Method of treating autoimmune and inflammatory diseases using B cells
12544439 ยท 2026-02-10
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P37/06
HUMAN NECESSITIES
A61K2239/38
HUMAN NECESSITIES
International classification
Abstract
GITRL.sup.+ IgD.sup.low/ B cells as well as methods of making and using said cells are described herein. Also described are methods for treating an autoimmune disease or an inflammatory condition in a subject in need thereof of using said B cells. The B cells may be GITRL.sup.+ IgD.sup.lowCCR7.sup.+ CXCR5.sup.+ B cells or GITRL.sup.+ IgD.sup.low CCR7.sup.+ CXCR5.sup.+ CD23.sup.+ CD24.sup.+ B cells.
Claims
1. A method for treating an autoimmune disease or an inflammatory condition in a subject in need thereof comprising: Administering to the subject in need thereof a therapeutically effective amount of glucocorticoid-induced tumor necrosis factor receptor ligand positive (GITRL.sup.+), immunoglobulin D low (IgD.sup.low), CCR7.sup.+, CXCR5.sup.+ B cells.
2. The method of claim 1, wherein administration of the GITRL.sup.+ IgD.sup.low B cells stimulates proliferation of CD4.sup.+ Foxp3.sup.+ T regulatory cells.
3. The method of claim 1, wherein the B cells are GITRL.sup.+ IgD.sup.low CCR7.sup.+ CXCR5.sup.+ CD23.sup.+ CD24.sup. B cells.
4. The method of claim 1, wherein the B cells are administered using adoptive cell transfer.
5. The method of claim 1, wherein the B cells are administered by infusion.
6. The method of claim 1, wherein the autoimmune disease or inflammatory condition is selected from the group consisting of multiple sclerosis, Guillian-Barr syndrome, autism, thyroiditis, Hashimoto's disease, Graves' disease, rheumatoid arthritis, ankylosing spondylitis, polymyalgia rheumatica, psoriasis, vitiligo, eczema, scleroderma, fibromyalgia, Wegener's granulomatosis, peripheral neuropathy, diabetic neuropathy, celiac disease, Crohn's disease, ulcerative colitis, type I diabetes, leukemia, lupus erythematosus, and hemolytic dysglycemia.
7. The method of claim 1, wherein the B cell is an engineered B cell that comprises a suicide gene selected from the group consisting of Herpes simplex virus thymidine kinase (HSV-TK), human inducible caspase 9 (icasp9), mutant human thymidylate kinase (mtmpk), and human CD20.
8. The method of claim 1, wherein the B cell is derived from a chronic lymphocytic leukemia cell line.
9. The method of claim 1, wherein the B cell does not express 2-microglobulin.
10. The method of claim 1, wherein the B cell does not express class II major histocompatibility complex transactivator (CIITA).
11. The method of claim 1, wherein the autoimmune disease or inflammatory condition is multiple sclerosis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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INCORPORATION BY REFERENCE
(24) All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
(25) The present disclosure broadly relates to a GITRL positive B cell as well as methods for making and using such a B cell.
(26) In some aspects, provided herein is an engineered GITRL positive, IgD.sup.low/ B cell. The B cell may be engineered to include a non-natural mutation that increases, decreases, or otherwise modulates expression of one or more markers or cytokines to provide a B cell with improved stimulation of homeostatic T regulator cell expansion as described herein. The B cell may also be engineered to decrease or eliminate in vivo reproduction, proliferation, and expansion of the B cells. In some aspects, provided herein is a GITRL+, IgD.sup.low/, CCR7.sup.+ B cell that cannot reproduce, proliferate or expand in vivo following administration to a subject.
(27) In some aspects, provided herein is a treatment for patients experiencing abnormally increased immune system reactivity or an autoimmune disease. The treatment includes administration of GITRL positive B cells to a subject in need thereof.
(28) Certain embodiments of the disclosure are described in Ray et al. 2019 (Ray, A., Khalil, M., Pulakanti, K. L., Burns, R. T., Gurski, C. J., Basu, S., Wang, D., Rao, S. and Dittel, B. N. 2019. Mature IgD.sup.low/ B cells maintain tolerance by promoting regulatory T cell homeostasis. Nat. Commun. doi.org/10.1038/s41467-018-08122-9), which is incorporated herein in its entirety.
(29) As used herein and in the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. As well, the terms a (or an), one or more and at least one can be used interchangeably herein. It is also to be noted that the terms comprising, including, and having can be used interchangeably.
(30) As used herein, the terms approximately or about in reference to a number are generally taken to include numbers that fall within a range of 5% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context.
(31) Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
(32) The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); and Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986).
(33) By modulation of a target gene, we mean to include over-expression, increased function, reduced-expression, reduced function, or gene knockout. In some embodiments, the target gene is over-expressed. In some embodiments, expression of the target gene is reduced. In some embodiments, the target gene is knocked out. By modulation of the gene, we also mean to include modification or manipulation of the regulatory regions of the target gene.
