Patent Application
20220339191
This invention was made with Government support under project number Z01EY000350-18 by the National Eye Institute of the National Institutes of Health. The Government has certain rights in the invention.
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 2,692 Byte ASCII (Text) file named “749447_ST25.TXT,” created on Jun. 12, 2020.
Uveitis, age-related macular degeneration (AMD), graft-vs-host disease (GVHD), and multiple sclerosis (MS) are diseases that initiate or progress as a result of adverse immunological activity. These diseases can result in blindness, paralysis, and significant morbidity that impacts quality of life. Uveitis is comprised of a diverse group of potentially sight-threatening intraocular inflammatory diseases of infectious or autoimmune etiology, where autoreactive lymphocytes contribute to ocular pathology by attacking and damaging uveal tissue. Similarly, autoimmune processes contribute significantly to the progression of retinal degeneration associated with AMD, though the processes that initiate AMD have not been definitively identified. MS is caused in part by lymphocytes that attack and/or destroy myelinated neurons, thereby interfering with synaptic transmission and communication between neurons. In GVHD, the allogeneic transplant views the recipient's body as foreign, and the transplant attacks the body. Although steroids are effective therapy for uveitis or MS, serious adverse effects preclude their prolonged use. Similar to uveitis and MS, there may be adverse effects associated with the use of steroids and immunosuppressants to treat GVHD. Further, there currently is no effective cure for AMD, and current treatments are directed to the slowing of progressive retinal degeneration. Therefore, there remains an unmet need for safe and effective long-term therapies for the aforesaid diseases.
The invention provides an isolated population of mammal cells comprising about 75% or higher B-1a regulatory cells expressing cell surface inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and C-X-C chemokine receptor type 4 (CXCR4), and secreting interleukin-27 (IL-27).
The invention also provides methods of preparing the population of mammal cells of an embodiment of the invention, comprising (a) isolating cluster of differentiation 5 positive (CD5+) expressing cells from a sample of mammal peripheral lymphoid tissue, mammal cord blood, mammal peritoneal fluid, or mammal bone marrow using fluorescence-activated cell sorting (FACS) to provide isolated CD5+ expressing cells; (b) culturing the isolated CD5+ expressing cells in a cell culture media to provide cultured cells; (c) activating the cultured cells with a BCR (B cell receptor) or a TLR (Toll-like receptor) agonists to provide activated cells; and (d) exposing the activated cells to IL-27.
The invention further provides methods of suppressing the immune system of a mammal, the method comprising administering the population of mammal cells of an embodiment of the invention to a mammal.
The invention further provides methods of treating a mammal with graft-versus-host disease, the method comprising administering the population of mammal cells of an embodiment of the invention to a mammal with graft-versus-host disease.
The invention provides methods of preventing or reducing the severity of graft-versus-host disease in a mammal, the method comprising administering the population of mammal cells of an embodiment of the invention to a mammal before the mammal receives an allogeneic transplant.
The invention provides methods of preventing or reducing the severity of graft-versus-host disease in a mammal, the method comprising (a) mixing the population of mammal cells of an embodiment of the invention with a transplant material to form a transplant mixture, and (b) administering the transplant mixture to a mammal.
C57BL/6J mice were injected (i.v) with LPS (50 μg/mouse) and frequency of IL-27-producing B2 cells in the spleen was assessed every day until day 4 post-injection. The B2 cells were isolated at various time points from the spleen and analyzed by intracellular cytokine staining assay.
Regulatory B-cells (Bregs) suppress autoimmune diseases through production of IL-10 or IL-35 alone or in combination with inhibitory cell-surface receptors. However, Bregs described thus far (e.g., U.S. Pat. No. 9,629,897) are antigen-specific and derive from B2-lymphocyte lineage. The invention provides an isolated population of human cells comprising a non-naturally occurring, concentrated population of regulatory B-cells of B-1a lineage that produce and secrete interleukin-27 (i27-Bregs).
Interleukin-27 (IL-27) is a member of the IL-12 cytokine family. IL-27 is a heterodimeric cytokine that is composed of two distinct protein subunits encoded by ebi3 (Epstein-Barr virus-induced gene 3) and IL-27p28. IL-27 is expressed by cells and interacts with IL-27 receptor (IL-27R). IL-27R consists of two proteins, IL-27a (IL-27 alpha) and gp130. IL-27 induces differentiation of the diverse populations of T cells in the immune system. Natural activation of B-1a regulatory cells upon inflammatory stimuli triggers IL-27 production and the coincident exodus of i27-Bregs to the spleen where they reprogram conventional lymphocytes to acquire immune-regulatory functions.
The population of cells of the invention can comprise about 25% or more B-la regulatory cells (e.g., about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, or about 90% or more B-1a regulatory cells). Populations of B-1a cells at such relatively high proportions compared to other cell types within the population of cells do not exist in the human body or in nature. B-1a cells within the human body are detected at low numbers in peripheral lymphoid tissues (<2%). Within this minority population of less than 2%, i27-Bregs comprise less than 2%—only up to about 4/10,000 of a naturally occurring human cell population (i.e., 0.02×0.02 =0.0004).
The population of cells of the invention expresses the cell surface inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and C-X-C chemokine receptor type 4 (CXCR4). The population of cells can have the receptors on their surfaces or be capable of having the receptors on their surfaces.
LAG-3 (or cluster of differentiation 223 (CD223)) is a protein encoded by the LAG3 gene in humans. LAG3 is an immune checkpoint receptor.
PD-1 (or cluster of differentiation 279 (CD279)) is a protein encoded by the PDCD1 gene in humans. PD-1 is also an immune checkpoint receptor. PD-1 promotes apoptosis of antigen-specific T-cells in lymph nodes and reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).
CXCR4 (or fusin or cluster of differentiation 184 (CD184)) is a protein encoded by the CXCR4 gene in humans. CXCR4 is an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1 or CXCL12), a molecule with chemotactic activity for lymphocytes.
The population of cells optionally also expresses the cell surface inhibitory receptor glucocorticoid-induced TNFR-related protein (GITR or tumor necrosis factor receptor superfamily member 18 (TNFRSF18) or activation-inducible TNFR family receptor (AITR)). GITR is a protein encoded by the TNFRSF18 gene in humans. GITR has been shown to have increased expression upon T-cell activation.
The population of cells of the invention optionally also expresses the cell surface inhibitory receptor OX40 (or tumor necrosis factor receptor superfamily member 4 (TNFRSF4) or cluster of differentiation 134 (CD134)). OX40 is a protein encoded by the TNFRSF4 gene in humans. OX40 is not constitutively expressed on resting naïve T cells.
The population of cells of the invention optionally also expresses the cell surface inhibitory receptor cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or cluster of differentiation 152 (CD152)). CTLA4 is a protein encoded by the CTLA4 gene in humans. CTLA4 is an immune checkpoint and downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation.
The population of cells of the invention can be from a mammal. The term “mammal” includes, but is not limited to, the order Rodentia, such as mice, and the order Logomorpha, such as rabbits, the order Carnivora, including Felines (cats) and Canines (dogs), the order Artiodactyla, including Bovines (cows) and Swines (pigs), the order Perssodactyla, including Equines (horses), Primates, Ceboids, or Simioids (monkeys), and the order Anthropoids (humans and apes). More preferably, the population of cell are from a human.
The invention provides methods of preparing the population of cells (e.g., human cells) comprising (a) isolating cluster of differentiation 5 positive (CDS+) expressing cells from a mammal tissue or fluid sample to provide isolated CD5+ expressing cells, (b) culturing the isolated CDS+expressing cells in a cell culture media to provide cultured cells, (c) activating the cultured cells with a BCR (B cell receptor) or a TLR (Toll-like receptor) agonist to provide activated cells; and (d) exposing the activated cells to IL-27. In this regard, the isolating of the CD5+ (CD5 is expressed on the surface of T cells and B-1a cells) expressing cells can be carried out by any suitable method, for example by using fluorescence-activated cell sorting (FACS), microfluidic cell sorting, or magnetic cell sorting.
