Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/428—Thiazoles condensed with carbocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
  • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/13—B-cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/414—Nervous system antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

• Degenerative diseases are medical conditions causing cells, tissues, or organs to deteriorate.
• Huntington’s disease are among many neurodegenerative diseases that involve degeneration of regions of the central nervous system. Traumatic brain injury (TBI) is also an example of a disorder which might increase the risk of a developing a degenerative brain disease such as PD or AD. Rheumatoid arthritis and osteoarthritis are still other examples of degenerative diseases involving inflammation. For most of these, there is no effective treatment available. Treatments for some of these diseases or disorders are under investigation. Proposed treatments, however, are very often expensive and involve significant risks and complications. There is accordingly a need for better approaches for treatment of degenerative diseases including neurodegenerative disease such as ALS and TBI.
• compositions including B cells (e.g., isolated, purified, or modified B cells or a combination thereof) and uses thereof for the treatment of disease (e.g., neurodegenerative diseases, traumatic brain injury (TBI), spinal cord injury (SCI), and inflammatory and immune diseases as described herein).
• disease e.g., neurodegenerative diseases, traumatic brain injury (TBI), spinal cord injury (SCI), and inflammatory and immune diseases as described herein.
• 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.
• the neurodegenerative disease is ALS (e.g., sporadic or familial ALS). In yet another preferred embodiment, the neurodegenerative disease is Parkinson’s Disease.
• the invention features a method of treating a subject having ALS, including administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount sufficient to reduce or ameliorate one or more symptoms of ALS.
• the invention features a method of treating a subject having ALS who exhibits one or more symptoms of ALS, including administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount that results in reduction or amelioration of one or more of the symptoms of ALS; monitoring one or more of the symptoms in the subject; and administering a second dose of B cells when the one or more symptoms begins to worsen.
• the one or more symptoms of ALS include difficulty lifting the front part 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; hand weakness; hand clumsiness; muscle cramps; fasciculations (muscle twitches) in one or both arms, in one or both legs, in one or both shoulders, or of the tongue; muscle cramps; spasticity (tight and stiff muscles); and difficulty chewing or swallowing (dysphagia), difficulty speaking or forming words (dysarthria), and difficulty breathing (dyspnea).
• the invention features a method 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., determining the level of a molecular marker of neurodegenerative diseases progression before and after treatment with a therapeutically effective amount of B cells).
• the level of a molecular marker may be determined according to methods known to those of skill in the art.
• the level of a molecular marker may be determined from a sample from the treated subject, e.g., from a sample of blood plasma or cerebrospinal fluid (CSF) of a treated subject.
• a sample from the treated subject e.g., from a sample of blood plasma or cerebrospinal fluid (CSF) of a treated subject.
• CSF cerebrospinal fluid
• Exemplary markers include T-tau (total tau), P-tau (hyperphosphorylated tau), Ab42 (amyloid beta 42), the ratio of Ab42/Ab40, YKL-40 (Chitinase-3-like protein 1 ), VLP-1 (visinin-like protein 1 ), NFL (neurofilament light), pNFH (phosphorylated neurofilament heavy subunit), Ng (neurogranin) and UCH-L1 (ubiquitin C-terminal hydrolase), TDP-43 (TAR DNA-binding protein 43), decreased a-synuclein and/or decreased levels of 3,4-dihydroxyphenylacetate (see, e.g., Robey and Panegyres. Cerebrospinal fluid biomarkers in neurodegenerative disorders. Future Neurol. 14(1 ). (2019), which is incorporated by reference in its entirety).
• 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.
• the inflammatory or immune disease is selected from cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis.
• cardiovascular disease e.g., coronary artery disease or aortic stenosis
• keratoconus keratoglobus
• osteoarthritis e.g., osteoporosis
• pulmonary arterial hypertension e.g., retinitis pigmentosa
• retinitis pigmentosa e.g., rheumatoid arthritis
• the invention features a method of treating a subject having a central nervous system (CNS) injury, including administering to the subject a therapeutically effective amount of isolated B cells.
• CNS injury is a traumatic brain injury (TBI) or a spinal cord injury (SCI).
• TBI traumatic brain injury
• SCI spinal cord injury
• the CNS injury includes both TBI and SCI.
• the invention features a method of treating a subject having a traumatic brain injury (TBI), including administering to the subject a therapeutically effective amount of isolated B cells.
• TBI traumatic brain injury
• the invention features a method of treating a subject having a spinal cord injury (SCI), including administering to the subject a therapeutically effective amount of isolated B cells.
• SCI spinal cord injury
• the TBI is damage to the brain resulting from external mechanical force.
• the SCI involves damage to the spinal cord resulting from external mechanical force.
• the TBI and/or SCI results from a head injury or a cerebral contusion (e g., resulting from a fall, a firearm wound, a sports accident, a construction accident, a vehicle accident, or an injury which penetrates the skull or brain of the subject).
• the subject having TBI and/or SCI suffers from one or more of a number of physical, cognitive, social, emotional and/or behavioral disorders. TBI and SCI may be co-occurring and may result from the same injury.
• the invention features a method of treating a subject having TBI who exhibits one or more symptoms of TBI, including administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount that results in reduction or amelioration of one or more of the symptoms of TBI; monitoring one or more of the symptoms in the subject; and administering a second dose of B cells when the one or more symptoms begins to worsen.
• the invention features a method of treating a subject having SCI (who exhibits one or more symptoms of SCI) including administering to the subject a therapeutically effective amount of B cells, wherein the therapeutically effective amount is an amount that results in reduction or amelioration of one or more of the symptoms of SCI; monitoring one or more of the symptoms in the subject; and administering a second dose of B cells when the one or more symptoms begins to worsen.
• the one or more symptoms of TBI and/or SCI include an inability to recall the traumatic event, confusion, difficulty learning and remembering new information, affective and executive dysfunction, trouble speaking coherently, unsteadiness, lack of coordination, and problems with vision or hearing; cognitive problems (e.g., amnesia, inability to speak or understand language, mental confusion, difficulty concentrating, difficulty thinking and understanding, inability to create new memories, or inability to recognize common things); behavioral problems (e.g., abnormal laughing and crying, aggression, impulsivity, irritability, lack of restraint (impulsiveness), or persistent repetition of words or actions); mood problems (e.g., anger, anxiety, apathy, or loneliness); whole body problems (e.g., blackout, dizziness, fainting, or fatigue); eye problems (e.g., dilated pupil, raccoon eyes, or unequal pupils); muscular problems (e.g., instability or stiff muscles); gastrointestinal problems (e.g., nausea or vomiting); speech problems (e.g.,
• the method includes monitoring the one or more symptoms of TBI and/or SCI between 1 and 7 days post-administration, between 7 and 28 days post administration, between 1 and 28 weeks post-administration, between 1 and 2 months post-administration, between 2 and 6 months post-administration, between 2 and 9 months post-administration, or between 6 months and a year or more post-administration.
• the invention features a method for monitoring the responsiveness of a patient having TBI and/or SCI to treatment with a therapeutically effective amount of isolated B cells by determining the level of a molecular marker of disease progression (e.g., determining the level of a molecular marker of neurodegenerative diseases progression before and after treatment with a therapeutically effective amount of B cells).
• the level of a molecular marker may be determined according to methods known to those of skill in the art.
• the level of a molecular marker of TBI and/or SCI may be determined from a sample for the treated subject, e.g., from a sample of blood plasma or cerebrospinal fluid (CSF) of a treated subject.
• exemplary molecular markers of the progression of TBI described herein include, without limitation, protein biomarkers for neuronal cell body injury (UCH-L1 , NSE), astroglial injury (GFAP,
• allogeneic B cells are administered.
• the allogeneic B cells are haploidentical allogeneic B cells, H LA-matched allogenic B cells, or genetically- modified B cells (e.g., B cells that have been genetically modified, for example by CRISPR, to reduce the immunogenicity of the B cell).
• autologous B cells are administered.
• xenogeneic B cells are administered.
• the method includes administering a second therapeutic composition.
• the second therapeutic composition is Edaravone, Riluzole, or an immunomodulatory composition (e.g., an anti-CD14 antibody, an anti-CDL40 antibody, or a composition including T reg cells).
• an immunomodulatory composition e.g., an anti-CD14 antibody, an anti-CDL40 antibody, or a composition including T reg cells.
• the second therapeutic composition is an antibiotic or a corticosteroid (e.g., prednisone).
• the B cells are mature naive B cells.
• the B cells are stimulated ex vivo.
• the B cells are stimulated ex vivo with a Toll-like receptor (TLR) agonist.
• TLR Toll-like receptor
• the TLR agonist is an endogenous ligand selected from a heat shock protein, a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta- defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A, low density lipoprotein (LDL), pancreatic elastase, a polysaccharide fragment of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and a CpG chromatin-lgG complex.
• a heat shock protein a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta- defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A,
• the TLR agonist is an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a glycoinositolphospholipid, a glycolipid, a GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, a mannuronic acid polymer, a bacterial outer membrane porin, zymosan, double- stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, an imidazoquinolines (e.g., imiquimod, resiquimod, loxoribine, bropirimine), an antiviral compound, an unmethylated CpG
• GPI glycosylphosphatidy
• the 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-1 b, IL-2, IL-4, IL- 6, TNFa, or IFNg).
• an immunomodulatory cytokine e.g., a pro-inflammatory cytokine, such as a pro-inflammatory cytokine selected from IL-1 b, IL-2, IL-4, IL- 6, TNFa, or IFNg.
• the B cells are B reg cells.
• the B reg cells express immunomodulatory cytokine IL-10.