(34) By a non-natural modification, we mean to include all manner of recombinant and transgenic manipulation to the B cell. For example, a B cell comprising an extra copy of the target gene has a non-natural modification. A B cell comprising a vector containing the target gene and a promoter is a non-natural modification. A B cell comprising a vector expressing an miRNA, a dsRNA, an hpRNA, a siRNA, or other RNA associated with RNA interference to silence or down regulate gene expression is a non-natural modification. We also mean to include modification or manipulation of the regulatory regions of the target gene or of any region that is contiguous with the target gene up to 5 KB on either side of the target sequence. Non-natural modification also covers CRISP/Cas9 mediated gene editing of a target gene. Also included are modifications to the target gene such as, but not limited to, insertions, deletions, non-sense mutations, substitutions, etc., that may increase or decrease the expression of the target gene or gene product thereof.
(35) By engineered B cell or engineered BD.sub.L, we mean a B cell or BD.sub.L that includes a non-natural modification as described herein. In some embodiments, the engineered B cell comprises an extra copy of the target gene. In some embodiments, the engineered B cell comprises a vector containing the target gene and a promoter. In some embodiments, the engineered B cell includes a non-natural modification or manipulation of the regulator region of the target gene or of any region that is contiguous with the target gene up to 5 KB on either side of the target gene sequence. In some embodiments, the engineered B cell comprises a vector expressing an RNA associated with RNA interference to silence or down regulate gene expression. In some embodiments, the engineered B cell comprises an RNA selected from the group consisting of miRNA, dsRNA, hpRNA, or siRNA specific to or complementary to the target gene, whereby said RNA downregulates or silences the gene. In some embodiments, a target gene of the engineered B cell has been modified using CRISPR/Cas9 mediated gene editing. In some embodiments, a target gene of the engineered B cell has been modified using lentiviral transduction mediated gene editing. In some embodiments, a target gene of the engineered B cell has been modified with an insertion, deletion, non-sense mutation, substitution, etc., that increases or decreases the expression of the target gene product thereof.
(36) As used herein, BD.sub.L refers to a B cell that is glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) positive (GITRL.sup.+) and stimulates homeostatic expansion of CD4+Foxp3+T regulator cells (Tregs). In some embodiments, the BD.sub.L are also immunoglobulin D low or silent (IgD.sup.low/), CD23.sup.+, B220.sup.+, CD21.sup.+, and/or CD93.sup.+. In some embodiments, the BD.sub.L also express one or more markers selected from the group consisting of CCR7, CXCR5, CD24, and CD86.
(37) As used herein, high means that the cells are characterized by higher expression of a particular gene compared to the average expression of said gene in well-defined B cell subsets with well-described expression levels as reporting in the art. See, for example, Allman et al. 2001 (Allman, D. et al. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J Immunol. 167, 6834-6840 (2001)), Allman et al. 2008 (Allman, D. & Pillai, S. Peripheral B cell subsets. Curr. Opin. Immunol. 20, 149-157 (2008)), and Meyer-Bahlburg et al. 2008 (Meyer-Bahlburg, A., Andrews, S. F., Yu, K. O., Porcelli, S. A. & Rawlings, D. J. Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation. J Exp. Med. 205, 155-168 (2008)). For example, GITRL high indicates that GITRL expression is higher than the average expression in a population of B cells. Similarly, low means that the cells are characterized by lower expression of a particular gene. For example, IgD low indicates that IgD expression is lower than the average expression in a population of B cells. Low can also mean that the expression levels are below the detection limit or that the cell is engineered to silence the particular gene of interest.
(38) In some embodiments, the BD.sub.L are genetically engineered to increase, introduce, or modulate the expression of one or more target genes or expression products. In some embodiments, the BD.sub.L are genetically engineered to reduce, silence, or delete the expression of one or more target genes or expression products. Target genes and expression products of interest that may be modulated in the BD.sub.L include, but are not limited to GITRL, IgD, class II major histocompatibility complex transactivator (CIITA), 2-microglobulin, CC-chemokine receptor 7 (CCR7), C-X-C motif chemokine receptor 5 (CXCR5), CD20, human leukocyte antigen G (HLA-G), human leukocyte antigen E (HLA-E), CD86, suicide genes, and a Cre recombinase.
(39) In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases GITRL expression.
(40) In some embodiments, the engineered BD.sub.L include a non-natural mutation that silences, downregulates, or reduces expression of IgD. In some embodiments, the engineered BD.sub.L include a non-natural mutation that is a deletion of the IgD gene.
(41) In some embodiments, the engineered BD.sub.L include a non-natural mutation that silences, downregulates, or reduces expression of CIITA. In some embodiments, the engineered BD.sub.L include a non-natural mutation that is a deletion of the CIITA gene. Without wishing to be bound by any particular theory embodiment, deletion, silencing, downregulation or reduction in CIITA will disrupt the ability of resulting B cells to express classical and nonclassical major histocompatibility complex (MHC) class I molecules on their cell surface. Resulting B cell lacking surface MHC expression will not be cleared by the immune system when administered to a subject in need thereof.
(42) In some embodiments, the engineered BD.sub.L include a non-natural mutation that silences, downregulates, or reduces expression of 02-microglobulin. In some embodiments, the engineered BD.sub.L include a non-natural mutation that is a deletion of the .sub.2-microglobulin gene. Without wishing to be bound by any particular theory embodiment, deletion, silencing, downregulation or reduction in 02-microglobulin will disrupt the ability of resulting B cells to express classical and nonclassical major histocompatibility complex (MHC) class I molecules on their cell surface. Resulting B cell lacking surface MHC expression will not be cleared by the immune system when administered to a subject in need thereof.