The mammal tissue or fluid sample can be from any suitable source, such as mammal peripheral lymphoid tissue, mammal cord blood, mammal peritoneal fluid, mammal bone marrow, induced pluripotent cells (iPSC), or any other sample containing B-1a cells. In at least some embodiments, the use of peritoneal fluid or cord blood as the sample may be desirable because these sources typically have a higher percentage of B-1a cells than other samples (e.g., peripheral lymphoid tissue). In some embodiments, the preferred source of the tissue or fluid may be from the donor subject that will be treated with the population of cells of the invention.
Any suitable cell culture media that can support the growth of B-1a cells can be used. For example, Roswell Park Memorial Institute medium (RPMI 1640) culture medium can be used.
The cultured cells are exposed to a BCR agonist or a TLR agonist. Any suitable BCR agonist or a TLR agonist that can activate the cells can be used. Examples of BCR agonists include anti-CD40 and anti-IgM antibodies. Examples of TLR agonists include TLR9 and TLR4 agonists. As is the case for all lymphocytes, the B-1a cells have to be activated to elicit biological activity and thus the CD5+B-1a cells activated with a BCR agonist or TLR agonist. CD40 is a costimulatory protein found on antigen presenting cells and is required for B cell activation following interaction of the B cell receptor with antibody to IgM. However, maximum secretion of IL-27 by the activated B-1a cell requires IL-27 signals provided by binding of IL-27 to its cognate receptor on the B-1a cell and further upregulation of the IL-27 receptor.
As used herein, the terms “Toll-like receptor” and “TLR” refer to any member of a family of highly-conserved mammalian proteins which recognize pathogen-associated molecular patterns and act as key signaling elements in innate immunity. TLR polypeptides share a characteristic structure that includes an extracellular domain that has leucine-rich repeats, a transmembrane domain, and an intracellular domain that is involved in TLR signaling.
The terms “Toll-like receptor 4” and “TLR4” refer to nucleic acids or polypeptides sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a publicly-available TLR4 sequence. A suitable TLR 4 agonist is LPS.
The terms “Toll-like receptor 9” and “TLR9” refer to nucleic acids or polypeptides sharing at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a publicly-available TLR9 sequence. Suitable TLR9 agonists are oligonucleotides containing CpG motifs (CpG ODNs).
The activated cells are exposed to IL-27. The exposure to IL-27 facilitates expansion of the i27-Bregs and creates an efficient ongoing increase in the proportion and amount of i27-Bregs.
The inventive methods are useful for the treatment of a disease in a mammal. The treatment may result in desirable suppression of the immune system.
The inventive methods are useful for the treatment, suppression, or prevention of GVHD. Patients can receive a solid organ or allogeneic bone marrow or hematopoietic stem cell transplant. In order to prevent or reduce the severity of GVHD, the population of mammal cells of the invention are administered to a mammal before the mammal receives an allogeneic transplant. Alternatively, GVHD can be prevented or suppressed by mixing the i27-Breg population of cells of the invention with a transplant material to form a transplant mixture, and then administering the transplant mixture to the mammal. In this regard, the transplant material can include allogeneic lymphocytes. In an embodiment, the transplanted cells are cells (e.g., heart cells, pancreatic cells, retinal cells) derived from iPS cells.
The population of mammal cells of the invention can be mixed with the transplant material ex vivo. “Ex vivo” refers to methods conducted within or on cells or tissue in an artificial environment outside an organism with minimum alteration of natural conditions. In contrast, the term “in vivo” refers to a method that is conducted within living organisms in their normal, intact state, while an “in vitro” method is conducted using components of an organism that have been isolated from its usual biological context.
The population of mammal cells can be administered in the form of a pharmaceutically acceptable (e.g., physiologically acceptable) composition. The composition may comprise a carrier, preferably a pharmaceutically (e.g., physiologically acceptable) carrier, and the population of mammal cells. Any suitable carrier can be used within the context of the invention, and many such carriers are known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).
The population of mammal cells can be administered to a mammal (as earlier defined herein). Preferably the mammal is a mouse or a human.
The invention provides a method of suppressing the immune system in a mammal, which method comprises administering the population of mammal cells of the invention to a mammal in need thereof, thereby suppressing the immune system in the mammal. Thus, the invention provides for a method of suppressing autoimmunity in a mammal comprising administering an isolated IL-27-producing B-1a cell population to a mammal whereupon the in vivo IL-27 production in the mammal is increased to artificially high levels and autoimmunity is thereby suppressed in the mammal. IL-27 is rapidly cleared in vivo, however, the administration of the isolated IL-27-producing B-1a cell population allows for proliferation of i27-Bregs and sustained IL-27 secretion in vivo. This provides distinct advantages over therapies that may rely upon direct administration of IL-27.
IL-27 and IL-35 are the two immune-suppressive members of the IL-12 family of cytokines. Although IL-35 or IL-27 show substantial promise in suppressing autoimmune diseases, a major disadvantage of using cytokines as biologics, especially heterodimeric cytokines, is their relatively short half-life, transient biological activities and unpredictable pharmacokinetic characteristics. Another important impediment relates to the issue of dosing. Because association of the IL-35 or IL-27 subunit proteins is not strong (non-covalent), IL-35 and IL-27 subunit proteins readily dissociate making it difficult to ascertain the effective dose of bioactive p35:Ebi3 or p28:Ebi3 heterodimer administered or required to ameliorate disease. Therapeutic use of i27-Bregs provides several therapeutic advantages over the use of biologics such as IL-10, IL-27 or IL-35, which are the most effective cytokines produced by Breg or Treg cells: (i) the ex-vivo generated i27-Bregs proliferated in-vivo and thereby sustained production of IL-27 in recipient host tissues; (ii) the ex-vivo generated i27-Bregs proliferated in-vivo and reprogram recipient lymphocytes into IL-10-, IL-27, IL-35-producing Bregs and Tregs, and can thereby sustained production of these immune suppressive cytokines in recipient host tissues; (iii) disease suppression by innate i27-Bregs does not require prior activation by autoantigen that elicits disease, providing potential therapeutic advantage over disease-specific Breg/Treg therapies used for autoimmune diseases.
The term “autoimmunity,” as used herein, refers to the failure of an organism (e.g., a mammal, such as a human or mouse) to recognize its own constituent parts as self, which results in an immune response against the organism's own cells and tissues. In other words, autoimmunity is an adaptive immune response directed against “self” antigens and is marked by the production of proinflammatory cytokines that mediate pathology by damaging host tissues or by production of “autoantibodies” that can cause complement mediated diseases.
“Autoimmune disease” refers to any one of a group of diseases or disorders in which tissue injury is associated with a humoral and/or cell-mediated immune response to body constituents or, in a broader sense, an immune response to self. The pathological immune response may be systemic or organ specific. For example, the immune response directed against self may affect joints, skin, the brain, the myelin sheath that protects neurons, the kidneys, the liver, the pancreas, the thyroid, the adrenals, the eyes (e.g., uveitis), and ovaries. Immune complex formation plays a role in the etiology and progression of autoimmune disease. Increased immune complex formation correlates with the presence of antibodies directed to self (autoantibodies). The presence of autoantibodies can contribute to tissue inflammation either as part of an immune complex or unbound to antigen (free antibody). In some autoimmune diseases, the presence of free autoantibody contributes significantly to disease pathology. Another aspect of the etiology and progression of autoimmune disease is the role of proinflammatory cytokines. Under normal circumstances, proinflammatory cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-1 (IL-1) play a protective role in the response to infection and cellular stress. However, the pathological consequences which result from chronic and/or excessive production of TNF-a and IL-1 are believed to underlie the progression of many autoimmune diseases such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, uveitis, and psoriasis. Other proinflammatory cytokines involved in autoimmune disease include interleukin-6, interleukin-8, and granulocyte-macrophage colony stimulating factor (see, e.g., U.S. Pat. No. 8,080,555).