• the B reg cells further express one or more additional immunomodulatory cytokines selected from IL-2, IL-4, IL-6, IL-35, TNF-a, TGFb, PD-L1 FasL, and TIM1.
• the B reg cells express one or more cell surface markers selected from B220, CD1d, CDS, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• the B reg cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD.
• the B reg cells express CD25 and CD71. In some embodiments, the B reg cells do not express CD73.
• the B reg cells include at least 80% (e.g., at least 85%, 90%, 95%, or 98%) CD19+ B cells. In some embodiments, the B reg cells include less than 10% (e.g., less than 5%) CD138+ plasma B cells.
• the B cells are neuroprotective, anti-inflammatory, and/or
• 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 cranial pressure (ICP) monitoring catheter.
• ICP intracranial cranial pressure
• the B cells are administered once daily, once weekly, twice weekly, once every 14 days, once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, or once yearly.
• the B cells are administered at least twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times.
• the therapeutically effective amount of B cells includes at least 0.5 X 10 6 B cells per administration, 0.5 X 10 7 B cells per administration, 1 x 10 8 B cells per administration, at least 2 x 10 8 B cells per administration, or at least 1 x 10 9 B cells per administration. In some embodiments, the therapeutically effective amount of B cells includes 1 x 10 8 B cells to 1 x 10 9 B cells per administration, 1 x 10 8 B cells to 5 x 10 8 B per administration, or 2 x 10 8 B cells to 4 x 10 8 B cells per administration.
• the pharmaceutically-acceptable excipient is an aqueous solution (e.g., a saline solution).
• the invention features, a method of treating a disease or condition in a subject in need thereof, the method including administering to the subject a pharmaceutical composition including a modified B cell and one or more phamnaceutically-acceptable excipients, wherein the modified B cell has been stimulated ex vivo with a Toll-like receptor (TLR) agonist and/or an immunomodulatory cytokine.
• TLR Toll-like receptor
• 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
• the immune or inflammatory disease is selected from cystic fibrosis, cardiovascular disease (e.g., coronary artery disease or aortic stenosis), keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis.
• the modified B cell is an allogeneic B cell.
• the allogeneic B cells is a haploidentical allogeneic B cell, an H LA-matched allogenic B cell, or a genetically- modified B cell (e.g., a B cell that has been genetically modified, for example by CRISPR, to reduce the immunogenicity of the B cell).
• modified B cell is an autologous B cell.
• modified B cell is a xenogeneic B cell.
• the invention features, a method of producing a modified B cell, the method including:
• TLR Toll-like receptor
• step i) further includes isolating a CD19+ mature naive B cell.
• isolating the CD19+ mature naive B cell is performed by immunoprecipitation with a CD19 antibody or antigen-binding fragment thereof.
• the CD19 antibody or antigen binding fragment thereof remains bound to the modified the modified B cell.
• the TLR agonist is an endogenous ligand selected from a heat shock protein, a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta- defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A, low density lipoprotein (LDL), pancreatic elastase, a polysaccharide fragment of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and a CpG chromatin-lgG complex.
• a heat shock protein a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta- defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A,
• the TLR agonist is an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a
• a triacylated lipopeptide selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a
• GPI glycosylphosphatidylinositol
• glycoinositolphospholipids a glycolipids, a GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, a mannuronic acid polymer, a bacterial outer membrane porin, zymosan, double- stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, an imidazoquinolines (e.g., imiquimod, resiquimod, loxoribine, bropirimine), an antiviral compound, an unmethylated CpG
• the immunomodulatory cytokine is a pro-inflammatory cytokine (e.g., a pro-inflammatory cytokine selected from IL-1 b, IL-2, IL-4, IL-6, TNFa, or IFNg).
• a pro-inflammatory cytokine selected from IL-1 b, IL-2, IL-4, IL-6, TNFa, or IFNg.
• the modified B cell is a B reg cell.
• the B reg cell expresses immunomodulatory cytokine IL-10.
• the B reg cell further expresses one or more additional immunomodulatory cytokines selected from IL-2, IL-4, IL-6, IL-35, TNF-a, TGFb, PD- L1 FasL, and TIM1.
• the B reg cell expresses one or more cell surface markers selected from B220, CD1d, CDS, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• the B reg cell expresses B220, CD19, CD20, CD24, CD138, IgM, and IgD.
• the B reg cell expresses CD25 and CD71. In some embodiments, the B reg cell does not express CD73.
• the modified B cell is neuroprotective, anti-inflammatory, and/or immunomodulatory.
• B lymphocytes are mature, terminally-differentiated cells, with a naturally limited lifespan of 5-6 weeks in vivo. Their application in a neurodegenerative setting as well as in the disrupted microenvironment of a brain contusion is expected to lead to elimination of the transplanted cells after even less time. This is advantageous because longer survival of transplanted cells could represent a significant safety concern, particularly considering that the microenvironment of the central nervous system contains a number of B cell-trophic factors.
• 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 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, Lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.
• the TLR agonist is an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a glycoinositolphospholipids, a glycolipids, a GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, a mannuronic acid polymer, a bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an
• B reg cells further express one or more additional immunomodulatory cytokines selected from IL-2, IL-4, IL-6, IL-35, TNF-a, TGFb, PD-L1 FasL, and TIM1.
• B reg cells express one or more cell surface markers selected from B220, CD1d, CDS, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• B reg cells express B220, CD19, CD20, CD24, CD138, IgM, and IgD.
• TBI traumatic brain injury
• the TLR agonist is an endogenous ligand selected from a heat shock protein, a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A, low density lipoprotein (LDL), pancreatic elastase, a polysaccharide fragment of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and a CpG chromatin-lgG complex.
• a heat shock protein a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A,
• the TLR agonist is an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a glycoinositolphospholipids, a glycolipids, a GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, a mannuronic acid polymer, a bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an
• imidazoquinolines an antiviral compound, an unmethylated CpG oligodeoxynucleotide, and profilin.
• B reg cells further express one or more additional immunomodulatory cytokines selected from IL-2, IL-4, IL-6, IL-35, TNF-a, TGFb, PD-L1 FasL, and TIM1.
• B reg cells express one or more cell surface markers selected from B220, CD1d, CDS, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• a pharmaceutical composition comprising a modified B cell and one or more
• TLR Toll-like receptor
• TLR agonist is an endogenous ligand selected from a heat shock protein, a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A, low density lipoprotein (LDL), pancreatic elastase, a polysaccharide fragment of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and a CpG chromatin-lgG complex. 70.
• the TLR agonist is an endogenous ligand selected from a heat shock protein, a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, H
• the TLR agonist is an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein, lipoarabinomannan, an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a glycoinositolphospholipids, a glycolipids, a GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, a mannuronic acid polymer, a bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, an imidazoquinolines, an antiviral compound, an unmethylated CpG oligodeoxynucleotide, and profilin.
• GPI glycosylphosphatidylinositol
• B reg cell expresses one or more cell surface markers selected from B220, CD1d, CDS, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• pharmaceutically-acceptable excipient is an aqueous solution.
• a method of treating a disease or condition in a subject in need thereof comprising administering to the subject the pharmaceutical composition of any one of paragraphs 68-84.
• 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, Lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.
• ALS amyotrophic lateral sclerosis
• Parkinson’s disease Alzheimer’s disease
• CTE chronic traumatic encephalopathy
• Huntington’s disease infantile neuroaxonal dystrophy
• progressive supranuclear palsy progressive supranuclear palsy
• Lewy body dementia Spinocerebellar ataxia
• spinal muscular atrophy and motor neuron disease.
• the disease or condition is selected from cystic fibrosis, cardiovascular disease, keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis.
• a method of producing a modified B cell comprising:
• TLR Toll-like receptor
• the TLR agonist is an endogenous ligand selected from a heat shock protein, a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A, low density lipoprotein (LDL), pancreatic elastase, a polysaccharide fragment of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and a CpG chromatin-lgG complex.
• a heat shock protein a necrotic cell or a fragment thereof, an oxygen radical, a urate crystal, an mRNA, a beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A,
• TLR agonist is an exogenous ligand selected from Pam3CSK4, a triacylated lipopeptide, a glycosylphosphatidylinositol (GPI)-anchored protein,
• lipoarabinomannan an outer surface lipoprotein, a lipopolysaccharide, a cytomegalovirus envelope protein, a glycoinositolphospholipids, a glycolipids, a GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, a mannuronic acid polymer, a bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, an
• imidazoquinolines an antiviral compound, an unmethylated CpG oligodeoxynucleotide, and profilin.
• pro-inflammatory cytokine is selected from IL-1 b, IL-2, IL-4, IL-6, TNFa, or IFNg.
• the B reg cell further expresses one or more additional immunomodulatory cytokines selected from IL-2, IL-4, IL-6, IL-35, TNF-a, TGFb, PD-L1 FasL, and TIM1.
• B reg cell expresses one or more cell surface markers selected from B220, CD1d, CD5, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• FIG.1A - FIG. 1 B shows B cell application induces complex changes in the molecular microenvironment of a wound.
• FIG. 1A is a schematic representation of the average duration of the major stages of wound healing in the wild-type murine wound model.
• FIG. 1 B shows heatmaps summarizing the expression dynamics over time in proteins with significantly altered expression in response to B cell application.
• Heatmaps show fold change expression after B cell treatment at 0, 1 , 4, 10 d post-injury. Red - up-regulation; Green - down- regulation. Particularly notable were the down-regulation of multiple proteins associated with inflammation and inflammatory cells at 4 days post-injury and the substantial up-regulation of proteins associated with cell proliferation, protection from apoptosis (cell death) and oxidative stress, and tissue remodeling (formation of hair follicles and muscle) at 4-10 days post injury.