(43) In some embodiments, the engineered BD.sub.L include a non-natural mutation that modulates expression of CCR7. In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases expression of CCR7.
(44) In some embodiments, the engineered BD.sub.L include a non-natural mutation that modulates expression of CXCR5. In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases expression of CXCR5.
(45) In some embodiments, the engineered BD.sub.L include a non-natural mutation that silences, downregulates, or reduces expression of CD20. In some embodiments, the engineered BD.sub.L include a non-natural mutation that is a deletion of the CD20 gene.
(46) In some embodiments, the engineered BD.sub.L include a non-natural mutation that introduces the HLA-G gene into the B cell. In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases expression of HLA-G. In some embodiments, the engineered BD.sub.L include a non-natural mutation that introduces the HLA-E gene into the B cell. In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases expression of HLA-E. Without wishing to be bound by any particular theory or embodiment, the resulting B cells that express HLA-G and/or HLA-E will be protected from immune system clearance by Natural Killer cells when administered to a subject in need thereof.
(47) In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases CD86 expression.
(48) In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases CD24 expression.
(49) In some embodiments, the engineered BD.sub.L include a non-natural mutation that introduces a Cre recombinase gene into the B cell. In some embodiments, the engineered BD.sub.L include a non-natural mutation that increases expression of a Cre recombinase gene.
(50) In some embodiments, the BD.sub.L are derived from a B cell line such as a chronic lymphocytic leukemia line (CCL line) or other cancerous line of cells. Other suitable cell lines include, but are not limited to, Hs505.T, OSU CLL, or other lines created through Epstein-Barr virus transformation of patient tumors. There are several examples of commercially available cell lines or others described in the scientific literature. The cells would be grown in culture and cells expressing high levels of GITRL and low levels of IgD would be selected. In some embodiments, the cells are genetically engineered to overexpress GITRL and to lower or silence IgD expression.
(51) In some embodiments, engineered BD.sub.L as described herein are made by transfecting a population of B cells with one or more nucleic acids to modulate a target gene of interest. The nucleic acid may be part of a viral or plasmid vector. Suitable vectors and transfection methods are known and used in the art. Suitable vectors include, but are not limited to, viral vectors, such as lentiviral, baculoviral, adenoviral, or adeno-associated viral vectors, yeast systems, microvesicles, gene guns, and gold nanoparticles. Suitable viral delivery vectors include, without limitation, adeno-viral/adeno-associated viral (AAV) vectors, lentiviral vectors, and Herpes Simplex Virus 1 (HSV-1) vectors. In some embodiments, the nucleic acid or vector may be delivered to the cell via liposomes, nanoparticles, exosomes, microvesicles, or a gene gun.
(52) Gene editing using the precise targeting process of Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) combined with the Cas9 nuclease to make a double stranded break (collectively referred to as CRISPR/Cas9 or CRISPR/Cas9 system) may be used to modulate a target gene as described herein. The site of the break is targeted by short guide RNA (sgRNA or gRNA) often about 20 nucleotides in length. The break can be repaired by non-homologous end joining (NHEJ) or homology-directed recombination to make a desired mutation or modification at the cut site. Suitable CRISPR/Cas9 nucleases and vectors are known in the art. See for example US20180273961, U.S. Pat. No. 8,771,945, Reem et al. (Reem N. T., Van Eck J. (2019) Application of CRISPR/Cas9-Mediated Gene Editing in Tomato. In: Qi Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana Press, New York, NY), and Brooks et al. (Brooks C., Nekrasov V., Lippman Z. B., Van Eck J. (2014).
(53) In some embodiments, the expression of the target gene is modulated by transformation of the B cell with multiple copies of the gene, use of modified or natural promoters designed to overexpress or downregulate the gene, use of constitutive or tissue-specific promoters designed to focus expression in specific tissues or in a non-specific manner, and use of vectors to carry a copy or copies of the gene. One may also wish to transform B cells with regulatory elements that will modify the native expression of the target gene or modify existing regulatory elements.
(54) In some embodiments, prior to administration to a subject the engineered B cell is modified or killed so that it is unable to reproduce, proliferate, or expand in vivo causing toxicity to the subject, e.g., proliferation that would cause a malignancy. Suitable methods and reagents to reduce reproduction, proliferation, and expansion of B cells in vivo are known and used in the art. In some embodiments, the B cells are treated with radiation or chemicals to prevent them from further dividing once administered to a patient. In some embodiments, the B cells are engineered to include a suicide gene to kill the engineered B cell prior to or after administration to the subject. A skilled artisan will recognize that the strategy used to kill the B cells prior to infusion will be determined in conjunction with regulatory approving agencies for a therapeutic clinical trial.
(55) In some embodiments, the B cells are treated with a chemical selected from the group consisting of doxorubicin, 5-Fluorouracil, paclitaxel, vinblastine, staurosporine, or combinations thereof. In some embodiments, the B cells are treated with about 0.1 to about 0.5 g/ml doxorubicin. In some embodiments, the B cells are treated with about 100-200 nM paclitaxel. In some embodiments, the B cells are treated with about 50 to about 100 nM vinblastine. In some embodiments, the B cells are treated with about 0.5 to about 5 mM staurosporine.