The inventive cell population and methods can be used to suppress autoimmunity associated with any autoimmune disease. There are more than 80 autoimmune diseases known in the art, examples of which include multiple sclerosis (MS), insulin-dependent diabetes mellitus, systemic lupus erythematosus (SLE), psoriasis, autoimmune hepatitis, thyroiditis, insulitis, uveitis, orchitis, myasthenia gravis, idiopathic thrombocytopenic purpura, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), encephalomyelitis, systemic autoimmune diseases (e.g., rheumatoid arthritis (RA), scleroderma, and juvenile arthritis).
Autoimmunity is “suppressed” if one or more symptoms of an autoimmune disease is reduced or alleviated in a mammal (e.g., a human) affected by an autoimmune disease. Improvement, worsening, regression, or progression of a symptom may be determined by any objective or subjective measure, many of which are known in the art. A person of ordinary skill in the art will appreciate that the symptoms of autoimmune diseases vary based on the disease and location of the abnormal immune response. Symptoms that are common to several autoimmune diseases include, for example, fatigue, muscle and/or joint pain, muscle weakness, fever, swollen glands, inflammation, susceptibility to infections, weight loss or gain, allergies, digestive problems, blood pressure changes, and vertigo.
The inventive cell population and methods can be used to decrease or suppress inflammation in the pancreas.
The inventive cell population and methods can be used to decrease or suppress the symptoms of AMD.
As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect.
Preferably, the pharmacologic and/or physiologic effect is therapeutic, i.e., the effect partially or completely cures a disease and/or adverse symptom attributable to the disease. To this end, the inventive method comprises administering a “therapeutically effective amount” of the isolated IL-27-producing B-1a cell population. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the IL-27-producing B-1a cell population to elicit a desired response in the individual.
Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents an autoimmune disease or symptom thereof. In this respect, the inventive method comprises administering a “prophylactically effective amount” of the isolated IL-27-producing B-1a cell population to a mammal that is predisposed to, or otherwise at risk of developing, an autoimmune disease. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of disease onset or prevention of disease flare-ups).
The isolated IL-27-producing B-1a cell population or composition comprising an isolated IL-27-producing B-1a cell population of the invention can be administered to a mammal using any suitable administration techniques, many of which are known in the art, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition preferably is suitable for parenteral administration. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
When the inventive method comprises administering an isolated IL-27-producing B-1a cell population to a mammal, the isolated IL-27-producing B-1a cell population is administered to the mammal at a dose sufficient to induce the generation of B-cells that produce IL-27 and suppress autoimmunity in the mammal. Therapeutic or prophylactic efficacy can be monitored by periodic assessment of treated patients. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
A typical amount of cells administered to a mammal (e.g., a human) can be, for example, in the range of 500,000 to 100 million cells, although amounts below or above this exemplary range can be suitable in the context of the invention. For example, the daily dose of cells can be about 500,000 to about 50 million cells (e.g., about 5 million cells, about 15 million cells, about 25 million cells, about 35 million cells, about 45 million cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 100 million cells (e.g., about 20 million cells, about 30 million cells, about 40 million, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, or a range defined by any two of the foregoing values), more preferably about 10 million cells to about 50 million cells (e.g., about 12 million cells, about 25 million cells, about 35 million cells, about 45 million cells, or a range defined by any two of the foregoing values).
The invention can be utilized in combination with other existing therapies for autoimmune diseases. For example, the cell population of the invention can be administered in combination with immunosuppressive or immunomodulating agents or other anti-inflammatory agents for the treatment or prevention of an autoimmune disease, such as the autoimmune diseases disclosed herein. In this respect, the inventive method can be used in combination with disease-modifying anti-rheumatic drugs (DMARD) (e.g., gold salts, sulphasalazine, antimalarias, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide, and glucocorticoids), a calcineurin inhibitor (e.g., cyclosporin A or FK 506), a modulator of lymphocyte recirculation (e.g., FTY720 and FTY720 analogs), an mTOR inhibitor (e.g., rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573, or TAFA-93), an ascomycin having immuno-suppressive properties (e.g., ABT-281, ASM981, etc.), corticosteroids, cyclophosphamide, azathioprene, methotrexate, leflunomide, mizoribine, mycophenolic acid, mycophenolate mofetil, 15-deoxyspergualine, or an immunosuppressive homologue, analogue or derivative thereof, immunosuppressive monoclonal antibodies (e.g., monoclonal antibodies to leukocyte receptors such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40. CD45, CD58, CD80, CD86, or their ligands), other immunomodulatory compounds, adhesion molecule inhibitors (e.g., LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists, or VLA-4 antagonists), a chemotherapeutic agent (e.g., paclitaxel, gemcitabine, cisplatinum, doxorubicin, or 5-fluorouracil), anti-TNF agents (e.g. monoclonal antibodies to TNF such as infliximab, adalimumab, CDP870, or receptor constructs to TNF-RI or TNF-RII, such as ENBRELTM (Etanercept) or PEG-TNF-RI), blockers of proinflammatory cytokines, IL-1 blockers (e.g., KINERET™ (Anakinra) or IL-1 trap, AAL160, ACZ 885, and IL-6 blockers), chemokine blockers (e.g., inhibitors or activators of proteases), anti-IL-15 antibodies, anti-IL-6 antibodies, anti-CD20 antibodies, NSAIDs, and/or an anti-infectious agent.
The invention can be utilized in combination with administration of B-cells that produce interleukin-35 (IL-35). The B-cells that produce IL-35 (i35-Bregs) can be administered sequentially (before or after) or simultaneously with the cell population of the invention to a mammal.
Embodiments of the invention may be beneficial alone or in combination, with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the invention are provided below as embodiments numbered 1-26. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. As such, the invention provides for all combinations of these embodiments and is not limited to combinations of embodiments explicitly provided below.
(1) An isolated population of mammal cells comprising about 75% or higher B-1a regulatory cells:
(a) expressing cell surface inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and C-X-C chemokine receptor type 4 (CXCR4); and (b) secreting interleukin-27 (IL-27).
(2) The population of mammal cells of embodiment (1), wherein the regulatory cells further express cell surface inhibitory receptor glucocorticoid-induced TNFR-related protein (GITR).
(3) The population of mammal cells of embodiment (1) or (2), wherein the regulatory cells further express cell surface inhibitory receptor OX40.
(4) The population of mammal cells of any one of embodiments (1)-(3), wherein the regulatory cells further express cell surface inhibitory receptor cytotoxic T-lymphocyte-associated protein 4 (CTLA4).
(5) A method of preparing the population of mammal cells of any one of embodiments (1)-(4), comprising
(6) A method of suppressing the immune system in a mammal, the method comprising administering to a mammal the population of mammal cells of any one of embodiments (1)-(4).
(7) The method of embodiment (6), further comprising sequentially or simultaneously administering B-cells that produce interleukin-35 (IL-35) to the mammal.
(8) The method of embodiment (6) or (7), wherein administration treats a disease in the mammal.
(9) The method of any of one of embodiments (6)-(8), wherein the mammal has an autoimmune disease.
(10) The method of embodiment (9), wherein the autoimmune disease is a disease of the eye.
(11) The method of embodiment (9), wherein the autoimmune disease is a disease of the central nervous system.
(12) The method of embodiment (9), wherein the autoimmune disease is a disease of the brain.
(13) The method of embodiment (9), wherein the autoimmune disease is uveitis.
(14) The method of embodiment (9), wherein the autoimmune disease is encephalomyelitis.
(15) The method of any of one of embodiments (6)-(8), wherein the mammal has multiple sclerosis.
(16) The method of any of one of embodiments (6)-(8), wherein administration suppresses inflammation of the pancreas.
(17) The method of embodiment (6) or (7), wherein the mammal has received an allogeneic bone marrow or hematopoietic stem cell transplant.