• FIG. 2A - FIG. 2H show average expression of proteins by functional family over time in wounds treated with saline (controls, regular wound healing) or after B cell treatment.
• This analysis illustrates the overall effect of B cells as a homeostatic agent, rather than an inducer or inhibitor of protein expression.
• B cell application was associated with the maintenance of steady levels of expression of proteins that normally either decline or increase during the course of injury and healing, significantly reducing the inflammatory peak observed in normal healing, preventing the reduction in anti-apoptotic factors (arrows) and oxidative stress protectants and increasing proliferation (FIG.2A - FIG.2B), reducing the decline in anti-oxidative stress protectants and in cell proliferation, and maintaining cell migration at lower levels (FIG. 2C - FIG.
• FIG. 2D maintaining steady levels of proteins associated with remodeling and secondary skin structures
• FIG. 2E - FIG. 2F maintaining steady levels of proteins associated with remodeling and secondary skin structures
• FIG. 2G - FIG. 2H maintaining steady levels of proteins associated with remodeling and secondary skin structures
• FIG. 4 shows the gating strategy and analysis of B cell-treated and control wound cell suspensions via flow cytometry.
• Live cells were gated into 3 main categories: B cells (CD19+/B220+ lymphocytes), non-B cell leukocytes (CD140a-/B220- leukocytes) which included a mix of neutrophils, monocytes and macrophages, dendritic cells and T cells, and fibroblasts (CD140a+/B220-). These cell categories were evaluated for markers of activation and cytokine production.
• FIG. 5 shows the dynamics of activation markers and key cytokines in B cells retrieved from the wound bed after defined exposure intervals to the wound microenvironment.
• B cells were exposed in vivo to the wound niche or injected under uninjured skin (control equivalent location). Control B cells maintained on ice immediately after isolation for the same time duration are shown for comparison. After time intervals including 18 hours, 2 days, 4 days, and 10 days, the wounds were treated with Brefeldin A for 4 hours to induce retention of cytokines within cells. B cells were then retrieved by excising and dissociating the tissue, and further characterized by flow cytometry both for surface markers and intracellular cytokines.
• B cells exposed to the wound microenvironment transiently upregulate multiple immunomodulatory cytokines, peaking at 2 days post-application.
• Some immunomodulatory cytokines, including TGFb and IL-6 remain elevated at 4 days, and IL-10 up to 10 days.
• N 3-6 animals/group.
• FIG. 6 is a heatmap summary of the average values for each marker in B cells exposed to the wound microenvironment, subcutaneous control, or maintained on ice (no exposure).
• FIG. 7 shows the dynamics of activation markers and key cytokines in the aggregate infiltrating non-B cell leukocytes in the wound.
• infiltrating leukocytes produced more anti-inflammatory cytokines IL-10, TGFb, and IL-35, and less pro-inflammatory TNFa and IL-2 when B cells were present in the wound. This impact was most pronounced at 4 days post injury and B cell application and persisted up to 10 days.
• N 3-6 animals/group.
• FIG. 8 is a heatmap summary of the average values for each marker in infiltrating non-B cell leukocytes in the wound microenvironment, illustrating the pattern of increased anti-inflammatory cytokine (IL-10 and TGFb) production in the presence of B cells.
• IL-10 and TGFb anti-inflammatory cytokine
• FIG. 9 shows the dynamics of activation markers and key cytokines in the CD140a+ fibroblast population of the wound and subcutaneous tissue. Fibroblasts in the wound produced significantly more IL-10 and TGFb at 10 days post-injury when wounds were exposed to B cells. Moreover, wound fibroblasts produce less of the pro-inflammatory cytokine TNFa when B cells were applied, both at 4 days and 10 days post-injury.
• FIG. 11 shows functional TLR signaling as well as IL-10 production are necessary components of the regenerative function of exogenous B cells in wound healing.
• FIG. 12 shows an unsupervised hierarchical cluster analysis of identified proteins expressed in skin wound samples. Only identified proteins that were found consistently present across all samples were included in the analysis.
• A Hierarchical clustering using complete linkage of 3809 proteins (rows) consistently expressed in all animals in both B cell and saline treated wounds at 4 different time points after injury (columns). The pseudocolor scale depicts normalized, log-transformed fold change expression values for each protein. The dendrogram shows 15 protein clusters derived from this analysis, with the color of each cell in (A) mapped to the mean expression value of the cluster at the respective time points. Proteins cluster by their pattern of expression over time.
• B Heatmap of hierarchical cluster from (A) showing all 3809 proteins.
• FIG. 13 shows the distribution of significantly altered proteins in response to B cell treatment at each assessed time point during wound healing.
• FIG. 14 shows experimental paradigm for assessing 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 above the left parieto-temporal cortex, and the bone flap was removed.
• a single infusion of 2x10 6 B cells was delivered intraparenchymally into the ipsi lateral hemisphere just prior to injury. Mice were then subjected to CCI or sham injury. After recovery, motor function, motor and spatial learning and memory performance, anxiety, and depression-like behavior were assessed using multiple assays.
• the animals were euthanized, and the brains were collected for evaluation of total lesion volume.
• FIG. 15 shows effect of acute B cell treatment on vestibulo-motor function and striatal learning.
• Rotarod assessment showed a significant protective effect of B cells administered at the time of CCI. Notably, the latency to fall over repeated trials increased in B cell treated mice as well as in sham- lesioned animals, suggesting a motor learning component. No such improvement was observed in controls treated with T cells or saline. After the second trial, no significant difference was observed between CCI injured mice that received B cell treatment and sham-lesioned B cell-treated animals.
• FIG. 16 shows effect of a single acute B cell application on learning and memory.
• A-D Morris water maze assessment. Learning curves showed a significant improvement in CCI mice treated with B cells over saline-treated CCI animals (p ⁇ 0.05). No significant difference was observed between B cell treated injured animals and either of the sham-lesioned conditions after the third trial (p >0.98) (A). Visible platform trials showed no difference between the treatment conditions with or without injury (B).
• C The probe trial showed that the B cell -treated CCI injured animals spent above-chance time in the target quadrant, not significantly different from sham-lesioned mice. By contrast, control CCI injured mice treated with either T cells or saline only spent chance-level time exploring the target quadrant and differed significantly from the sham-lesioned animals (p ⁇ 0.05). Dashed line indicates chance level.
• D Morris water maze assessment. Learning curves showed a significant improvement in CCI mice treated with B cells over saline-treated CCI animals
• FIG. 17 shows effect of B cell treatment on anxiety and depression-like behavior after CCI.
• A Elevated plus maze assay of anxiety-like behavior. No overall significant differences were observed between treatment groups with the exception of a modest difference in the time spent in the closed arm between CCI injured mice that received B cells versus animals that received equal numbers of T cells at the time of injury (*p ⁇ 0.05).
• B Forced swim assay of depressive-like behavior. No effect of lesion or treatment was observed in this assay. All data are shown as mean ⁇ SEM. * p ⁇ 0.05.
• FIG. 18 shows effect of B cell treatment on histological outcome after CCI.
• A Representative coronal sections through the lesion site at day 35 post injury. In B cell-treated animals a portion of the hippocampus was often spared in the lesioned hemisphere (arrow). Sections shown are located approximately -2.2 mm from bregma.
• B The total volume of the brain lesion in mice treated with B cells was significantly reduced by 40-60% at 35 days post-TBI as compared to saline and T cell controls.
• C Lesion areas in transverse brain sections along the rostro-caudal axis of the brain. Results illustrate a consistently reduced lesion size in lesioned brains that received B cell treatment.
• FIG. 20 shows B cell survival and persistence in the brain.
• A Representative example of intravital imaging of a WT C57BI6/J mouse at multiple time points after CCI and intraparenchymal application of 5 x 10 6 B luc cells.
• FIG. 21 shows B cell localization at the injury site after CCI.
• the pre-labeled B cells can be visualized at the injury site.
• the black square indicates the area imaged in B.
• B Confocal microscopy image of a coronal section through the lesion site showing B220+ B cells clustered at the injection site (arrow). No cell proliferation, indicated by KI67 immunolabeling, was observed immediately after injury.
• C Four days after B cell injection and CCI, the labeled B cells can still be observed at the injury site, although the intensity of the vital stain coloration had diminished by this time point compared to immediately after injection.
• the black square indicates the area imaged in D.
• D Confocal image of a coronal section through the injection site four days post injury and B cell administration.
• the B220+ B cells can still be found in large numbers clustered at the injury site. Abundant cell proliferation can be observed throughout the region, however no co-staining of B220 and KI67 was observed.
• E Magnified view of the area boxed in D.
• F In sham-lesioned animals, the needle track through the cortex can still be found 35 days post-treatment outlined by astroglial scarring. No B220+ B cells can be observed at this time point at the original site of injection.
• G High-magnification confocal image of the area boxed in F. In all confocal images, cell nuclei are counterstained with DARI n - 4 animals per time point.
• FIG. 22 shows an overview of experimental design. Weight and Neuroscore assessments were performed twice weekly, at the same time of day, by an experimenter who was blinded to the treatment conditions.
• FIG. 23 shows normalized weight (percentage of value at day 76 for each individual animal) over time in the B cell and saline treatment groups, measured twice weekly.
• the graph shows a composite measure of normalized weight and survival in which individuals that died received weight values of 0.
• the results also illustrate a delay in the decline for transgenic SOD1 animals that received B cells (arrow).
• N 32 per treatment condition.
• FIG. 24A and FIG. 24B show an analysis of peak weight in SOD1-G93A animals.