(56) In some embodiments, the B cells are treated with a total dose of radiation between about 30 and about 40 Gy. In some embodiments, the B cells are treated with a radiation dose between 0.2-0.3 Gy/h for about 7 days. In some embodiments, the B cells are treated with a radiation dose between 0.4-0.6 Gy/hr for about 3 days.
(57) Suicide genes could also be employed to kill the BD.sub.L cell populations and several examples of this can be found in the literature. See, for example Marin et al. (Comparison of Different Suicide-Gene Strategies for the Safety Improvement of Genetically Manipulated T Cells, Hum Gene Ther Methods, 2012, (6):276-286). Marin describes the use of 4 different strategies to incorporate suicide genes into immune cells. Any of these strategies could be employed to modify engineered B cells, including BD.sub.L cell populations, prior to infusion into patients to induce cell suicide prior to or after administration to the subject. Suitable suicide genes include, but are not limited to, Herpes simplex virus thymidine kinase (HSV-TK), human inducible caspase 9 (iCasp9), mutant human thymidylate kinase (mTMPK), and human CD20. In some embodiments, the suicide genes are codon-optimized prior to introduction into the engineered B cell. In some embodiments, the suicide genes are cloned in from with 2A-truncated and codon optimized CD34 and transduced into the B cell with a retroviral vector.
(58) Treatment
(59) According to the methods of the present invention, BD.sub.L are administered to a subject in need of thereof. Subjects in need of treatment include those already having or diagnosed with a disease as described herein or those who are at risk of developing a disease as described herein.
(60) A disease or injury of the present invention may include, but is not limited to, conditions associated with aberrant, uncontrolled, or inappropriate inflammation, and autoimmune diseases.
(61) Autoimmune disease may refer to, but is not limited to, multiple sclerosis, Guillain-Barry syndrome, autism, thyroiditis, Hashimoto's disease, Graves' disease, rheumatoid arthritis, ankylosing spondylitis, polymyalgia rheumatica, psoriasis, vitiligo, ecxema, scleroderma, fibromyalgia, Wegener's granulomatosis, peripheral neuropathy, diabetic neuropathy, Celiac's disease, Crohn's disease, ulcerative colitis, type I diabetes, leukemia, lupus erythematosus, and hemolytic dysglycemia.
(62) As used herein, the terms treat and treating refers to therapeutic measures, wherein the object is to slow down (lessen) an undesired physiological change or pathological disorder resulting from a disease or injury as described herein. For purposes of this invention, treating the disease or injury includes, without limitation, alleviating one or more clinical indications, decreasing inflammation, reducing the severity of one or more clinical indications of the disease or injury, diminishing the extent of the condition, stabilizing the subject's disease or injury (i.e., not worsening), delay or slowing, halting, or reversing the disease or injury and bringing about partial or complete remission of the disease or injury. Treating the disease or injury also includes prolonging survival by days, weeks, months, or years as compared to prognosis if treated according to standard medical practice not incorporating treatment with BD.sub.L.
(63) Subjects in need of treatment can include those already having or diagnosed with a disease or injury as described herein as well as those prone to, likely to develop, or suspected of having a disease or injury as described herein. Pre-treating or preventing a disease or injury according to a method of the present invention includes initiating the administration of a therapeutic (e.g., BD.sub.L) at a time prior to the appearance or existence of the disease or injury, or prior to the exposure of a subject to factors known to induce the disease or injury. Pre-treating the disorder is particularly applicable to subjects at risk of having or acquiring the disease injury. As used herein, the terms prevent and preventing refer to prophylactic or preventive measures intended to inhibit undesirable physiological changes or the development of a disorder or condition resulting in the disease or injury. In exemplary embodiments, preventing the disease or injury comprises initiating the administration of a therapeutic (e.g., BD.sub.L) at a time prior to the appearance or existence of the disease or injury such that the disease or injury, or its symptoms, pathological features, consequences, or adverse effects do not occur. In such cases, a method of the invention for preventing the disease or injury comprises administering BD.sub.L to a subject in need thereof prior to exposure of the subject to factors that influence the development of the disease or injury.
(64) As used herein, the terms subject or patient are used interchangeably and can encompass any vertebrate including, without limitation, humans, mammals, reptiles, amphibians, and fish. However, advantageously, the subject or patient is a mammal such as a human, or a mammal such as a domesticated mammal, e.g., dog, cat, horse, and the like, or livestock, e.g., cow, sheep, pig, and the like. In exemplary embodiments, the subject is a human. As used herein, the phrase in need thereof indicates the state of the subject, wherein therapeutic or preventative measures are desirable. Such a state can include, but is not limited to, subjects having a disease or injury as described herein or a pathological symptom or feature associated with a disease or injury as described herein.
(65) In some cases, a method of treating or preventing a disease or injury as described herein comprises administering a pharmaceutical composition comprising a therapeutically effective amount of BD.sub.L as a therapeutic agent (i.e., for therapeutic applications). As used herein, the term pharmaceutical composition refers to a chemical or biological composition suitable for administration to a mammal. Examples of compositions appropriate for such therapeutic applications include preparations for parenteral, subcutaneous, transdermal, intradermal, intramuscular, intracoronarial, intramyocardial, intrapericardial, intraperitoneal, intravenous (e.g., injectable), intraparenchymal, intrathecal, or intratracheal administration, such as sterile suspensions, emulsions, and aerosols. Intratracheal administration can involve contacting or exposing lung tissue, e.g., pulmonary alveoli, to a pharmaceutical composition comprising a therapeutically effective amount of BD.sub.L. In some cases, pharmaceutical compositions appropriate for therapeutic applications may be in admixture with one or more pharmaceutically-acceptable excipients, diluents, or carriers such as sterile water, physiological saline, glucose or the like. For example, BD.sub.L described herein can be administered to a subject as a pharmaceutical composition comprising a carrier solution.