(18) The method of embodiment (6) or (7), wherein the mammal has received an allogeneic solid organ transplant.
(19) The method of embodiment (17) or (18), wherein the mammal has graft-versus-host disease (GVHD).
(20) The method of any of one of embodiments (6)-(8), wherein the mammal has age-related macular degeneration (AMD).
(21) A method of treating a mammal with graft-versus-host disease, the method comprising administering the population of mammal cells of any one of embodiments (1)-(4) to a mammal with graft-versus-host disease.
(22) The method of embodiment (21), wherein the mammal received an allogeneic bone marrow or hematopoietic stem cell transplant prior to the administration of the population of mammal cells.
(23) The method of embodiment (21), wherein the mammal received an allogeneic solid organ transplant prior to the administration of the population of mammal cells.
(24) A method of preventing or reducing the severity of graft-versus-host disease in a mammal, the method comprising administering the population of mammal cells of any one of embodiments (1)-(4) to a mammal before the mammal receives an allogeneic transplant.
(25) The method of embodiment (24), wherein the allogeneic transplant is an allogeneic bone marrow or hematopoietic stem cell transplant.
(26) The method of embodiment (24), wherein the allogeneic transplant is an allogeneic solid organ transplant.
(27) A method of preventing or reducing the severity of graft-versus-host disease in a mammal, the method comprising (a) mixing the population of mammal cells of any one of embodiments (1)-(4) with a transplant material to form a transplant mixture; and (b) administering the transplant mixture to a mammal.
(28) The method of embodiment (27), wherein the transplant material comprises allogeneic lymphocytes.
(29) The population of mammal cells of any one of embodiments (1)-(4) or the method of any one of embodiments (5)-(28), wherein the mammal is a human.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
The following materials and procedures were used in Examples 1-5.
Mice and human PBMC and human cord blood CD19+ cells. Six- to 8-week-old C57BL/6J and IL-27RαKO mice were purchased from Jackson Laboratory (Bar Harbor, Maine). Female mice were used, and the mice were randomized for all the studies described. Human peripheral blood mononuclear cells (PBMC) were obtained from the National Institutes of Health (NIH) Blood Bank administered by the NIH Department of Transfusion
Medicine. Primary human umbilical cord blood CD19+ B cells were purchased from STEMCELL™ Technologies (Vancouver, Canada).
Isolation of mouse and human B cells. PBMC of normal human subjects were isolated from buffy coats by density gradient centrifugation by using a commercially available lymphocyte separation medium (Mediatech Inc., Manassas, Virginia). Human CD19+B cells were sorted using anti-CD19 antibody-conjugated magnetic beads (Miltenyl Biotec, Bergisch Gladbach, Germany). Mouse B2 cells were isolated from the spleen using B cell Isolation kit (130-090-862), CD19 MicroBeads (130-052-201), and Plasma Cell Isolation Kit (130-092-530) (all available from Miltenyl Biotec). B1 cells were isolated from the peritoneal cavity of C57BL/6J mice. Some of the mice were immunized with LPS in the presence or absence of IL-27. For the B-1a cells, the isolation was performed in a two-step procedure using the B-1a Cell Isolation Kit; Catalog # 130-097-413) as recommended by the manufacturer. Briefly, B-1a cells from the peritoneal cavity were negatively selected over a MACS™ magnetic cell column consisting of magnetic beads labeled with a cocktail of biotin-conjugated non-B-1a antibodies and the B-1a cells. The B-1a cells were then positively selected with magnetic beads conjugated with B-1a-specific antibodies.
Immunofluorescence Staining and Confocal Imaging Analysis. CD19+ B cells were activated in vitro for 48 h by stimulation with LPS or anti-CD40/anti-IgM antibodies in presence or absence of IL-27. The cells were fixed, blocked with 5% goat serum, and then incubated with fluorescence labelled anti-p28 (Invitrogen, Waltham, Mass.) or anti-Ebi3 antibody (Santa Cruz Biotechnology, Dallas, Texas). Cells were washed, incubated in ALEXA FLUOR™ 568-, ALEXA FLUOR™ 488-, or ALEXA FLUOR™ 647-conjugated secondary antibody (Invitrogen) containing 4′,6-diamidino-2-phenylindole (DAPI), and examined on a laser scanning confocal microscope (FV1000, Olympus Corporation, Tokyo, JP, or LSM700, Carl s AG) (see Oh et al., J. Biol. Chem., 287: 30436-30443 (2012)).
Experimental autoimmune uveitis (EAU). EAU was induced by active immunization of C57BL/6J and IL-27RaKO mice with interphotoreceptor retinoid binding protein (IRBP)651-670-peptide in a 0.2 ml emulsion (1:1 v/v with complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis strain H37RA (2.5 mg/ml). Mice also received Bordetella pertussis toxin (1 μg/mouse) concurrently with immunization. Mice were treated by intraperitoneal injection of IL-27 (100 ng/mouse) or phosphate-buffered saline (PBS) on day −1 of immunization and every other day until day 12 post-immunization. For each study, 8 mice were used per group, and the mice were matched by age and sex. Clinical disease was established and scored by fundoscopy and histology (see Wang et al., Nat. Med., 20: 633-641 (2014), and Oh et al., I Immunol., 187: 3338-3346 (2011)). Eyes were examined for disease severity using a binocular microscope with coaxial illumination. Eyes for histology were enucleated 21 days post-immunization, fixed in 10% buffered formalin and serially sectioned in the vertical pupillary-optic nerve plane. All sections were stained with hematoxylin and eosin.
Fundoscopy. Funduscopic examinations were performed at day 10 to 21 after EAU induction. Briefly, following systemic administration of systemic anesthesia (intraperitoneal injection of ketamine (1.4 mg/mouse) and xylazine (0.12 mg/mouse)), the pupil was dilated by topical administration of 1% tropicamide ophthalmic solution (Alcon Inc., Fort Worth, Texas). The fundus image was captured using Micron III retinal imaging microscope (Phoenix Research Labs Pleasanton, California) for small rodent or a modified Karl Storz veterinary otoendoscope coupled with a Nikon D90 digital camera (see Oh et al., (2012), supra, and Paques et al., Invest Ophthalmol. Vis. Sci., 48: 2769-2774 (2007)). To avoid a subjective bias, evaluation of the fundus photographs was conducted without knowledge of the mouse identity by a masked observer. At least 6 images (2 posterior central retinal view, 4 peripheral retinal views) were taken from each eye by positioning the endoscope and viewing from superior, inferior, lateral and medial fields and each individual lesion was identified, mapped, and recorded. The clinical grading system for retinal inflammation was used (see Xu et al., Exp. Eye Res., 87: 319-326 (2008), and Chan et al., J. Autoimmun., 3: 247-255 (1990)).
Imaging mouse retina by Spectral-domain Optical Coherence Tomography (SD-OCT). Optical coherence tomography (OCT) is a noninvasive procedure that allows visualization of internal microstructure of various eye structures in living animals. An SD-OCT system with 820 nm center wavelength broadband light source (Bioptigen Inc., Morrisville, North Carolina) was used for in vivo non-contact imaging of eyes from control or EAU mice. Mice were anesthetized, and the pupils were dilated as described above. Mice were then immobilized using adjustable holder that could be rotated easily allowing for horizontal or vertical scan scanning. Each scan was performed at least twice, with realignment each time. The dimension of the scan (in depth and transverse extent) was adjusted until the optimal signal intensity and contrast was achieved. Retinal thickness was measured from the central retinal area of all images obtained from both horizontal and vertical scans from the same eye, using the system software, and averaged. A known method was used to determine the retinal thicknesses in the system software (see Gabriele et al., Invest. Ophthalmol. Vis. Sci., 52: 2250-2254 (2011)).
