trial (Figure 16B). In the probe trial, B cell-treated injured mice performed similarly to the sham-injured group (Fig. S16C). In contrast, CCI-injured mice receiving either T cells or saline performed worse than the sham-injured ones (p < 0.05). The swimming patterns of B cell-treated injured mice, similar to those of the sham-injured group, showed evidence of a spatial exploration strategy, whereas the exploration strategy was non-spatial in the T cell- and saline-treated injured groups (Fig. S16D).

[0170] In the Y-maze test (FIG. 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 mice treated with B cells. There were no significant differences between any of the injury and treatment groups 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 (FIG. 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 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 to animals receiving the same number of T cells (p < 0.05).

[0172] The sham-lesioned and lesioned groups did not differ from each other in the forced swim test, suggesting that the lesion paradigm employed in this study does not affect the depressive-like behavior measured in this assay (Figure 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 in association with B cell treatment had a neuropathological correlate, the brains of all animals that underwent behavioral assessment were harvested 35 days after injury for histological examination (Figure 18). Analysis of brain tissue damage in the CCI group showed cavitation of the injury area in all mice that underwent CCI, while the sham-injured group showed no brain tissue loss (Figure 18A). Quantitative analysis of the injury volume across all groups (Figure 18B) showed a significant effect of CCI, as expected (p < 0.0001). Brain tissue loss was significantly reduced in CCI mice treated with B cells compared to the saline-treated injury group (p < 0.001) or the T cell-treated injury group (p < 0.0001). There was no significant difference in injury volume between the T cell-treated CCI-injured group and the saline-treated CCI-injured group. Analysis of the distribution of the 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 mainly 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 to 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, respectively. Unbiased analysis of confocal images collected from a location proximal to the cavitation lesion formed at the site of the initial CCI injury showed a significant effect of B cell treatment delivered at the time of injury compared to saline-treated and T cell-treated controls (Figure 19). Glial scar formation, indicated by strong immunolabeling for GFAP and significant hypertrophy of astrocytes, was significantly reduced in animals treated with B cells, as indicated by reduced immunolabeling for the microglial activation marker CD68 (Figures 19E, 19F).

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

[0176] To investigate whether the transferred B cells could proliferate in situ, thereby having the potential to establish a longer-lived population in the brain parenchyma, coronal sections of the brain through the injection site were taken and immunolabeled for B cell and proliferation markers. Labeling of B cells ex vivo with toluidine blue prior to injection showed that after delivery, the cells were distributed only within a radius of approximately 1 mm around the injection site (Figure 21A). Confocal imaging confirmed that injected B cells remained mostly 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 day 0 and day 4 time points, while adjacent non-B cells showed high levels of Ki67 immunopositivity 4 days after injury in all animals examined (n = 4 animals per time point; Figures 21D, 21E). This finding was consistent with the fact that no B cells could be 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). At day 35, the original injection site is destroyed by cavitation in CCI-injured animals, but can be easily detected in sham-injured controls due to gliosis surrounding the injury caused by the needle (Figure 21F). By day 35, no B220-positive B cells were found at the injection site of the B cell-treated sham-injured ones (Figures 21F, 21G).

[0177] conclusion This study describes, to our knowledge, the 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 can significantly reduce learning and memory impairments and reduce the amount of brain tissue loss after injury. This result suggests a previously unknown protective effect of endogenous B cells in a brain contusion model.

[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 with changes in emotional behavior (ibid.). In this CCI injury paradigm, the impact force was applied directly to the cortex, so this region reliably and inevitably degenerates in all animals regardless of treatment condition, while differences between treatment conditions were observed in the compensation of subcortical structures, especially the hippocampus. The observed behavioral benefits of treatment with B cells also correlated with the functions supported by these structures, suggesting a link between the localization of the introduced lymphocytes, the compensation of tissues around the injection site, and functional neuroprotection. Supporting this hypothesis, in the present 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 in situ lifespan. The molecular mechanisms underlying the observed neuroprotective effects of B cells are not fully understood, but it is possible that they involve diffusible factors emanating from the cells, which overcome the strict localization of the applied cells and reach distal regions. This hypothesis is supported by the observation that in animals treated with B cells during CCI, injury volume was significantly reduced and potentially deleterious reactive phenomena, including astrogliosis and microglial activation, were significantly reduced at the injury site compared to controls.