(66) Formulations may be designed or intended for oral, rectal, nasal, topical or transmucosal (including buccal, sublingual, ocular, vaginal and rectal) and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrathecal, intraocular intraparenchymal, intrathecal and epidural) administration. In general, aqueous and non-aqueous liquid or cream formulations are delivered by a parenteral, oral or topical route. In other embodiments, the compositions may be present as an aqueous or a non-aqueous liquid formulation or a solid formulation suitable for administration by any route, e.g., oral, topical, buccal, sublingual, parenteral, aerosol, a depot such as a subcutaneous depot or an intraperitoneal, intraparenchymal or intramuscular depot. In some cases, pharmaceutical compositions are lyophilized. In other cases, pharmaceutical compositions as provided herein contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
(67) The preferred route may vary with, for example, the subject's pathological condition or weight or the subject's response to therapy or that is appropriate to the circumstances. The formulations can also be administered by two or more routes, where the delivery methods are essentially simultaneous or they may be essentially sequential with little or no temporal overlap in the times at which the composition is administered to the subject.
(68) Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations, but nonetheless, may be ascertained by the skilled artisan from this disclosure, the documents cited herein, and the knowledge in the art.
(69) In some cases, BD.sub.L may be optionally administered in combination with one or more additional active agents. Such active agents include anti-inflammatory, anti-cytokine, analgesic, antipyretic, antibiotic, and antiviral agents, as well as growth factors and agonists, antagonists, and modulators of immunoregulatory agents (e.g., TNF-, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-, IFN-, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including antibodies reactive against any of the foregoing, and antibodies reactive against any of their receptors). Any suitable combination of such active agents is also contemplated. When administered in combination with one or more active agents, BD.sub.L can be administered either simultaneously or sequentially with other active agents. For example, a subject with multiple sclerosis may simultaneously receive BD.sub.L and anti-CD20 depletion therapy for a length of time or according to a dosage regimen sufficient to support recovery and to treat, alleviate, or slow multiple sclerosis disease progression. In some embodiments, the BD.sub.L are administered prior to, simultaneously with, or following the administration of a second cell therapy such as to improve or enhance delivery, survival or function of the administered cells.
(70) In some embodiments, BD.sub.L is administered with a preconditioning therapy. Preconditioning therapies are known and used in the art. For example, preconditioning therapies are used with cell transplantation and chimeric antigen receptor T cell (CAR-T) therapies. See, for example, Almasbak et al. (CAR T Cell Therapy: A game changer in cancer treatment, Journal of Immunology Research, 2016) and Lo et al. (Preconditioning therapy in ABO-incompatible living kidney transplantation, Transplantation, 2016, 100(4):933-942). Preconditioning may include administration of a chemotherapeutic agent. Without wishing to be bound by any particular theory or embodiment, preconditioning treatment creates space for the engraftment of BD.sub.L and homeostatic expansion of CD4+ Foxp3+ T regulator cells.
(71) In some embodiment, BD.sub.L is administered with an anti-CD20 antibody therapy. Anti-CD20 therapy is known and used to typically to treat a variety of autoimmune disorders. Anti-CD20 therapy is also a known treatment for multiple sclerosis. Suitable anti-CD20 antibodies and treatment methods are known in the art. See, for example, Du et al. (Next-generation anti-CD20 monoclonal antibodies in autoimmune disease treatment, Auto Immun Highlights, 2017, 8(1):12). Suitable anti-CD20 therapies include, but are not limited to, rituximab, ocrelizumab, veltuzumab, obinutuzuman, and ofatumumab.
(72) In some embodiments, BD.sub.L are administered to a subject in need thereof using an infusion, topical application, surgical transplantation, or implantation. In an exemplary embodiment, administration is systemic. In such cases, BD.sub.L can be provided to a subject in need thereof in a pharmaceutical composition adapted for intravenous administration to subjects. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The use of such buffers and diluents is well known in the art. Where necessary, the composition may also include a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a cryopreserved concentrate in a hermetically sealed container such as an ampoule indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. In some cases, compositions comprising BD.sub.L are cryopreserved prior to administration.
(73) Therapeutically effective amounts of BD.sub.L are administered to a subject in need thereof. An effective dose or amount is an amount sufficient to effect a beneficial or desired clinical result. With regard to methods of the present invention, the effective dose or amount, which can be administered in one or more administrations, is the amount of BD.sub.L sufficient to elicit a therapeutic effect in a subject to whom the cells are administered. In some cases, an effective dose of BD.sub.L is about 110.sup.5 cells/kilogram to about 1010.sup.9 cells/kilogram of body weight of the recipient (e.g., 110.sup.5 cells/kilogram, 510.sup.5 cells/kilogram, 110.sup.6 cells/kilogram, 510.sup.6 cells/kilogram, 110.sup.7 cells/kilogram, 510.sup.7 cells/kilogram, 110.sup.8 cells/kilogram, 510.sup.8 cells/kilogram, or 110.sup.9 cells/kilogram). In some embodiments, an effective dose of BD.sub.L is between about 210.sup.6 cells/kilogram and 510.sup.6 cells/kilogram of the body weight of the subject. Effective amounts will be affected by various factors that modify the action of the cells upon administration and the subject's biological response to the cells, e.g., severity of the autoimmune disease, the patient's age, sex, and diet, the severity of inflammation, time of administration, and other clinical factors.