• Statistics: A: Gehan-Breslow-Wilcoxon test; B: unpaired t-test. N 32 animals per group.
• FIG. 25 shows neuroscore values over time.
• FIG. 27 shows end-point motor neuron evaluation in the lumbar spinal cord.
• A B: Lumbar spinal cord sections were stained with H&E and all motor neurons (large cell bodies, at least one nucleolus) as well as impaired, abnormal neurons showing morphological characteristics of injury/degeneration (arrows) were counted by experimenters blinded to the treatments.
• C total numbers of motor neurons were significantly reduced in transgenic animals but did not differ with treatment within this group.
• D When the percentage of degenerating, pyknotic motor neurons was specifically analyzed, a significant benefit of B cell treatment became apparent.
• Statistics (left): two-way ANOVA; (right): unpaired t-test.
• N 19-24 animals per group. Note that collection of tissue samples was not possible in all tested animals.
• neurodegenerative disease refers to a neurologic disease, disorder, or condition characterized by the progressive loss of structure or function of neurons, including death of neurons, e.g., in the central nervous system (CNS). Many similarities appear that relate these diseases to one another on a sub-cellular level. Moreover, there are many parallels between different neurodegenerative disorders including atypical protein assemblies as well as induced cell death.
• Parkinson’s disease Alzheimer’s disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia, Huntington’s disease, infantile neuroaxonal dystrophy, progressive supranuclear palsy, Lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.
• CTE chronic traumatic encephalopathy
• Huntington s disease, infantile neuroaxonal dystrophy, progressive supranuclear palsy, Lewy body dementia, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron disease.
• TBI traumatic brain injury
• TBI may further be 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 (all-spectrin breakdown products), axonal injury (NF proteins), white matter injury (MBP), post-injury neurodegeneration (total Tau and phospho-Tau), post-injury autoimmune response (brain antigen-targeting autoantibodies) (see, e.g.,
• TBI may be co-incident with SCI and may result from the same injury or accident.
• SCI spinal cord injury
• a spinal contusion e.g., resulting from a fall, a firearm wound, a sports accident, a construction accident, a vehicle accident, or an injury which penetrates the spinal cord of the subject.
• SCI is diagnosed according to clinical guidelines known to those of skill in the art. SCI may be-coincident with TBI and may result from the same injury or accident.
• inflammatory disease or“immune disease” refers to a disease, disorder, or condition having an inflammatory or an immune component to the etiology, pathogenesis, progression, or symptomology of the disease.
• inflammatory or immune disorders may include dysregulation of an inflammatory or immune pathway and/or an abnormal inflammtory or immune response to a stimulus.
• exemplary inflammatory or immune disorders include cystic fibrosis,
• cardiovascular disease cardiovascular disease, keratoconus, keratoglobus, osteoarthritis, osteoporosis, pulmonary arterial hypertension, retinitis pigmentosa, and rheumatoid arthritis
• immunomodulatory composition or method may increase an activity of a cell involved in an immune response, e.g., by increasing pro-inflammatory markers such as cytokines, and/or may decrease an activity of a cell involved in an immune response, e.g., by decreasing pro-inflammatory markers such as cytokines.
• B cell refers to a type of white blood cell of the small lymphocyte subtype.
• B cells unlike the other two classes of lymphocytes, T cells and natural killer cells, express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it will initiate an antibody response. B cells function in the humoral immunity component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigen (they are also classified as professional antigen- presenting cells (APCs)) and secrete cytokines. In mammals, B cells mature in the bone marrow.
• APCs professional antigen- presenting cells
• mature B cells refers to a B cell that has 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 may interact with exogenous antigen and or T helper cells. The stages of B cell maturation have been well-characterized in the scientific literature and are known to those of skill in the art.
• nonaive B cell refers to a B cell that has not been exposed to an antigen.
• Breg cell or“B regulatory cell” refers to a type of B cell which participates in immunomodulation and in suppression of immune responses.
• Breg cells of the disclosure are mature, naive B cells, expressing characteristic cell surface markers.
• Breg cells may express one or more of B220, CD1d, CDS, CD19, CD20, CD21 , CD22, CD23, CD24, CD25, CD27, CD38, CD44, CD48, CD71 , CD73, CD138, CD148, CD274, IgM, IgG, IgA, and IgD.
• Breg cells may express cell surface markers including but not limited to B220, CD19, CD20, CD24, IgM, IgD, and CD138.
• Breg cells Upon introduction 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-alpha, TGF-beta, interferon-gamma.
• immunomodulatory cytokines including but not limited to IL-2, IL-4, IL-6, IL-10, IL-35, TNF-alpha, TGF-beta, interferon-gamma.
• 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 include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.
• pro-inflammatory cytokine refers to a cytokine secreted from immune cells that promotes inflammation. Immune cells that produce and secrete pro-inflammatory cytokines include T cells (e.g., Th cells) macrophages, B cells, and mast cells.
• Pro-inflammatory cytokines include interleukin-1 (IL-1 , e.g., IL-1 b), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, e g., TNFa), interferon gamma (IFNg), and granulocyte macrophage colony stimulating factor (GMCSF).
• IL-1 interleukin-1
• IL-6 interleukin-6
• IL-8 IL-10
• IL-12 IL-13
• IL-18 tumor necrosis factor
• TNFa tumor necrosis factor
• IFNg interferon gamma
• GMCSF granulocyte macrophage colony stimulating factor
• TLR Toll-like receptor
• TLR agonist refers to ligands that bind to and activate Toll-like receptors (TLRs), leading to downstream TLR cell signaling.
• TLR agonists are known to those of skill in the art and include endogenous and exogenous ligands.
• Exemplary endogenous ligands which are TLR agonists include heat shock proteins, necrotic cells or a fragment thereof, oxygen radicals, urate crystals, mRNA, beta-defensin, fibrin, fibrinogen, Gp96, Hsp22, Hsp60, Hsp70, HMGB1 , lung surfactant protein A, low density lipoprotein (LDL), pancreatic elastase, polysaccharide fragment of heparan sulfate, soluble hyaluronan, alpha A-crystallin, and CpG chromatin-lgG complex.
• Exemplary exogenous ligands which are TLR agonists include Pam3CSK4, triacylated lipopeptide,
• glycosylphosphatidylinositol (GPI)-anchored protein lipoarabinomannan, outer surface lipoprotein, lipopolysaccharide, cytomegalovirus envelope protein, glycoinositolphospholipids, glycolipids, GPI anchor, Herpes simplex virus 1 or a fragment thereof, lipoteichoic acid, mannuronic acid polymer, bacterial outer membrane porin, zymosan, double-stranded RNA, single-stranded RNA, Poly(l).Poly(C), taxol, flagellin, modulin, imidazoquinolines, antiviral compounds, unmethylated CpG
• treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of the disease or disorder (such as those described herein), alleviation of symptoms of such diseases, diminishment of any direct or indirect pathological consequences of the diseases, as well as altering an immune response. Additionally, treatment refers to clinical intervention relating to any of the diseases or conditions described herein.
• compositions utilized in the methods described herein can also be administered system ically or locally.
• a dosage is administered in a lotion, a cream, an ointment, or a gel.
• the method of administration can vary depending on various factors (e.g., the composition being administered, and the severity of the condition, disease, or disorder of immune dysregulation being treated).
• a subject to be treated according to this invention is a mammal.
• the mammal could be, for example, a primate (e.g., a human), a rodent (e.g., a rat or a mouse), or a mammal of another species (e.g., farm or other domesticated animals).
• a primate e.g., a human
• rodent e.g., a rat or a mouse
• a mammal of another species e.g., farm or other domesticated animals.
• the mammal may be one that suffers from any of the diseases or disorders disclosed herein.
• the subject is a human.
• an“effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result or a specifically state purpose.
• An “effective amount” can be determined empirically and by known methods relating to the stated purpose.
• a cell population may consist essentially of cells having a common phenotype such as the phenotype of B cell (e.g., a B reg cell) if any other cells present in the population do not alter or have a material effect on the overall properties of the cell population and therefore it can be defined as a cell line.
• a cell population typically comprises at least 60%, or between 60% and 99%, or between 70% and 90%, of B cells (or subpopulations of B cells such as B reg cells). Collection and Isolation of B Cells
• bone marrow is preferably obtained from the posterior superior ilium.
• the B cells obtained may be immediately used after isolation and relative purification, may be stored for subsequent use, or may be cultured for a period of time before use.
• the B cell population in the bone marrow contains pre-pro-B cells, pro-B cells, pre-B cells, immature B cells, and some mature B cells.
• B cell encompasses pre-pro-B cells, pro-B cells, pre-B cells, immature B cells, and mature B cells. From blood or other tissues, B cells can be isolated using standard techniques known to one of ordinary skill in the art.
• Antibodies may be linked to various molecules that provide a label or tag that facilitates separation.
• primary antibodies may be linked to magnetic beads that permit separation in a magnetic field.
• primary antibodies may be linked to fluorescent molecules that permit separation in a fluorescent activated cell sorter. Fluorescent and magnetic labels are commonly used on primary and/or secondary antibodies to achieve separation.
• Secondary antibodies which bind to primary antibodies may be labeled with fluorescent molecules that permit separation of cells in a fluorescence activated cell sorter.
• metallic microbeads may be linked to primary or secondary antibodies. In this manner, magnets may be used to isolate these antibodies and the cells bound to them.
• B cells can be isolated either straight from whole blood or buffy coat without density gradient centrifugation or erythrocyte lysis, or from peripheral blood mononuclear cells (PBMCs) after density gradient centrifugation. Both positive selection and depletion strategies can be pursued for direct isolation and isolation of B cells according to standard methods.