[0180] Thus, B cells can be used as a therapeutic strategy for patients with brain contusion. Unlike any other existing cell-based therapy, B cells are easily available from peripheral blood or other blood bank products, which is a key advantage for developing a fast-acting off-the-shelf therapeutic. Indeed, a fast-acting, minimally manipulated autologous B cell therapy may have high applicability to clinical practice. This is especially true in cases of severe brain injury, where surgery is often performed to remove hematomas or pierced 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 damaged brain.

[0181] Unlike other cell types used in therapy, such as stem cells, B lymphocytes are mature, terminally differentiated cells with a limited natural life span of 5-6 weeks in vivo. The transplanted cells are expected to disappear in a shorter time after application in a microenvironment disturbed by cerebral contusion. This is beneficial because the longer survival of transplanted cells may raise significant safety concerns, especially considering that the microenvironment of the central nervous system 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, reaching undetectable levels by day 17. For CCI, the 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 cell proliferation in situ, 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 proliferation of nearby cells, no proliferation of B cells 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 shows the SOD1 mouse model of ALS. G93A Illustrates the safety and efficacy of intravenous (iv) administration of B cells in mice.

[0184] Male and female transgenic SOD1 G93A Mice were treated with 5 x 10 immunizations on a 10-week schedule 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 once a week with saline containing 100 B cells (or saline control). G93A We found that treatment with B cells delayed the onset of symptoms (p < 0.0001), as shown by the attainment of peak body weight and significantly extended survival time (p < 0.05) in mice. Treatment with B cells was associated with a significant reduction in the relative number of damaged / degenerated motor neurons in the lumbar spinal cord at the endpoint (p < 0.05), even though the total number of motor neurons was not altered by treatment. Treatment with B cells was not associated with any observable adverse effects (behavioral and phenotypic) in non-transgenic control wild-type littermates that received the same treatment, and the controls continued to gain weight over the duration of the repeated treatment. Taken together, these results demonstrate that B cell therapy may provide a viable method to reduce neuroinflammation in ALS.

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

[0186] Test Plan To evaluate the efficacy of B cell immunotherapy in ALS, we used 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 with transgenic expression of the G93A mutant form of human SOD1 exhibit a phenotype similar to ALS in humans. The mice progressively become paralyzed in one or both limbs, 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 mice die at approximately 20-24 weeks of age. Thus, this model shows a rapid and aggressive progression of the disease. The model is highly stable and reproducible, allowing the evaluation of therapeutic options for this neurodegenerative disorder.

[0187] A total of 15-17 females and males (per 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 weekly intravenous infusions of B cells (or saline control) delivered by retro-orbital injection (Figure 22). For feasibility, the study was performed 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 line (often referred to as G1H) has been reported by Jackson Laboratory to carry a high copy number of the SOD1 transgene. All animals are individually genotyped by Jackson Laboratory prior to shipping, and only heterozygous animals carrying a high copy number of the SOD1 transgene (top third of the distribution) are sold. Control animals are littermates without the SOD1 transgene (non-carriers).

[0191] Donor animals for B cell isolation were C57BL / 6J mice, 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] Isolation of B cells: All cell isolation procedures were performed under aseptic 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 that shared half the genetic background of the recipient (as well as their siblings). The spleens were dissociated into splenocyte suspensions, and a commercial kit (Miltenyi B Cell Isolation Kit, mouse; 130-095-873, Miltenyi Biotec) was used to isolate all B cells by negative immunomagnetic selection, as in our previously published protocol (DeKosky et al. (2013) Nat Rev Neurol 9, 192-200; Stocchetti et al. (2017) Lancet Neurol 16, 452-464). The resulting cells were >98% CD19+ B cells and typically >85-90% CD19+ / B220+ / IgM+ / IgD+, with approximately 5% CD138+ plasma cells, and <1% other cell populations, as confirmed by flow cytometry analysis after isolation (DeKosky et al. (2013) Nat Rev Neurol 9, 192-200). This was designated 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) for a total of 10 times, delivered by retro-orbital injection. Animals were anesthetized with 3% isoflurane mixed 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 method of administration was chosen because it has a much lower risk of failure compared to tail vein injections for cell transplantation, especially when repeated administration is involved.