(74) Therapeutically effective amounts for administration to a human subject can be determined in animal tests and any art-accepted methods for scaling an amount determined to be effective for an animal for human administration. For example, an amount can be initially measured to be effective in an animal model (e.g., to achieve a beneficial or desired clinical result). The amount obtained from the animal model can be used in formulating an effective amount for humans by using conversion factors known in the art. The effective amount obtained in one animal model can also be converted for another animal by using suitable conversion factors such as, for example, body surface area factors.
(75) It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the BD.sub.L. For example, a BD.sub.L dosage for a particular subject with an autoimmune disease can be increased if the lower dose does not elicit a detectable or sufficient improvement in one or more clinical indications of the autoimmune disease. Conversely, the dosage can be decreased if the autoimmune disease is treated or eliminated.
(76) In some cases, therapeutically effective amounts of BD.sub.L can be determined by, for example, measuring the effects of a therapeutic in a subject by incrementally increasing the dosage until the desired symptomatic relief level is achieved. A continuing or repeated dose regimen can also be used to achieve or maintain the desired result. Any other techniques known in the art can be used as well in determining the effective amount range. Of course, the specific effective amount will vary with such factors as the particular disease state being treated, the physical condition of the subject, the type of animal being treated, the duration of the treatment, route of administration, and the nature of any concurrent therapy.
(77) Following administration of BD.sub.L to an individual subject afflicted by, prone to, or likely to develop a disease or injury described herein, a clinical symptom or feature associated with the disease or injury is observed and assessed for a positive or negative change. For example, for methods of treating multiple sclerosis in a subject, positive or negative changes in the subject's disease progression during or following treatment may be determined by any measure known to those of skill in the art including, without limitation, MRI and Expanded Disability Status Scale (EDSS) scoring. EDSS scoring is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability over time.
(78) In some embodiments, BD.sub.L are administered to a subject in need thereof using adoptive cell transfer. As used herein adoptive cell transfer refers to treatment of a subject by first administering to a subject a lymphodepleting agent to reduce a subjects natural lymphocyte population followed by administration of the immunotherapeutic cells of interest. Suitable lymphodepleting agents are known in the art and include, but are not limited to, cyclophosphamide and fludarabine.
(79) In any of the methods of the present invention, the donor and the recipient of the BD.sub.L can be a single individual or different individuals, for example, allogeneic or xenogeneic individuals. As used herein, the term allogeneic refers to something that is genetically different although belonging to or obtained from the same species (e.g., allogeneic tissue grafts or organ transplants). Xenogeneic means the cells could be derived from a different species.
(80) The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention.
(81) The invention will be more fully understood upon consideration of the following non-limiting Examples.
EXAMPLES
(82) Our published studies suggest that a specific subset of B cells of unknown phenotype play a critical role in immune tolerance. Using a partial B cell depletion strategy to enrich for B cells with regulatory activity, we found that B cells exhibiting an IgD low (L) (BD.sub.L) phenotype induce Treg expansion and promote recovery from EAE, an experimental autoimmune disorder. Both genetic and developmental studies led us to conclude that BD.sub.L are a new subset of mature B cells. Importantly, human B cells with an IgD.sup.low/ phenotype exhibit BD.sub.L regulatory activity by the induction of Treg proliferation. The ability to modulate Treg numbers to either suppress or enhance immune responses is a goal for the treatment of disease. Thus the ability to harness the regulatory function of BD.sub.L is of therapeutic interest.