Electroretinogram (ERG). Before the ERG recordings, mice were dark-adapted overnight, and experiments were performed under dim red illumination. Mice were anesthetized with a single intraperitoneal injection of ketamine (1.4 mg/mouse) and xylazine (0.12 mg/mouse) and pupils were dilated with MIDRIN™ P containing of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Santen Pharmaceutical Co., Osaka, Japan). ERGs were recorded using an electroretinography console (Espion E2; Diagnosys LLC, Lowell, Massachusetts) that generated and controlled the light stimulus. Dark-adapted ERG was recorded with single-flash delivered in a ganzfeld dome with intensity of −4 to 1 log cd·s/m2 delivered in 6 steps. Light-adapted ERG was obtained with a 20 cd/m2 background, and light stimuli started at 0.3 to 30 cd·s/m2 in 5 steps. Gonioscopic prism solution (Alcon Labs, Fort Worth, Tex.) was used to provide good electrical contact and to maintain corneal moisture. A reference electrode (gold wire) was placed in the mouth, and a ground electrode (subcutaneous stainless steel needle) was positioned at the base of the tail. Signals were differentially amplified and digitized at a rate of 1 kHz. Amplitudes of the major ERG components (a- and b-wave) were measured (Espion software; Diagnosys LLC, Lowell, Massachusetts) using automated and manual methods. Immediately after ERG recording, imaging of the fundus was performed as previously described.
Retinal cells isolation. To characterize inflammatory cells that cross the blood-retina barrier during EAU, mice were anesthetized and perfused with lx PBS. Enucleated eyes were put in Petri dishes containing culture medium (Roswell Park Memorial Institute medium (RPMI 1640)) for immediate isolation of the retina under a dissecting microscope. The eye was cut along the limbus of the eye and the lens and cornea were carefully removed. Then, the retina was peeled off and the attached optic nerve was removed before digesting the freshly isolated retina with collagenase (1 mg/ml) in RPMI 1640 medium containing 10 μg/ml DNase (Sigma-Aldrich, St. Louis, Mo.) for 2 hours at 37° C. During incubation, the cells were pipetted intermittently every 30 minutes and the digestion reactions was quenched with 5-10 fold volumes of 10% fetal bovine serum (FBS) in RPMI 1640 medium. The cells were washed twice in complete RPMI 1640 medium and the cells were counted using the VI-CELL' XR cell viability analyzer (Beckman Coulter, Brea, California).
Cell co-culture. Uveitogenic cells isolated from the lymph nodes and spleen of mice with EAU, B-1a, macrophages, and dendritic cells were isolated from EAU immunized mice on day 17. B-1a, macrophages, and dendritic cells were isolated by magnetic column beads (Miltenyi Biotech). Co-culture experiments were performed in a Trans-well system (Coming Incorporated, Corning, New York) in RPMI 1640 medium with 10% FBS. After seeding uveitogenic cells or B-1a cells (5 x105) in the bottom well, macrophages or dendritic cells (5x105) were seeded in the upper chamber (pore size: 0.4 p.m) and re-stimulated with IRBP651-670 (20 μg/ml). Cells were collected for analysis with flow cytometry and thymidine incorporation assay after 72 h of the co-culture. For functional analysis of human B-1a cells, CD19+CD20+CD27+CD43+B1 cells from healthy controls were purified by cell sorting and stimulated with anti-CD40 (10 μg/m1) plus anti-IgM (5 μg/m1) in the presence or absence of rhlL-27 (100 ng/ml) for 72 h.
Experimental Autoimmune Encephalomyelitis (EAE). EAE was induced by subcutaneous immunization with 200 pg myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) (Sigma-Aldrich) in CFA emulsion, containing 2.5 mg/ml of heat killed, pulverized Mycobacterium tuberculosis strain H37RA. The mice also received two doses of 0.3 μg Bordetella pertussis toxin (Sigma-Aldrich) on day 0, and day 2 post-immunization by intraperitoneal (i.p.) injection in 100 μl of RPMI 1640 medium containing 0.1% normal mouse serum. Some mice received IL-27 (100 ng/mouse) concurrently with immunization with MOG35-55 and every other day until day 12 post-immunization. The control or IL-27-treated group (n=12) was euthanized 17 days post-immunization. The mice were monitored, and disease severity was assessed daily by a masked observer. Clinical signs of EAE were graded according to the following scale: 0, No clinical symptoms; 1, clumsiness, incontinence or atonic bladder, flaccid tail; 2, mild paraparesis (trouble initiating movement); 3, moderate paraparesis (hind limb weakness); 4, complete front and hind limb paralysis; 5, moribund state (see Liu et al., J. Immunol., 180: 6070-6076 (2008)). Spinal cords and brains were harvested 17 days post-immunization and stained with hematoxylin and eosin (H&E).
For adoptive transfer studies, mice with EAE, treated with or without IL-27, were sacrificed on day 10 post-immunization and used as donors in passive induction of EAE by adoptive transfer of encephalitogenic cells. Spleen cells were isolated, stimulated with MOG35-55 peptide (20 μg/ml) and anti-CD40 (10 μg/ml) antibody for 3 days in the presence or absence of IL-27 and transferred intravenously (i.v.) to naive syngeneic recipient mice (10×106 cells/mouse; n=12). Twenty days after adoptive cell transfer, disease was assessed and brain or spinal cord tissue was collected from recipient mice, fixed in 10% buffered formalin, and sectioned for histopathological examination. Central Nervous System (CNS) infiltrates were collected from the brain and spinal cord and lymphocytes/mononuclear cells were isolated by collagenase digestion followed by percoll gradient for analysis.
Adoptive transfer of B-1a cells. B-1a cells were isolated from the peritoneal cavity of donor mice and sorted using magnetic beads. The B-1a cells were cultured in complete RPMI 1640 with LPS (1 μg/ml) for 48 h, washed (2×) to remove residual LPS and adoptively transferred (5×105) into C57BL/6J and IL-27RaKO mice.
In vivo model of LPS-induced inflammation. C57BL/6J mice were injected with LPS (50 μg/mouse) and some mice received IL-27 (100 ng/mouse) 1 h before LPS injection by i.v. route. The mice in the control and IL-27-treated group (n=5) were euthanized 24 h post-injection and spleen cells were subjected to fluorescence-activated cell sorter (FACS) analysis.
Proliferation assay. Uveitogenic cells or B-1a cells were harvested from IRBP immunized C57BL/6J or IL-27RaKO mice at day 17 post-immunization. The cells were re-stimulated in vitro with IRBP peptide for 72 h in the presence or absence of B-1a, dendritic cell, and macrophages. For in vitro studies, CD19+B cells were stimulated with anti-CD40 antibodies (10 μg/ml) and anti-IgM antibodies (5 μg/ml) in the presence or absence of IL-27. Cells were pulsed with 3H-thymidine (0.5 μCOO μl/well) for the last 24 h in culture. Presented data are mean CPM±S.E.M. of responses of 5 replicate cultures.
Detection of Cytokine-expressing Lymphocytes by FACS. CD19+ B cells (>98%) were stimulated with LPS (2 μg/ml) or activated with anti-CD40 antibodies (10 μg/m1) and anti-IgM antibodies (5 μg/ml) as described above. For intracellular cytokine detection, cells were re-stimulated for 5 h with phorbol myristate acetate (PMA) (50 ng/ml)/ionomycin (500 ng/ml). GOLGIPLUG™ (BD Pharmingen, San Diego, California) was added in the last three hours and intracellular cytokine staining was performed using BD CYTOFIX/CYTOPERM™ kit as recommended (BD Pharmingen). FACS analysis was performed on a MACSQUANT™ analyzer (Miltenyi Biotec) using protein-specific monoclonal antibodies and corresponding isotype control antibodies (BD Pharmingen) (see Amadi-Obi et al., Nat. Med., 13: 711-718 (2007), and Wang et al., Nat. Med., 20: 633-641 (2014)). FACS analysis was performed on samples stained with monoclonal antibodies conjugated with fluorescent dyes (including CD19, CD20, CD24, CD27, CD38, CD43, CD138, and CD11b). Cells were color compensated and quadrant gates were set using isotype controls with less than 0.3% background. Live cells were subjected to side-scatter (SSC) and forward scatter (FSC) analysis.