• PBMCs peripheral blood mononuclear cells
• the bone marrow can be subjected to a density gradient centrifugation.
• the buffy coat layer containing the bone marrow derived cells is removed from the gradient following the centrifugation.
• the cells are washed and resuspended in the antibody binding buffer and is then incubated with primary antibodies directed toward stem cells, T cells, granulocytes and monocytes/macrophages (called lineage depletion) followed by positive selection using antibodies toward B cells.
• Different B-cell subpopulations can be distinguished on the basis of differential expression of various surface markers and collected accordingly.
• B cells may be treated or stimulated by exposing them to one or more TLR agonists or immunomodulatory cytokine as is described herein. Production of IL-10 producing B reg cells using such ex vivo stimulation is taken useful in the methods and therapeutic strategies described herein.
• the number of cells to be administered will be related to the area or volume of affected area to be treated, and the method of delivery.
• B cell number for administration is 10 4 to 10 14 B cells, depending on the volume of tissue or organ to be treated. Other ranges include 10 5 to 10 12 B cells and 10 6 to 10 10 B cells.
• a pharmaceutical composition including B cells may include 10 4 to 10 14 B cells, 10 5 to 10 12 B cells, or 10 6 to 10 10 B cells in a single dose.
• Individual injection volumes can include a non-limiting range of from 1 ml to 1000 ml, 1 mI to 500 mI, 10 mI to 250 mI, or 20 mI to 150 mI.
• Total injection volumes per animal range from 10 mI to 10 ml depending on the species, the method of delivery and the volume of the tissue or organ to be treated.
• the B cells described herein may be incorporated into a vehicle for administration into a patient, such as a human patient suffering from a disease or condition described herein.
• Pharmaceutical compositions containing B cells can be prepared using methods known in the art.
• such compositions can be prepared using, e.g., 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, e.g., in the form of aqueous solutions.
• the B cells described herein can be administered in any physiologically compatible carrier, such as a buffered saline solution or a solution containing one or more electrolytes (e.g., one or more of sodium chloride, magnesium chloride, potassium chloride, sodium gluconate, or sodium acetate trihydrate).
• a physiologically compatible carrier such as a buffered saline solution 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).
• the B cells may be administered in a PlasmaLyte infusion buffer.
• PlasmaLyte is a family of balanced crystalloid solutions with multiple different formulations available worldwide according to regional clinical practices and preferences. It closely mimics human plasma in its content of electrolytes, osmolality, and pH.
• PlasmaLyte solutions also have additional buffer capacity and contain anions such as acetate, gluconate, and even lactate that are converted to bicarbonate, CO 2 , and water.
• the advantages of PlasmaLyte include volume and electrolyte deficit correction while addressing acidosis.
• the infusion buffer is PlasmaLyte A.
• PlasmaLyte A is a sterile, nonpyrogenic isotonic solution for injections (e.g., intravenous) administration. Each 100 mL of
• PlasmaLyte A contains 526 mg of Sodium Chloride (NaCI); 502 mg of Sodium Gluconate (C 6 H 1 1 NaO 7 ); 368 mg of Sodium Acetate Trihydrate, (C 2 H 3 NaO 2 .3H 2 O); 37 mg of Potassium Chloride (KCI); and 30 mg of Magnesium Chloride (MgCl2*6H20). It contains no antimicrobial agents.
• the pH is adjusted with sodium hydroxide. The pH is about 7.4 (e.g., 6.5 to 8.0).
• Suitable pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid media, for example, Dulbeccos modified eagle's medium (DMEM), sterile saline, sterile phosphate buffered saline, Leibovitz's medium (L15, Invitrogen, Carlsbad, Calif.), dextrose in sterile water, and any other physiologically acceptable liquid.
• DMEM Dulbeccos modified eagle's medium
• sterile saline sterile saline
• sterile phosphate buffered saline sterile phosphate buffered saline
• Leibovitz's medium L15, Invitrogen, Carlsbad, Calif.
• dextrose in sterile water, and any other physiologically acceptable liquid
• Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
• Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the solution is preferably sterile and fluid to the extent that easy syringability exists.
• the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosol, and the like.
• Solutions of the invention can be prepared by using a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above, followed by filtered sterilization, and then incorporating the B cells as described herein.
• a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
• sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.
• human serum may be used to stabilize pharmaceutical compositions including cells.
• naive B cells placed at the site of an injury detect local inflammatory signals and damage-associated molecular patterns (DAMPs) via TLR- and B cell receptor (BCR)-dependent pathways, and adopt a regulatory phenotype associated with production of anti-inflammatory cytokines, preferably IL-10, but also IL-4, IL-35, and TGF-b, that act on adjacent immune cells and fibroblasts and bias their phenotype towards an anti-inflammatory, pro- regenerative one.
• DAMPs damage-associated molecular patterns
• BCR B cell receptor
• Mouse spleens were collected in ice cold EasySepTM buffer (STEMCELL Technologies) containing 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA) in phosphate- buffered saline (PBS). Spleens were dissociated mechanically through a 40 pm cell strainer and the splenocyte suspension was processed for negative B or T cell selection through immunomagnetic separation, using commercially available cell isolation kits (STEMCELL Technologies) according to the manufacturer's instructions.
• FBS fetal bovine serum
• EDTA ethylenediaminetetraacetic acid
• Silicone splints with an inner diameter of 7 mm were attached around the wounds using Vetbond tissue adhesive (3M). The splinted wounds were then covered with TegadermTM transparent dressing (3M). Cell suspensions in PBS or equal volumes of PBS solution alone (saline control) were applied directly onto the wound bed using a manual pipette. Each mouse also received two localized subcutaneous injections under the dorsal skin with equal doses of B cells or saline solution. Each treated wound or subcutaneous site received 15-20 ⁇ 10 6 B cells in 20 ml PBS.
• mice were lightly anesthetized using 3% isoflurane in O 2 , and 10-20 pi of a working solution of brefeldin A (GolgiPlugTM, BD Pharmingen) in PBS was applied to each of the treated wound and subcutaneous sites in order to facilitate the
• tissue biopsies including the wounds and the subcutaneous injection sites were collected. Tissue biopsies were enzymatically dissociated for 30 minutes at 37°C with gentle rocking 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 hyaluronidase from bovine testes (Millipore Sigma), 0.4 mg/ml, 400 U/mg DNAse 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 for another 30 minutes in the same solution at 37°C with gentle rocking.
• the digested tissues from individual wound and subcutaneous samples were then pooled for each mouse, and passed through a 100 pm cell strainer followed by a 40 pm cell strainer, resulting in single-cell suspensions.
• the solubilized protein was digested in a two-step process starting with overnight digestion at room temperature with 3 mg of Lys-C (Wako) followed by 6 h of digestion with 3 mg of trypsin (sequencing grade, Promega) at 37°C.
• the digest was acidified with trifluoroacetic acid (TEA).
• TAA trifluoroacetic acid
• the digested peptides were desalted with C18 solid-phase extraction (SPE) (Sep-Pak, Waters).
• SPE solid-phase extraction
• the concentration of the desalted peptide solutions was measured with a BCA assay, and peptides were dried under vacuum in 50-mg aliquots, which were stored at -80 °C until they were labeled with the TMT reagents.
• Peptides were separated on an in-house-pulled, in-house- packed microcapillary column (inner diameter, 100 pm; outer diameter, 360 pm). Columns were packed first with ⁇ 0.5 cm of Magic C4 resin (5 pm, 100 A, Michrom Bioresources) followed by ⁇ 0.5 cm of Maccel C18 AQ resin (3 pm, 200 ⁇ ; Nest Group) and then to a final length of 30 cm with GP-C18 (1.8 pm, 120 ⁇ ; Sepax Technologies). Peptides were eluted with a linear gradient from 11 to 30% ACN in 0.125% formic acid over 165 min at a flow rate of 300 nL/min while the column was heated to 60 °C. Electrospray ionization was achieved by applying 1 ,800 V through a PEEK T-junction at the inlet of the microcapillary column.
• MS3 analysis was performed using synchronous precursor selection (MultiNotch MSS) that was enabled to maximize sensitivity for quantification of TMT reporter ions.
• MS2 precursors Up to ten MS2 precursors were simultaneously isolated and fragmented for MSS analysis.
• the isolation window was set to 2.5 m/z, and fragmentation was carried out by HCD at a normalized collision energy of 50%.
• Fragment ions in the MS3 spectra were detected in the Orbitrap at a resolution of 60,000 at m/z >110.
• the AGC target was set to 5 x 10 4 ions and the maximum ion injection time to 250 ms.
• Fragment ions in the MS2 spectra with an m/z of 40 m/z below and 15 m/z above the precursor m/z were excluded from being selected for MSS analysis.
• MS2 data were made using the Sequest algorithm to search the Uniprot database of mouse protein sequences, including known contaminants such as trypsin.
• the probability of a peptide-spectral match to be correct was calculated using a posterior- error histogram, the probabilities of all peptides assigned to one specific protein were combined through multiplication, and the data set was re-filtered to a protein assignment FDR of ⁇ 1 % for the entire data set of all proteins identified across all of the samples analyzed. Peptides that matched to more than one protein were assigned to the protein containing the largest number of matched redundant peptide sequences following the law of parsimony.
• TMT reporter ion intensities were extracted from the MSS spectra by selecting the most intense ion within a 0.003-m/z window centered at 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 of the reporter ions was >386 and the isolation specificity for the precursor ion was >0.75. Protein intensities were calculated by summing the TMT reporter ions for all of the peptides assigned to a protein.