(83) Anti-CD20 IgG.sub.1 B cell depletion retains regulatory activityIn our previous studies, total B cell depletion with anti-CD20 IgG2a prior to EAE induction led to significantly reduced Treg numbers and the inability to recover from EAE.sup.3, indicating that the protective B cell population was depleted. These data suggested that a specific B cell population that facilitates Treg homeostasis and EAE resolution could be identified.sup.3. To that end, the strategy we chose was to partially deplete B cells with anti-CD20 that contains the same antigen recognition domain, but with the IgG.sub.2a Fc region swapped for IgG.sub.1.sup.12. Administration of anti-CD20 IgG.sub.1 lead to a significant reduction in the total number of splenic B cells that was due to 85% loss of FO B cells, while sparing the MZ subset (
(84) Following anti-CD20 IgG.sub.1 administration, interestingly, IgD expression was lost while IgM expression remained (
(85) Representative CD4.sup.+ T cell and Treg flow cytometry is shown in
(86) Anti-CD20 IgG.sub.1 depleted B cells are enriched for IgD.sup.low/ Given that B cell regulatory activity resided within the retained FO B cell subset they were further analyzed by flow cytometry to uncover an enriched phenotype. This was accomplished by utilizing a B cell gating strategy utilizing differential expression of IgM, CD21 and CD23.sup.13,14. First, B220.sup.+ B cells and differential IgM and CD21 expression was used to define three groups: IgM.sup.hiCD21.sup.hi containing T2-MZP and MZ B cells, IgM.sup.intCD21.sup.int containing T3 and FO B cells and IgM.sup.hiCD21.sup.low/ containing T1 and T2 B cells (
(87) We then further refined the expression pattern of BD.sub.L in nave mice and found that they expressed slightly lower levels of the B cell marker B220, as compared to FO and MZ B cells (
(88) BD.sub.L regulatory activity is IL-10-independent We next demonstrated that BD.sub.L exhibit B cell regulatory activity by driving Treg expansion after transfer into MT mice similarly to total B cells (
(89) We next tested the regulatory potential of BD.sub.L in EAE. To control for the possibility that IgG.sub.1 anti-CD20 treatment modulated the function of the non-depleted cells, FACS purified splenic B cell subsets from nave mice were utilized. Adoptive transfer of BD.sub.L into MT mice prior to EAE induction resulted in complete recovery similar to WT (
(90) To demonstrate that BD.sub.L regulatory activity is not restricted to EAE, we performed the same experiment in a contact hypersensitivity (CHS) model that does not require B cells for induction.sup.15. As with EAE, MT mice exhibited a more severe disease course than WT mice that was normalized to WT levels in mice that received BD.sub.L (
(91) BD.sub.L are a new B cell subset separate from FO and MZ B cells To determine whether BD.sub.L could be differentiated from FO and MZ B cells, we performed RNA-seq comparing splenic BD.sub.L to FO and MZ B cells. Using principle component (PC) analysis plotted in 3D, we obtained the expected result that FO and MZ B cells cluster differentially in all three dimensions (
(92) When we compared BD.sub.L and FO B cells, the subsets clustered similarly in PC1 and PC2 (
(93) We then utilized flow cytometry to validate increased cell surface expression of proteins indicated in the RNA-seq. Cell surface proteins validated are CD93 (
(94) BD.sub.L regulatory function is GITRL-dependentIn our previous studies using total splenic B cells, we found that their regulatory activity was GITRL-dependent.sup.3. Consistent with this finding, we found that Tnfs18 (GITRL) message was upregulated 2.5 fold (p=0.001) in BD.sub.L in the RNA-seq analysis. When we examined the cell surface expression of GITRL, we found that all splenic B cell subsets expressed low levels of GITRL (
(95) The BD.sub.L subset develops in parallel with FO B cells in the spleenWe next determined whether BD.sub.L develop independently from FO B cells. This was accomplished by determining the timing of BD.sub.L emergence as compared to FO B cells following sublethal irradiation.sup.18. As shown in
(96) BD.sub.L are a stable mature B cell subset We next examined the stability of BD.sub.L upon adoptive transfer by repeating the sublethal irradiation experiment (
(97) To determine whether the BD.sub.L phenotype is stable long-term, we tracked their presence in EAE following adoptive transfer. On EAE day seven, five % of spleenocytes were B220 (
(98) Human BD.sub.L cells are present in spleen and peripheral bloodWe next determined whether a similar population of BD.sub.L human B cells exist that also promote Treg proliferation. To accomplish this splenic human B cells were FACS purified by gating on total B cells (CD4.sup.CD19.sup.+) (
(99) Discussion In this study, we sought to identify a definitive cell surface phenotype that could be used to identify B cells with the capacity to promote Treg proliferation that we have named BD.sub.L. Extensive cell surface phenotying and functional assays revealed that BD.sub.L are IgD.sup.low/. These cumulative data led to the conclusion that BD.sub.L are a B cell subset that plays an essential role in immune tolerance by regulating the homeostatic expansion of Treg.
(100) The ability of B cells to regulate inflammation was indicated as early as 1974 in models of delayed-type hypersensitivity, sarcoma and autoimmunity.sup.24-26. These studies fell short of definitively demonstrating a regulatory role for B cells. When MT mice became available, we demonstrated that B cells were required for EAE recovery.sup.1. Subsequently, a regulatory role for B cells in a model of inflammatory bowel disease was demonstrated.sup.27, which were shown to regulate via IL-10.sup.28. B cells were also shown to regulate EAE severity via IL-10.sup.4. These studies were impactful on the field and led to a plethora of papers demonstrating B cell regulation via IL-10 in a variety of disease models and in humans.sup.29,30. Given the capacity for all B cell subsets to produce IL-10, in retrospect, it is not surprising that a number of B cell phenotypes were described that regulate via IL-10 production.sup.29-32. Recently, the phenotype of B cells that regulate via IL-10 was identified as a population of LAG-3.sup.+ regulatory plasma cells that develop from numerous B cell subsets in a BCR-dependent manner.sup.33. In humans, the most definitive IL-10 producing B cell phenotype was reported as CD19.sup.+ CD24.sup.hiCD38.sup.hi, which includes both immature and CD5CD1d.sup.hi B cells.sup.34. Thus B cells that regulate via IL-10 are not a unique B cell subset.