Characterization of Regulatory B (Breg) and T (Treg) cells. Primary B cells isolated from the brain, spinal cord, retina, peritoneal cavity, blood, spleen, or draining lymph node (LN) of unimmunized, EAE or EAU mice were sorted for CD19+cells and used for surface and intracellular FACS analysis. Some cells were reactivated with LPS, IRBP651-670-peptide and anti-CD40 antibody, MOG35-55-peptide and anti-CD40 (see Wang et al., Nat. Med., 20: 633-641 (2014), and Choi et al., Front Immunol., 8: 1258 (2017)). For intracellular cytokine detection, cells were re-stimulated for 5 h with PMA (50 ng/ml) and ionomycin (500 ng/ml). GOLGIPLUG™ (BD Pharmingen) was added in the last hour, and intracellular cytokine staining was performed using the BD BD CYTOFIX/CYTOPERM™ kit as recommended (BD Pharmingen). FACS analysis was performed on a MACSQUANT™ analyzer (Miltenyi Biotec) using protein-specific monoclonal antibodies and corresponding isotype control antibodies (BD Pharmingen) as previously described by using protein-specific monoclonal antibodies and corresponding isotype control antibodies (BD Pharmingen). Dead cells were stained with dead cell exclusion dye (Fixable Viability Dye EFLUOR™ 450, Thermo Fisher Scientific), and live cells were subjected to side-scatter (SSC) and forward-scatter (FSC) analyses. Breg and Treg cells were characterized by analysis of the expression of CD4, CD19, CDS, CD27, CD38, CD138, B220, CD1d, IL-10, p28, p35 or Ebi3. FACS analysis was performed on cells stained with monoclonal antibodies conjugated with fluorescent dyes, dead cells were excluded, and each tube of cells was color-compensated. Quadrant gates were set using isotype controls with less than 0.5% background. CRISPR/Cas9-mediated gene deletion. sgRNA was generated and cloned into lentiCRISPR v2, pMD2.G using a known technique (see Sanjana et al., Nat. Methods, 11: 783-784 (2014)). The sgRNAs were selected by CRISPRSCAN, an online tool racking sgRNA sites by their on-target binding efficiency and probabilities of off-target hits. For IL-27, three sgRNAs were selected and cloned into a Lentiviral vector carrying the SpCas9 sgRNA scaffold driven by the U6 promoter. The sgRNA sequences were: sgp28 targeting site 1, 5′-GCTTCCTCGCTACCACACT-3′ (SEQ ID NO: 1), site 2; 5′-GGGCCATGAGGCTGGAT CTC-3′(SEQ ID NO: 2); site 3 5′-GATGGTATCCCAGGGGCAGG-3′(SEQ ID NO: 3). For Ebi3 targeting, the same Lentiviral vector was used for cloning of three sgRNAs: site 1; 5′-GTCGGGGATGGTGCATCGGG-3′(SEQ ID NO: 4); site 2 5′-TCTCTGATGGGTCACTAACT-3′(SEQ ID NO: 5); site 3 5′-CAGGAGCAGTCCACGGCCAC-3′(SEQ ID NO: 6). For deletion of IL-27, purified B-1a cells or macrophages were transduced with lentiviral clones expressing the sgRNAs. Two days after infection, cells were activated with LPS for 48 h and analyzed by FACS or ELISA.
Detection of cytokine secretion by ELISA. CD19+B cells or B-1a cells were activated in vitro in presence or absence of LPS, anti-CD40 plus anti-IgM and/or IL-27. Supernatants were collected after 48 h in culture. IL-27 and IL-35 were quantified using mouse IL-27- or IL-35-specific heterodimeric ELISA kit (BioLegend, San Diego, California). IL-17 or IL-10 were quantified using kits from R&D systems as recommended by manufacturer.
RNA extraction, NanoString analysis, and PCR. Total RNA was isolated from the peritoneal cavity or spleen using RNEASY™ plus mini kit (Qiagen, Hilden, Germany). cDNA synthesis, RT-PCR and qPCR analyses were performed according to known techniques (see Amadi-Obi et al., Nat. Med., 13: 711-718 (2007)). Each gene-specific primer pair used for RT-PCR analysis spans at least an intron. The following primers and probes used for qPCR were purchased from Applied Biosystems (Foster City, Calif.): IRF8 (Mm_00492567), IRF4 (Mm_00516431), BCL6 (Mm_00477633), Blimpl (Mm_00476128), Pax5 (Mm_00435501), Lag-3 (Mm_01185091), PD-1 (Mm_00435532), IL-27 (Mm_004461162), IL-12a (Mm_00434169), IL-10 (Mm_00439614), IL-27Ra (Mm_00497259), p21 (Mm 00817699), p27 (Mm_00438168), Cdkl (Mm 00772472), Cdk2 (Mm_00443947), Cdk4 (Mm_00726334), and mRNA expression was normalized to the levels of GADPH (Mm_99999915) genes. For NanoString nCounter analysis, 100 ng total of RNA per sample was used. A custom nCounter Gene Expression CodeSet immunology panel was used. Data was normalized using housekeeping genes and analyzed with nSolver Analysis software, version 3.
Immunoprecipitation and immunoblotting. Whole cell lysates were prepared according to a known technique (see Li et al., Invest. Ophthalmol. Vis. Sci., 40: 976-982 (1999)). Cleared lysates or cellular supernatants were immunoprecipitated with antibody that was pre-coupled to protein G-sepharose beads according to a known technique (see Oh et al., J. Biol. Chem., 286: 30888-30897 (2014)). Immunoprecipitates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blots were probed with specific antibodies. The following antibodies were used for immunoprecipitation and/or Western blotting: p28 (Invitrogen), Ebi3, and β-actin (Santa Cruz Biotechnology). Pre-immune serum was used in parallel as controls and signals were detected with HRP conjugated-secondary F(ab')2 (Zymed Labs, San Francisco, Calif.) using ECL system (Amersham, Arlington Heights, Ill).
Western blotting analysis. Preparation of whole cell lysates and performance of Western blot analysis were performed according to known techniques (see Wang et al., Nat. Med., 20: 633-641 (2014) and Egwuagu et al., J. Immunol., 168: 3181-3187 (2002)). Cell extracts (20-40 μg/lane) were fractionated on 10% gradient SDS-PAGE in reduced condition and Western blot analysis was performed using antibodies specific to pSTAT1, pSTAT3, STAT1, STAT3, p28, p35, Ebi3, IL-27Ra, GP130, IRF8 or β-Actin (Santa Cruz Biotechnology and Cell Signaling Technology, Danvers, Mass.). Pre-immune serum was used in parallel as controls and signals were detected with HRP-conjugated secondary F(ab′)2 Ab (Zymed Laboratories) using the ECL-PLUS system (Amersham). Each Western blotting analysis was repeated at least three times.
Chromatin Immunoprecipitation (ChIP) analyses. ChIP assays were performed using EZ-CHIP™ chromatin immunoprecipitation kits (Millipore Sigma, Darmstadt, Germany). B cells were activated with LPS in presence or absence of IL-27 and DNA-protein complexes were cross-linked for 10 min by addition of fresh formaldehyde (Sigma-Aldrich) to the culture medium at a final concentration of 1%, followed by quenching in 135 mM glycine. The cells were then washed in cold PBS (2×), lysed (EZ-CHIPTM lysis buffer) and sonicated (5×) in 15s bursts (output 5 on Sonic Dismembrator Model 1000, Thermo Fisher Scientific). Lysates were then cleared with Protein G-agarose for 1 h, pelleted, and incubated overnight with control IgG or anti-STAT1 or STAT3 antibody (Cell Signaling Technology). Prior to antibody incubation, input samples were removed from the lysate and stored at −80 ° C. until extraction. Immunoprecipitation was performed according to the manufacturer's instructions (EZ-CHIP™). The immunoprecipitated and input DNA were subjected to PCR and qPCR using primers to detect STAT1 and STAT3 binding activities. The primers (5′-CTGAAACCCCAGCTTCCTGCCA-3′ (SEQ ID NO: 7) and 5′-CATCTCCTGGGTAGGGGGGTCTTATACT-3′(SEQ ID NO: 8)) for IL-27p28 gene promoter are from -134 to -303 with STAT binding motif GGAAGGGAAATTACGTT (SEQ ID NO: 9), while the primers (5′-CTGATTCTGTCTCTGTTTCTCTCAGTT-3′ (SEQ ID NO: 10) and 5′-GTGGGGAAAGGCCTTGAGGTAGA-3′ (SEQ ID NO: 11)) for EBI3 gene promoter region are from -1 to -150 with STAT binding motif CCTCAAGGCCTTTCC (SEQ ID NO: 12).