• the cell suspensions were washed and resuspended in PBS and stained using a Zombie UV fixable viability kit (Biolegend, Inc.) for 30 minutes in the dark at 4°C with gentle rocking. The stained cells were then washed and resuspended in PBS containing 1 % FBS, 0.01 % sodium azide (RICCA Chemical, Arlington, TX) and 5% FcR blocking reagent (Miltenyi Biotec, Inc) for 10 minutes in the dark at 4°C.
• a Zombie UV fixable viability kit Biolegend, Inc.
• Blocked cells were then incubated for 30 minutes in the dark at 4°C with the following fluorophore-conjugated primary surface antibodies: 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 -conj ugated 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 CD 140a (clone APA5) (both from BD Biosciences, San Jose, CA).
• Brilliant Violet 785-conjugated rat anti-mouse CD19 (clone 6D5)
• LSRFortessa X-20 flow cytometer (BD Biosciences, San Jose, CA) equipped with BD FACS DIVATM software and 355 nm, 405 nm, 488 nm, 561 nm and 640 nm lasers. 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).
• APC-conjugated rat anti mouse CD45R/B220 (clone RA3-6B2; BioLegend.lnc.), PE-conjugated rat anti-mouse CD31 (clone MEC 13.3; BD Biosciences), Alexa Fluor® 488-conjugated mouse anti- tubulin b3 (clone TUJ1 ; BioLegend, Inc.), Alexa Fluor® 488-conjugated rat anti-mouse F4/80 (clone BM8; BioLegend, Inc.), PE-conjugated rat anti-mouse CD11 b (clone M1/70; BioLegend, Inc.), rabbit polyclonal anti-Ki67 (Abeam), and rabbit monoclonal anti-activated caspase 3 (clone C92-605; BD Pharmigen).
• Unbound primary antibody was removed by 3 rinses for 5 min each in TBS. If unconjugated primary antibodies were used, antigenic sites were visualized by incubating the sections for 2 hours at room temperature with Alexa Fluor 488®- conjugated F(ab')2- goat anti-rabbit IgG (Thermo Fisher Scientific), diluted 1 :200 in blocking solution.
• Sections were counterstained by incubation with 2 mg/ml of 4', 6-diamidino- 2-phenylindoledihydrochloride (DAPI; Sigma Aldrich) in PBS for 3 min at room temperature. The sections were washed 3 times for 7 min in TBS and embedded using Fluoromount (Novus Biologicals).
• Antibody controls included incubation of the tissue sections with isotype antibodies and omission of the primary antibody when a secondary antibody was used for visualization. No unspecific signal was detected in the control samples.
• Wound biopsies at the end point of the wound healing time course were collected using a 10-mm biopsy punch and fixed in 4% paraformaldehyde in PBS for 24-48 hours at 4°C, after which the samples were dehydrated through graded ethanol and xylene washes and embedded in paraffin.
• Transverse sections through the wound bed were cut at a thickness of 5 pm and mounted onto microscopy slides.
• Serial sections were stained with hematoxylin and eosin and with Masson’s trichrome stain for visualizing collagen fibers.
• Stained slides were digitized at a resolution of 0.25 pm/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.
• B cell application induces complex changes in the molecular microenvironment of a wound
• FIG. 1A A schematic representation of the average duration of the major stages of wound healing in the wild-type murine wound model is shown in FIG. 1A .
• Heatmaps summarizing the expression dynamics over time in proteins with significantly altered expression in response to B cell application are shown in FIG. 1 B.
• a total of 213 proteins representing the aggregate of significantly altered proteins associated with B cell treatment (n - 111 ; p ⁇ 0.05, unpaired t-test) as well as those proteins with high fold change (top 20 up- or down-regulated proteins) for each time point, regardless of significance level (n 112), were classified according to processes relevant to wound healing.
• FIG. 2A through 2H Average expression of proteins by functional family over time in wounds treated with saline (controls, regular wound healing) or after B cell treatment are shown in Figs. 2A through 2H.
• This analysis illustrates the overall effect of B cells as a homeostatic agent, rather than an inducer or inhibitor of protein expression.
• B cell application was associated with the maintenance of steady levels of expression of proteins that normally either decline or increase during the course of injury and healing, significantly reducing the inflammatory peak observed in normal healing, preventing the reduction in anti-apoptotic factors (arrows) and oxidative stress protectants and increasing proliferation (FIG.2A - FIG.2B), reducing the decline in anti-oxidative stress protectants and in cell proliferation, and maintaining cell migration at lower levels (FIG. 2C - FIG.
• FIG. 2D maintaining steady levels of proteins associated with remodeling and secondary skin structures
• FIG. 2E - FIG. 2F maintaining steady levels of proteins associated with remodeling and secondary skin structures
• FIG. 2G - FIG. 2H maintaining steady levels of proteins associated with remodeling and secondary skin structures
• FIG. 12A An unsupervised hierarchical cluster analysis of identified proteins expressed in skin wound samples is depicted in FIG. 12. Only identified proteins that were found consistently present across all samples were included in the analysis. Hierarchical clustering using complete linkage of 3809 proteins (rows) consistently expressed in all animals in both B cell and saline treated wounds at 4 different time points after injury (columns) is shown in FIG. 12A.
• the pseudocolor scale depicts normalized, log- transformed fold change expression values for each protein.
• the dendrogram shows 15 protein clusters derived from this analysis, with the color of each cell in (FIG. 12A) mapped to the mean expression value of the cluster at the respective time points. Proteins cluster by their pattern of expression over time.
• FIG. 12B a heatmap of hierarchical cluster from (FIG.
• FIG. 12A showing all 3809 proteins is depicted.
• 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 probe the 15 hierarchical clusters. Bar graphs show the top biological function categories for each cluster.
• FIG. 3 An experimental paradigm for in vivo assessment of B cell application in acute wound healing is depicted in FIG. 3.
• a total of 4 full-thickness lesions were generated in the dorsal skin of a wild-type C57BI6 mouse and mature naive B cells purified from isogeneic animals were applied directly on the wound beds.
• Control animals received saline applications.
• B cells or saline control were also injected subcutaneously under intact skin to provide a similar microenvironment without the injury.
• the wound or cutaneous uninjured tissue was collected, dissociated, and processed for flow cytometry analysis. Scatterplots on the right show typical distributions of cell suspensions from each treatment category. Wound samples show a characteristic influx of leukocytes
• B cells CD19+/B220+ lymphocytes
• CD140a-/B220- leukocytes non-B cell leukocytes
• fibroblasts CD140a+/B220-
• B cells were exposed in vivo to the wound niche or injected under uninjured skin (control equivalent location). Control B cells maintained on ice immediately after isolation for the same time duration are shown for comparison. After time intervals including 18 hours, 2 days, 4 days, and 10 days, the wounds were treated with Brefeldin A for 4 hours to induce retention of cytokines within cells. B cells were then retrieved by excising and dissociating the tissue, and further characterized by flow cytometry both for surface markers and intracellular cytokines. B cells exposed to the wound microenvironment transiently upregulate multiple immunomodulatory cytokines, peaking at 2 days post-application.
• immunomodulatory cytokines including TGFb and IL-6 remain elevated at 4 days, and IL-10 up to 10 days.
• N 3-6 animals/group.
• FIG. 6 A heatmap summary of the average values for each marker in B cells exposed to the wound microenvironment, subcutaneous control, or maintained on ice (no exposure) is found in FIG. 6.
• the dynamics of activation markers and key cytokines in the aggregate infiltrating non-B cell leukocytes in the wound are shown in FIG. 7.
• FIG. 7 Overall, infiltrating leukocytes produced more anti-inflammatory cytokines IL- 10, TGFb, and IL-35, and less pro-inflammatory TNFa and IL-2 when B cells were present in the wound. This impact was most pronounced at 4 days post injury and B cell application and persisted up to 10 days.
• N 3-6 animals/group.
• FIG. 8 A heatmap summary of the average values for each marker in infiltrating non-B cell leukocytes in the wound microenvironment, illustrating the pattern of increased anti-inflammatory cytokine (IL-10 and TGFb) production in the presence of B cells is shown in FIG. 8.
• FIG. 9 The dynamics of activation markers and key cytokines in the CD140a+ fibroblast population of the wound and subcutaneous tissue is shown in FIG. 9. Fibroblasts in the wound produced significantly more IL-10 and TGFb at 10 days post-injury when wounds were exposed to B cells. Moreover, wound fibroblasts produce less of the pro-inflammatory cytokine TNFa when B cells were applied, both at 4 days and 10 days post-injury.
• FIG. 10 An additional heatmap summary of the average values for each marker in fibroblasts in wounds and subcutaneous tissue treated either with B cells or saline solution is shown in FIG. 10.
• Fibroblasts are among the most important sources of anti-inflammatory and pro-regenerative factors in wound healing and generate high levels of IL-10 and TGFb regardless of treatment. Nevertheless, fibroblasts from wounds treated with B cells continued to produce higher levels of both IL-10 and TGFb at 4 and 10 days post-injury, while in saline-treated wounds, the levels of these anti-inflammatory cytokines decreased. Interestingly, a significant effect of B cell application was observed in the reduction of pro-inflammatory cytokines in wound fibroblasts, including IL-6 and TNFa.
• TLR signaling as well as IL-10 production are necessary components of the regenerative function of exogenous B cells in wound healing
• TLR signaling as well as IL-10 production are necessary components of the regenerative function of exogenous B cells in wound healing as is shown in FIG. 11.