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

[0196] Efficacy assessment: Body weight and neurological scores (Neuroscore) were assessed twice weekly by an experimenter blinded to treatment conditions and were 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 (presymptomatic) When the mouse is suspended by the tail, the hind limbs show 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 (early symptoms) When mice are suspended by the tail, the hindlimbs show abnormal splay, i.e., the hindlimbs are folded or partially folded toward the lateral midline, or While suspended by the tail, hind limb tremors were observed; 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 (although joint movement is still evident). When the mouse is allowed to walk, the hindlimbs are used for forward movement, but the toes curl downward or point upward at least twice during the 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, they develop complete tonic paralysis of the hind limbs. 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 the mouse is hung by the tail, a tonic complete paralysis of the hind limbs is observed. 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 will 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 of maximum 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 cord was harvested from all animals, preserved in fixative, and processed for histological analysis. To visualize motor neurons in the anterior horn, 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, which were counted in 3-6 regions of interest of at least 500 x 500 μm, randomly collected from 2-3 longitudinal sections per animal. We also quantified healthy motor neurons (large, rounded cell bodies; nuclei with single, distinct nucleoli; presence of Nissl substance; see FIG. 27A) separately from damaged / degenerated motor neurons (shrunken cell bodies, high levels of hyperbasophilia, strong clumping of nuclear chromatin, pyknotic nuclei; see FIG. 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, malaise, piloerection, hunchback 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, independent of treatment with B cells (Figure 23). Moreover, in transgenic SOD1-G93A animals, no such adverse effects attributable to cell infusion treatment were observed in the period prior to the onset of symptoms (days 72-90 of treatment).

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

[0206] Peak body weight (i.e., the onset of symptoms, which correlates with weight loss) was delayed on average by approximately 28 days in naive B cell treatment conditions 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 FIG. 25). For this analysis, disease onset was defined as the time point at which an animal received a neurological score of 1 on three consecutive assessments and did not subsequently experience a decrease in neurological score. A progressive increase in neurological score over time was observed in all animals, although treatment with B cells was associated with a slower rate of progression (FIG. 25).

[0208] c. Survival analysis (shown in FIG. 26). As mentioned above, animals that were completely paralyzed and unable to return to their upright position within 10 seconds when 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; FIG. 26).

[0209] d. Histological analysis of spinal motor neurons (shown in FIG. 27). All histological sample processing and examination was performed by an experimenter blinded to the treatment conditions. As expected, a highly significant reduction in the total number of motor neurons in the anterior horn of the lumbar spinal cord was observed in transgenic SOD1 animals compared to WT controls. 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, but the percentage of these motor neurons that were dead or dying 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 once a week to mice 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 noted 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 can be administered to a subject with Parkinson's disease, such as any of the compositions described herein. 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 of skill 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 also be monitored by determining the levels of molecular markers of disease progression associated with neurodegenerative diseases, such as, for example, 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), reduced α-synuclein, and / or reduced 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 decrease in the rate of disease progression (e.g., 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 Immunologic 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 efficacy according to methods known to those of skill 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 mentioned in this specification (including patents and patent applications, including U.S. Provisional Patent Application Nos. 62 / 795,629, 62 / 837,765, and 62 / 965,032) are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

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

Claims

1. A pharmaceutical composition for treating a neurodegenerative disease in a subject in need thereof comprising isolated mature naive B cells, wherein a therapeutically effective amount of the isolated mature naive B cells is administered to the subject.

2. 2. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS).

3. 13. The pharmaceutical composition of claim 1, used in combination with a second therapeutic composition.

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

5. The pharmaceutical composition of claim 1, wherein the mature naive B cells are allogeneic B cells, autologous B cells, or xenogeneic B cells.

6. The method of claim 1, wherein the mature naive B cells are formulated for local administration; or The mature naive B cells are formulated to be administered systemically.

2. The pharmaceutical composition of claim 1.

7. The pharmaceutical composition of claim 1, wherein the mature naive B cells are formulated for intravenous, intraarterial, subcutaneous, intrathecal, or intraparenchymal administration.

8. The pharmaceutical composition of claim 1, wherein the mature naive 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.

9. The therapeutically effective amount of said mature naive B cells 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 The pharmaceutical composition of claim 1, comprising the B cells.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject is a human.

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