Electrophoretic mobility shift assay (EMSA). EMSA was performed according to well-known procedures (see Yu et al., J. Immunol., 157: 126-137 (1996)). The double stranded oligonucleotides containing motifs from the AP1-IRF-1 composite elements (AICE) 5′TGAnTCA/GAAA-3′ (SEQ ID NO: 13) were labeled by a fill-in reaction using Klenow polymerase (New England BioLabs, Beverly, Mass.) with [alpha-P32]dATP or (alpha-32P)dGTP (3000 Ci/mmol) (PerkinElmer Inc., Waltham, Mass.). Sorted CD19+ B cells were stimulated with LPS (1 μg/ml) in the presence or absence IL-27 (20 μg/ml) for three days and nuclear extracts were prepared in buffer containing the following protease inhibitors: 2 μM leupeptin, 2 μM pepstatin, 0.1 μM aprotinin, 1 mM [4-(2-aminoethyl)benzenesulfonyl fluoride, hydrochloride], 0.5 mM phenylmethyl-sulfonyl fluoride, and 1 μM E-64 [N-(N-1-trans-carboxyoxiran-2-carbonyl)-1-leucyflagmatine according to known procedures (see Yu et al., J. Immunol., 157: 126-137 (1996)). Protein levels were determined by the BCA method as recommended, and extracts were stored at —70 ° C. until use. DNA-protein binding reaction was performed in a 20-p.1 mixture containing 5 μg nuclear protein and 1 μg double-stranded poly(d1:C) (Boehringer Mannheim, Barcelona, Spain), 12 mM HEPES (pH 7.9), 60 mM KCI, 0.5 mM DTT, 12% glycerol, 2.5 mM MgCl. After a 15 min incubation on ice, samples were further incubated with 1 μl P32-labeled probe (15,000 cpm) at room temperature for 20 min and fractionated on 5% native polyacrylamide gel in 0.25×Tris-borate-EDTA buffer. For super-shift analysis, before the addition of 32P-labeled probes, extracts were pre-incubated with 1 μl antibodies specific to basic leucine zipper transcription factor (BATF) (Cell Signaling Technology), Jun B, Jun D, IRF-4, IRF-8 or IRF-1 (Santa Cruz Biotechnology).
Proximity ligation assay. The Proximity Ligation Assays (PLA) were performed with Duolink PLA kit (Sigma Aldrich, St. Louis, Mo.). Activated B cells were attached to slides, blocked for 1 h in blocking solution and then incubated overnight with primary mouse anti-p28 (rabbit) and anti-Ebi3 (mouse). A pair of oligonucleotide-labeled secondary Abs (PLA probes) that bind to the primary Abs were then added, incubated for 1 h, and then ligation solution containing hybridizing connector oligos was added. PLA probes in close proximity (within 40nm) then interacted and ligated to the connector oligos. The resulting closed, circular DNA template was amplified by DNA polymerase. Complementary detection oligos coupled to fluorochromes hybridized to repeating sequences in the amplicons and p28:Ebi3 heterodimers are then detected as discrete fluorescent spots by confocal microscopy (LSM 700, Carl Zeiss AG, Oberkochen, Germany).
RNA-Seq and Analysis. For RNA-Seq, mRNA was isolated by oligo-dT beads and a library was prepared using the standard Illumina, Inc. library protocol (kit RS-122-2101 TruSeq Stranded mRNA LT Sample prep kit, Illumina, Inc., San Diego, Calif.). Libraries were sequenced on the NovaSeq 6000 system (Illumina, Inc.). The relative abundances of genes were measured in Read Count using StringTie. The statistical analysis was performed to find differentially expressed genes using the estimates of abundances for each gene in samples. Genes with one more than zeroed Read Count values in the samples were excluded. To facilitate loge transformation, 1 was added to each Read Count value of filtered genes. Filtered data were loge-transformed and subjected to the trimmed mean of M-values (TMM) normalization method. Statistical significance of the differential expression data was determined using exact t-test using edgeR and fold change in which the null hypothesis was that no difference exists among groups. P values were adjusted for multiple testing using the false discovery rate (FDR) correction of Benjamin and Hochberg. For heatmaps, the R package heatmap and Broad institute tool Morpheus was used to normalized counts. Hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity to display the expression patterns of differentially expressed transcripts which are satisfied with |fold change|≥2 and independent t-test raw p <0.05.
Statistical analysis. Graphs were plotted and analyzed using GraphPad Prism 7.0, two-tailed unpaired Student's t test, non-parametric Mann-Whitney U-test or One way ANOVA depending on the experiments. Probability values of <0.05 were considered statistically significant. Some data are presented as mean +SEM. Asterisks denote p value as follows: *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
Sample sizes are indicated in figures or figure legends and refer to number of animals. In vitro assays using human cord blood or PBMC were repeated independently using cells from at least three unrelated donors. Results shown represent at least three independent experiments as noted in the legends. Optical coherence tomography, ERG and confocal image analyses were performed blindly. EAE and EAU scoring were performed by masked investigators. Essential immunotherapeutic effects of i27-Breg in EAU was validated and recapitulated in the EAE model. Mice were age/sex matched and randomized, consisting of equal numbers of males and females.
This example demonstrated that peritoneal B1 cells secrete IL-27 (i27-Bregs) and activation of i27-Bregs during inflammation triggers their exodus into secondary lymphoid tissues.
Immunohistochemical/confocal microscopy co-localized p28 and Ebi3 expression on activated mouse CD19+ B-cells (
FACS analysis of activated B-cells revealed a discrete population of IL-27-producing B-cells (about 7.73%) that increased (2.85-fold) in response to IL-27 (
To investigate whether B-cells can produce IL-27 in vivo, C57BL/6J mice were injected (i.v) with LPS, and the percentage of IL-27-producing B-1a or B2 cells in the peritoneal cavity or spleen was determined. As many as about 19.4% of B-1a cells in the peritoneal cavity of PBS-treated mice were producing IL-27 at the 24 h time-point while the percentage of these cells increased to ˜55.6% in mice injected with LPS (
The data show a time-dependent increase of CXCR3- and CXCRS-expressing B-1a cells in the spleen which coincided temporally with significant decrease of CXCR4-expressing B-1a cells in the peritoneal cavity (
Taken together, these observations suggest that differential regulation of chemokine receptors expression by B-1a cells in response to IL-27 promotes egress of B-1a cells from the peritoneal cavity and their subsequent trafficking to the spleen.
This example demonstrated that IL-27-producing B-1a cells (i27-Bregs) confer protection from severe uveitis.