• Full-thickness excision wounds (illustrated here at day 6 of healing) were treated at day 0 with B cells lacking the common TLR-signaling adaptor myeloid differentiation factor 88 (MyD88), IL-10, or WT B cells as control. Saline was also included as internal control in each tested animal. While the WT B cells consistently accelerated the wound closure by 2-3 days in WT animals, MyD88-/- or IL-10-/- B cells showed no benefit for wound closure, similar to saline application.
• MyD88-/- or IL-10-/- B cells showed no benefit for wound closure, similar to saline application.
• Example 2 B Cell Treatment Improves Outcome After TBI
• B lymphocytes are dynamic regulators of the immune system that have not been systematically studied in TBI.
• CCI mouse controlled cortical impact
• Mice were injected intraparenchymally at the lesion site with 2x10 6 mature naive syngeneic splenic B cells, then subjected to CCI.
• Control CCI mice received equal numbers of T cells or saline, and sham-injured mice (craniotomy only) were given B cells or saline. Sham-injured groups performed similarly in motor and learning tests.
• Injured mice administered B cells showed significantly improved post-injury Rotarod, Y maze, and Morris water maze (MWM) performance compared to saline- or T cell-treated CCI groups. Moreover, lesion volume in mice treated with B cells was significantly reduced by 40% at 35 days post- TBI compared to saline and T cell controls, and astrogliosis and microglial activation were decreased. In vivo tracking of exogenous B cells showed that they have a limited lifespan of approximately 14 days in situ and do not appear to proliferate. The data suggest proof of principle that local administration of B lymphocytes represent a therapeutic option for treatment of cerebral contusion, especially when clinical management involves procedures that allow access to the injury site.
• In vivo tracking of the exogenously applied B cells after intraparenchymal injection showed that the cells have a limited survival of approximately 2 weeks in situ, indicating that they would represent a safe, feasible option for the treatment of acute and sub-acute contusion TBI.
• Lymphocyte isolation Cell isolation was performed using negative immunomagnetic selection as described previously. 16 Briefly, mouse spleens were collected in ice cold buffer containing 2% fetal bovine serum (FBS) and 1 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS).
• FBS fetal bovine serum
• EDTA ethylenediaminetetraacetic acid
• Spleens were dissociated mechanically through a 40 pm cell strainer and the splenocyte suspension was processed for negative B or T cell selection through immunomagnetic separation and retention of non-target cells, using commercially available cell isolation kits (STEMCELL Technologies, Inc., Vancouver, Canada), according to the manufacturer’s instructions.
• the B cell isolation procedure was verified by flow cytometry analysis and typically resulted in an >98% pure population of mature naive CD45R + /CD19 + B lymphocytes, with under 1% contamination with other leukocytes, although some residual erythrocytes may be present.
• 16 Purified lymphocytes were re-suspended in sterile PBS at a concentration of 4 ⁇ 10 5 cells/ ml.
• Controlled cortical impact All surgical procedures, including the injury and the application of cells or saline, were performed by an experimenter blinded to the treatment conditions, who did not take part in the preparation of treatment doses for injection. Mice were anesthetized with 4.5% isoflurane (Baxter, Deerfield, IL) for 90 s in a mixture of 70% N2O and 30% O 2 using a FluotecS vaporizer (Colonial Medical, Windham, NH) and placed in a stereotaxic frame. Anesthesia was maintained with 4.5% isoflurane.
• isoflurane Boxter, Deerfield, IL
• a craniotomy was made using a portable drill and 5- mm trephine over the left parieto-temporal cortex, and the bone flap was discarded.
• An ipsilateral intraparenchymal injection was delivered at approximately -1 mm from bregma on the anterior/posterior axis, +2 mm medial/lateral, at a depth of 3 mm through the left parietal cortex.
• the selected cell dose has been optimized previously in a cutaneous injury model presenting similar lesion volume.
• 16 Cell application was performed immediately before CCI to ensure correct and consistent injection of cells while the brain structure was intact. Mice were immediately thereafter subjected to CCI using a pneumatic cylinder with a 3-mm flat-tip impounder, at a velocity of 6 m/s, a depth of 0.6 mm and a 100-ms impact duration. Sham-injured mice underwent anesthesia, craniotomy, and intraparenchymal injection with equal numbers of B cells or saline, but no CCI injury. The craniotomy was left open and the skin was closed over the skull using 6-0 nylon sutures (Fisher Scientific, Waltham, MA).
• Behavioral testing schedule Behavioral testing was conducted during the light phase of the circadian cycle by experimenters blinded to the treatment conditions. Prior to each test, mice were acclimatized to the room for at least 30 min. Mice were tested in a battery of assays, according to the schedule described in FIG. 14. Vestibulo-motor ability was assessed by wire grip assay on days 1 , 3, and 7 post injury. Rotarod testing was performed on days 7, 9, 10, 13, and 14 post injury. Animals were subjected to assessment of anxiety using an elevated plus maze assay on day 17 post injury. Morris water maze (MWM) testing was done on days 20, 21 , 22, 23, and 24 after injury, with a probe test on day 27. On day 29 post injury, mice were subjected to the forced swim test to assay depression-like behavior, and on day 30 to the Y-maze, an assay for hippocampus-dependent working memory.
• MVM Morris water maze
• Wire grip test Vestibulo-motor function was assessed using a 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 traverse the wire for 60 s. The latency to fall within the 60 s interval was measured, and a wire grip score was quantitated using a 5-point scale. Testing was performed in triplicate and an average value calculated for each mouse on each test day.
• Rotarod Mice were placed on an automated Rotarod apparatus (Harvard Apparatus, Holliston, MA) which accelerated from 4 to 40 r/min over 60 s. Maximum trial duration was 300 s, or until the mouse fell off the rotarod. Each mouse was assessed five times per day with 5 min rest intervals. The average latency to drop and the average r/min speed attained over the five trials was recorded for each day of testing.
• MWM The MWM was performed as previously described with minor modifications (Mannix et al. (2013) Ann Neurol 74, 65-75). Spatial learning was assessed at approximately the same time each day. Each mouse was subjected to seven hidden platform trials (one to two trials per day) using a random set of starting positions at any one of the four quadrants. One trial consisted of the average latency from each of the four starting positions. If a mouse failed to find the platform within 90 s, then it was placed on the platform for ⁇ 10 s. Probe trials were performed 24 h after the last hidden platform trial by allowing the mice to swim in the tank for 30 s with the platform absent, and recording the time spent in the target quadrant.
• Porsolt forced swim test Mice were placed in a cylindrical transparent glass tank of 30 cm (height) ⁇ 20 cm (diameter) filled with water (25°C) up to a height of 20 cm. A white Styrofoam box provided visual shielding on three sides. Mice were placed in the water for 6 min and swimming movements were recorded. Total active time (swimming, pawing/climbing the beaker wall) versus inactive time (passive flotation) was quantified for the last four minutes of the test.
• Y-maze spontaneous alternation test The Y maze test was conducted in an apparatus constructed of white opaque acrylic, consisting of three 40-cm long arms joined at 120° angles, with a wall height of 15 cm. Each arm was labeled with a different contrasting visual cue (black-on-white square, circle, star). Mice were placed in the center of the apparatus and allowed to explore the maze for 10 min. Their movements were recorded using a webcam positioned directly overhead and Photo Booth software (ANY-maze). Normal exploratory behavior in rodents involves a preference to enter a less recently visited arm of the maze (spontaneous alternation). An alternation score was calculated by dividing the number of three successive choices that included one instance of each arm by the total number of arm entries (i.e. opportunities for alternation). The apparatus was cleaned with 70% ethanol between trials.
• Elevated plus maze The apparatus consisted of two 130 ⁇ 8 cm platforms with a 8 ⁇ 8 cm square area at their intersection, elevated at 60 cm above ground. The closed arms of the platform had 10 cm walls, whereas the open arms had none. Each mouse was placed in the central area of the maze and video-recorded for 5 min. The apparatus was cleaned with 70% ethanol between trials. Video recordings were analyzed by ANY-Maze (Stoelting Co., Wood Dale, IL) software for mean speed and percent time in closed and open arms.
• IVIS imaging Splenic B cells were isolated from mice homozygous for the CAG-luc-eGFP L2G85 transgene, which show 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 pi PBS were injected into the left hemisphere of recipient WT C57BI6/J mice, as described above. The mice were imaged using an IVIS Lumina II system (Caliper Life Sciences, Waltham, MA) on the day of the surgery and at regular intervals thereafter for a total of 4 weeks.
• IVIS Lumina II system Caliper Life Sciences, Waltham, MA
• luciferase activity 100 mI of 30 mg/ml aqueous D-luciferin solution (Regis Technologies, Inc., Morton Grove, IL) was injected subcutaneously proximal to the injury site at least 6 minutes before imaging. Mice were imaged for 10 min, and identical parameters were maintained for every repeat imaging.
• Tissue sampling At 35 days after CCI and treatment, the 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.
• the brains were rapidly extracted on ice, frozen in liquid nitrogen vapor, and stored at -80°C.
• cryosectioning the brains were embedded in M-1 embedding matrix (Thermo Fisher Scientific, Waltham, MA), and sectioned coronally at a thickness of 16 pm using a cryostat. Sections were collected at 500 pm intervals along the rostro-caudal axis, and thaw-mounted onto SuperFrost Plus Gold slides (Fisher Scientific, Waltham, MA).
• Alexa Fluor® 594-conjugated rat anti-mouse CD45R/B220 (clone FRA3-6B2; Bio Legend, Inc., San Diego, CA)
• Alexa Fluor® 488-conju gated 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;
• Sections were counterstained by incubation with 2 mg/ml of 4', 6-diamidino-2- phenylindoledihydrochloride (DAPI; Sigma Aldrich).
• Antibody controls included incubation of the tissue sections with isotype antibodies and omission of the primary antibody when a secondary antibody was used for visualization. No unspecific signal was detected in the control samples.
• Stained tissue sections were imaged using a Zeiss LSM 710 laser scanning microscope (Carl Zeiss), and confocal images were collected using Zen software (Carl Zeiss).
• Lesion volume measurement Sections were stained with hematoxylin and high-resolution overview photographs of the slides were collected. Morphometric image analysis in Imaged (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 .
• Foreground objects were identified as sets of pixels with intensities exceeding the corresponding background levels by more than 25 for GFAP, respectively 50 for CD68 immunolabeling.
• the relative area labeled was calculated as the fraction of pixels within the region of interest marked as foreground, while the mean labeling intensity was calculated as the average intensity of all foreground pixels found within the region of interest. All image analyses were performed by an experimenter blinded to the treatment conditions.
• mice that received B cells spent significantly more time in the closed arms of the maze as compared to animals that received an equal number of T cells (p ⁇ 0.05).
• B lymphocyte application at the time of injury is associated with reduced lesion volume and glial scarring at 35 days post CCI
• FIG. 18 brains from all animals subjected to behavioral assessments were collected on day 35 after injury for histological examination.
• Analyses of brain tissue damage in the CCI groups showed cavitation of the lesioned area in all mice subjected to CCI, whereas sham- lesioned groups showed no loss of brain tissue (FIG. 18A).
• Quantitative analysis of the lesion volume across all groups showed a significant effect of CCI as expected (p ⁇ 0.0001 ).
• B cell-treated CCI mice had significantly less brain tissue loss as compared to injured groups treated with saline (p ⁇ 0.001) or T cells (p ⁇ 0.0001 ).
• B lymphocytes do not proliferate in situ and have a limited lifespan of approximately 2 weeks after application
• the CCI injury model produces highly reproducible lesions as well as very low mortality rates (Xiong et al., (2013) Nat Rev Neurosci 14, 128-142). While the impact is delivered to the cortical surface, with the dura intact, the neuropathological consequences of the injury are typically extensive, and include cortical, hippocampal, and thalamic degeneration (Id.) These pathologies are associated with long-term cognitive deficits and alterations in emotional behavior (Id.). In the present CCI injury paradigm, as the impact force was applied directly to the cortex, this region reliably and invariably degenerated in all animals, regardless of treatment condition, while differences between therapeutic conditions were observed in the sparing of subcortical structures, particularly the hippocampus.
• B lymphocytes were injected approximately 1 mm posterior from bregma and remained mostly localized between the caudoputamen and the hippocampus (Lein et al. (2007) Nature 445, 168-17).
• B cells accordingly may be used as a therapeutic strategy for patients with cerebral contusion.
• B cells can be readily obtained from peripheral blood or other blood bank products, an important advantage for the development of a rapid off-the-shelf therapeutic agent.
• a rapid, minimally-manipulated, autologous B cell therapy would be highly translatable into a clinical setting. This is especially true in the case of severe brain lesions, where surgery is often performed to remove hematomas or penetrating bone fragments, and either
• intraparenchymal or intraventricular catheters are placed to monitor intracranial pressure, (Stocchetti et al. (2017) Lancet Neurol 16, 452-464; Galgano et al. (2017) Cell Transplant 26, 1118-1130) thus providing a convenient route of administration for B cells into injured brain.
• B lymphocytes are mature, terminally-differentiated cells, with a naturally limited lifespan of 5-6 weeks in vivo. Their application in the disrupted microenvironment of a brain contusion is expected to lead to elimination of the transplanted cells after even less time. This is advantageous because longer survival of transplanted cells could represent a significant safety concern, particularly considering that the microenvironment of the central nervous system contains a number of B cell-trophic factors.
• This example illustrates the safety and efficacy of intravenous (i.v.) B cell administration in a standard murine model of ALS, the SOD1 G93A mouse.
• B cell treatment delayed symptom onset (p ⁇ 0.0001), as indicated by reaching a peak weight, and significantly extended survival (p ⁇ 0.05) in SOD1 G93A mice.
• Treatment with B cells was associated with a significant reduction in the relative numbers of injured/degenerating motor neurons in the lumbar spinal cord (p ⁇ 0.05) at endpoint, even as total numbers of motor neurons were not changed with treatment.
• B6SJL-Tg(SOD1 * G93A)1 Gur/J (SOD1-G93A) transgenic mice were purchased from The Jackson Laboratory (Bar Harbor, ME; Stock No: 002726). This founder line (often referred to as G1 H) is reported by Jackson Laboratory to have high SOD1 transgene copy number. All animals are individually genotyped by Jackson Laboratory prior to shipping and only heterozygous animals with high SOD1 transgene copy numbers (upper third of the distribution) are commercialized. Control animals are littermates without the SOD1 transgene (Noncarrier).
• Donor animals for B cell isolation were C57BL/6J mice also purchased from the Jackson Laboratory (Stock No: 000664).
• the resulting cells are >98% CD19+ B cells, and typically over 85-90% CD19+/B220+/lgM+/lgD+, including approximately 5% CD138+ plasma cells, and ⁇ 1 % other cell populations, as confirmed after isolation through flow cytometric analysis (DeKosky et al. (2013) Nat Rev Neurol 9, 192-200). This represented the naive B cell fraction (Treatment), and it was infused into animals the same day, after isolation.
• Treatment Starting at week 10 of life (day 72), all animals received a total of 10 weekly intravenous infusions of B cells (or saline control), delivered via retro-orbital injection. The animals were anesthetized with 3% isoflurane in oxygen and a 100 mI bolus of saline containing 5 million naive B cells (Treatment) or no cells (saline control) was injected into the retro-orbital venous sinus. Eye ointment was then applied to the treated eye. This method of administration was selected because it has a considerably lower risk of failure as compared to tail vein injection for cell transplantation, particularly with repeated administration.
• Weight and neurological scores were assessed twice weekly by an experimenter blinded to the treatment conditions, and were used to assess disease progression, as described e.g. in Hatzipetros, T. et al. (2015) J. Vis. Exp. (104), e53257,
• NeuroScore 1 (First symptoms): When the mouse is suspended by the tail, the hindlimb presents an abnormal splay, i.e., it is collapsed or partially collapsed towards lateral midline OR it trembles during tail suspension OR it is retracted/ clasped. When the mouse is allowed to walk, normal OR slightly slow gait is observed.
• NeuroScore 2 (Onset of paresis): When the mouse is suspended by the tail, the hindlimb is partially OR completely collapsed, not extending much. (There might still be joint movement). When the mouse is allowed to walk, the hindlimb is used for forward motion however the toes curl downwards at least twice during a 90 cm walk OR any part of the foot is dragging along. When the mouse is placed on its left AND right side, it is able to right itself within 10 sec from BOTH sides.
• NeuroScore 3 (Paralysis): When the mouse is suspended by the tail, there is rigid paralysis in the hindlimb OR minimal joint movement. When the mouse is allowed to walk there is forward motion however the hindlimb is NOT being used for forward motion. When the mouse is placed on its left AND right side, it is able to right itself within 10 sec from BOTH sides.
• Weight was used as a reliable and unbiased assessment of disease progression. The appearance of disease onset was retrospectively determined using the age of maximal body weight which is a reliable and objective measure of muscle denervation onset, as previously described (Turner et al. (2014) Neurobiol Aging 35, 906-915.). Histology: At euthanasia, the lumbar spinal cord was collected from all animals, preserved in fixative solution, and processed for histology. Spinal cords were sectioned longitudinally in the horizontal plane, at a thickness of 10 pm and stained with hematoxylin and eosin (H&E) to visualize motor neurons in the ventral horns.
• H&E hematoxylin and eosin
• Motor neurons easily identifiable based on large size and distinctive morphology, were counted in 3-6 regions of interest of at least 500 x 500 pm, randomly collected from 2-3 longitudinal sections per animal.
• We also quantified separately healthy motor neurons large, rounded cell body; nuclei with single prominent nucleoli; Nissl substance present; see FIG. 27A) and injured/degenerating motor neurons (shrunken cell body, hyperbasophilia, strong aggregation of nuclear chromatin, pyknotic nucleus; see FIG. 27B). All counts were performed by an experimenter blinded to the treatment conditions.
• Peak weight (shown in FIG. 24). Peak weight was defined as the time point after which the
• a composition including a therapeutically effective amount of B cells may be administered to a subject having Parkinson’s’ disease.
• Treatment of Parkinson’s’ disease may be evaluated using the methods described herein by 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 the therapeutic efficacy according to methods known to those of skill in the art. Methods for monitoring the response include assessment of motor function, pain, neuroinflammation, and death of nigral neurons (see, e.g., Peng Q. et al. The Rodent Models of Dyskinesia and Their Behavioral Assessment. Front Neurol. (2019)).
• B reg cells e.g., B reg cells
• Example 5 Administration of B cells to treat additional neurodegenerative diseases
• Alzheimer’s disease see, e.g., Esquerda-Canals G. et al. Mouse Models of Alzheimer's Disease. J Alzheimers Dis. (2017));
• the responsiveness to treatment may be monitored by a decrease in the rate of progression of the disease (e.g., a decrease in the rate of progression as measured by the severity of symptoms associated with the neurodegenerative disease). Alternately, responsiveness to treatment may be monitored by determining the level of a molecular marker of disease progression associated with neurodegenerative disease, such as, a molecular marker of disease progression provided in Example 4.
• Example 6 Administration of B cells to treat inflammatory or immune diseases
• 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));
• 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. E.L. 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

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