EAU is an animal model of human uveitis and is a predominantly T cell-mediated intraocular inflammatory disease induced by immunization with retinal proteins/peptides in CFA. The EAU model was used to investigate whether i27-Bregs contribute to regulating immunity during uveitis. EAU was induced in C57BL/6J mice by immunization with a peptide derived from interphotoreceptor-retinoid-binding protein (IRBP651-670), and the mice were treated with PBS (control) or IL-27 concurrent with immunization. Fundus images of PBS-treated mice revealed characteristic features of uveitis including blurred optic disc margins, enlarged juxta-papillary area, moderate to severe retinal vasculitis, and cellular infiltrate (
To investigate the potential therapeutic importance of i27-Bregs, peritoneal cavity B-1a cells (>80% i27-Bregs) were purified from WT donor CD45.2+mice with EAU, transferred 5×105 cells/mouse to naive syngeneic WT or IL-27RαKO CD45.1+ mice, and then induced EAU 24 h after prophylactic administration of the B-1α cells. Fundus images on day-17 post-immunization showed severe uveitis in IL-27Rα-deficient mice (
Further, it was found that plasmacytoid dendritic cells induce expansion of i27-Breg cells as confirmed by flow cytometry following co-culture of activated IL-27-producing B-1a and plasmacytoid dendritic cells (1:1) (see
This example demonstrated that i27-Bregs in the brain and spinal cord suppress neuroinflammation and encephalomyelitis.
For these studies, the EAE model was used that shares essential immunopathogenic features with multiple sclerosis (MS) and exhibits progressive and relapsing-remitting forms of the human disease. EAE was induced by immunization of C57BL/6J mice with MOG35-55-peptide/CFA. Control PBS-treated mice developed EAE characterized by infiltration of inflammatory cells into the brain and spinal cord, flaccid tail, paraparesis, front/hind limb paralysis, and moribund state (
The role of i27-Breg cells in suppressing EAE was further demonstrated in adoptive transfer studies using CD45.1+ and CD45.2+ congenic mouse strains. CD45.2+ mice were immunized with MOG35-55-peptide/CFA and treated with PBS or IL-27. Encephalitogenic cells were harvested from the spleen and LN 21 days after immunization, and 10×106 cells from PBS-treated or IL-27-treated CD45.2+ mice were adoptively transferred to unimmunized CD45.1+ mice and evaluated for EAE development and severity. Transfer of cells from PBS-treated mice induced disease with characteristic features of EAE, while CD45.1+ mice that received CD45.2+ cells from IL-27-treated mice developed mild
EAE with delayed onset (
EXAMPLE 4
This example demonstrated that innate IL-27-producing B-1a cells suppress EAE and EAU in antigen-independent manner.
Bregs are mostly antigen-specific and effective in suppressing diseases mediated by lymphocytes that recognize the same cognate autoantigen. Thus, it was investigated whether IL-27-producing B-1a cells induced by an irrelevant stimulus like LPS could suppress encephalitogenic lymphocytes that mediate EAE. CD45.2+ C57BL/6J mice were injected with LPS, and after 2 days purified B-1a cells were obtained from the peritoneal cavity (>80% B-1a i27-Bregs). The i27-Bregs then were transferred into naïve CD45.1+ congenic mice. EAE was induced in recipient CD45.1+ mice by immunization with MOG35(n=7) 24 h after adoptive transfer. Transfer of the ex-vivo generated B-1a i27-Bregs (5 x105 cells/mouse) suppressed EAE (
This example demonstrated that cross talk exists between IL-27-producing B-1a and lymphoid or myeloid cells in the CNS.
This study examined whether i27-Bregs that enter the CNS during EAE or EAU might be a source of IL-27 that contributes to immune-suppressive environment of the CNS. B-1a cells and macrophages from IRBP-immunized wild type were sorted, and it was found that co-culture of the cells for 3 days in a trans-well system significantly increased of IL-27-producing B-1a cells (
The data show that co-culture of B-1a cells and uveitogenic T-cells induced the expansion of CD4+ T-cells expressing the inhibitory receptor, LAG-3 (LAG-3+ CD4+ T-cells) (
This example demonstrated that IL-27 regulates B1 and B2 cells differently.
This study examines whether the B-1a cells that suppress EAU and EAE through production of IL-27 also suppress inflammation by expressing inhibitory molecules. B-1a cells were isolated from mouse peritoneal cavity by sorting. The cells were then stimulated for 48 h with LPS and demonstrated that they were B-1a cells by their capacity to produce IgM (
A previous report indicated that IL-35-producing B-cells are exclusively B2 CD138+ plasma cells (Shen et al., Nature, 507: 366-370 (2014)). However, this study shows that IL-27-producing B-cells derive from the B1 compartment. To understand mechanisms that skew activated B-cells toward the i27-Breg developmental program, the transcriptome of activated CD19+ B-cells stimulated with IL-27 was profiled. qPCR (
This example demonstrated that i27-Breg cells exist in humans and can be expanded in response to inflammatory stimuli.
This study examined whether i27-Breg cells existed in humans and expanded in response to inflammatory stimuli by culturing healthy human PBMC for 3 days with TLR agonist CpG and BCR (anti-CD40 or anti-IgM). Gating on human B-1 cells (CD19+CD20+CD27+CD43+) revealed that as high as 19.9% of BCR-activated B-cells in-human PBMC produced IL-27 (
This example demonstrates that human i27-Breg cells can be used to successfully treat humans suffering from a disease or at risk for suffering from a disease.
Human i27-Breg cells are administered, by injection or i.v., to a human suffering from a disease, such as uveitis, MS, AMD, and/or GVHD, or a human in need of the prevention of a disease, such as GVHD. Following administration of the human i27-Breg cells, the severity and/or the symptoms of the disease will be decreased and/or prevented.
This example demonstrates that i27-Breg have a unique transcriptome.
Peritoneal cavity B-1a cells enriched for i27-Breg cell by activation with BCR and IL-27 were used to determine the gene expression program required for the development of i27-Breg cells. Characterization of the highly enriched IL-27-producing B-1a cells (>83% i27-Bregs) revealed that while B-1a cells constitutively secrete natural IgM antibodies, the development into i27-Breg cell phenotype coincides with loss of capacity to produce IgM (
This example demonstrates that i27-Breg and i35-Breg in human cord blood and PBMC have distinct transcriptomic profiles.
Human PBMC and cord blood (CB) B cells produced IL-27 and in the PBMC ˜19.9% of activated B-1-like cells (CD19+CD20+CD27+CD43+) are i27-bregs (
This example demonstrates that innate i27-Bregs suppress CNS autoimmune disease through a BCR-independent mechanism.
Peritoneal cavity B-1 cells are to a large extent unresponsive to BCR-induced signals, but highly responsive to innate immune signals induced by pathogens and TLR agonists, suggesting that immune-suppressive activities of i27-Bregs. To address whether i27-Breg-mediated suppression of EAU or EAE requires prior activation by IRBP or MOG autoantigen, “sepsis” was induced in CD45.2+C57BL/6J mice by injection of LPS, sorted B-1a cells (>83.5% i27-Bregs) from the peritoneal cavity, and the i27-Breg-enriched cells (5×105/mouse) were adoptively transferred into naïve CD45.1+congenic mice. 24 h later the mice were challenged by EAE induction. Clinical evaluation of the mice revealed significant suppression of EAE (
Collectively, the above examples show an innate IL-27-producing Breg population exists in human cord blood, PBMC, as well as the brain, spinal cord, retina, and peritoneal cavity of mice suffering from experimental autoimmune encephalomyelitis (EAE) or experimental autoimmune uveitis (EAU), which are models of multiple sclerosis and uveitis, respectively. In vitro experimental systems including, confocal microscopy, FACS-based cell sorting, RNA-seq, Chip assay, and immunohistochemistry show that the IL-27-producing Breg has a unique transcriptome and is functionally distinct from other Bregs. Adoptive transfer of i27-Bregs ameliorated EAE and EAU by reprogramming resting B cells to i35-Breg cells that trafficked to the uvea, brain, and spinal cord and suppressed pathogenic T cells, thus demonstrating the efficacy of i27-Breg immunotherapy.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This patent application claims the benefit of co-pending U.S. Provisional Patent Application No. 62/863,054, filed Jun. 18, 2019, which is incorporated by reference in its entirety herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/038368 | 6/18/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62863054 | Jun 2019 | US |