(101) To define IL-10-independent B regulatory mechanisms, we utilized an adoptive transfer model of EAE that is does not require B cell effector functions.sup.2,3,35. This approach allowed us to discover that B cells induce the proliferation of Treg in a GITRL-dependent manner.sup.3. Our development of in vivo B cell regulatory assays allowed us to track the ability of purified B cell subsets to induce Treg proliferation.sup.3. This afforded us the opportunity to identify a definitive B cell regulatory phenotype. It is well known that the various IgG isotypes exhibit differential cell depletion. The effector function of the IgG subclass is determined by the Fc domain. The primary difference between the mouse IgG.sub.1 and IgG.sub.2a subclasses is that the later binds with high affinity to both FcRI and FcRIV, which are activating receptors that facilitate antibody-dependent cell clearance.sup.36-39. In mice, anti-CD20 IgG.sub.2a efficiently depletes all B cells in the spleen, while the IgG.sub.1 depletes the majority of FO B cells, but not the MZ subset (
(102) To the best of our knowledge, a subset of FO B cells expressing IgD.sup.low/ has not been previously described. Using functional assays, EAE and CHS disease models (
(103) Currently, there are four major populations of mature B cells including B1a that arise from the fetal liver and BM-derived Bib, MZ and FO subsets.sup.42,43. CD5.sup.+ B1a cells are the major producers of natural IgM and IgG3, aid in the control of infections, play a prominent role in autoimmunity and produce high levels of IL-10.sup.44,45. CD5.sup.CD11b.sup.+ B1b have the natural tendency to recognize protective antigens in bacteria.sup.46. MZ B cells are the first line of defense against blood-borne pathogens particularly those with repeating polysaccharides.sup.47. FO B cells produce the majority of high affinity isotype class switched antibodies in a T cell-dependent manner.sup.48. Here, we now provide evidence for the existence of BD.sub.L as a fifth mature B2 cell population that develops in the spleen from the T2 stage (
(104) Adoptive transfer studies demonstrate that a stable BD.sub.L phenotype does exist (
(105) Because BD.sub.L play an essential role in the maintenance of immunological tolerance they are clinically relevant. In autoimmunity, they could be potentially harnessed to increase Treg numbers.sup.50. Conversely, a reduction in BD.sub.L thereby decreasing Treg numbers could promote immune responses to clear cancer.sup.51. Using IgD.sup.low/ as the marker of BD.sub.L, we have been able to demonstrate the presence of BD.sub.L in humans (
(106) In this study, we provide evidence for the existence of BD.sub.L whose unique effector function is the maintenance of immune tolerance by promoting the homeostatic expansion of Treg in a GITRL-dependent manner. Our findings are an important first step in determining the therapeutic potential of BD.sub.L.
(107) Methods
(108) Mice. B10.PL, C57BL/6J (CD45.2), B6.129S2-Ighm.sup.tm1Cgn/J (C57BL/6MT), B6N(Cg)-Tnfsf18.sup.tm1.1(KOMP)Vlcg/J (GITRL.sup./) and B6.SJL-Ptprc.sup.a Pep.sup.b/BoyJ (CD45.1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MBP-TCR transgenic mice and generation of Foxp3.sup.EGFP, MT and Il10.sup./ mice on the B10.PL (H-2.sup.u) background was previously described.sup.1,3,61. MBP-TCR transgenic mice were genotyped by PCR using the following primers: Forward-5-TGC AGT ATC CCG GAG AAG GG-3 (SEQ ID NO:3); Reverse-5-TTC TCA TTT CCA TAG TTC TCA C-3 (SEQ ID NO:4). Notch2.sup.fl/flCD19cre.sup. and Notch2.sup.fl/flCD19cre.sup.+ mice were kindly provided by Dr. Shigeru Chiba, Tokyo University, Japan and Dr. Maeda Takahiro, Beckman Research of the City of Hope, CA.sup.16. Mice were housed and bred at the animal facility of the Medical College of Wisconsin (MCW) and animal protocols using all relevant ethical regulations were approved by the MCW Institutional Animal Care and Use Committee. All experiments were carried out with mice that were age- and sex-matched utilizing both genders.
(109) B cell depletion in vivo. Anti-mouse-CD20 mAb (18B12IgG1) and its corresponding isotype control (2B8 msIgG1) were provided by Biogen Inc. 250 g of antibody was i.v. injected into mice, once, or twice, 14 days apart.
(110) Peptide and antibodies. MBP Ac.sub.1-11 peptide (Ac-ASQKRPSQRSK (SEQ ID NO:5)) was generated by the Protein Core Laboratory of the Blood Research Institute, Versiti Wisconsin. The 2.4G2 hybridoma was obtained from American Tissue Culture Collection. Commercial antibodies utilized in this study are described in Table 1.
(111) TABLE-US-00001 TABLE 1 Commercial Antibodies Utilized Antibody.sup.1 Specificity.sup.2 Source.sup.3 Catalog.sup.4 Dilution.sup.5 B220-PE/CF594 Mouse BD Biosciences 562290 1:100 GITRL-Alexa Fluor 647 Mouse BD Biosciences 563542 1:100 GITRL-Purified Mouse Biolegend 120302 1:100 B220-Alexa Fluor 700 Mouse/Human Biolegend 103232 1:100 lgM-PE Dazzle 594 Mouse Biolegend 406529 1:100 lgD-PE Mouse Biolegend 405705 1:100 lgD-Alexa Fluor 488 Mouse Biolegend 405717 1:100 CD45.1-Alexa Fluor 647 Mouse Biolegend 110720 1:100 Ki-67-APC Mouse Biolegend 652405 1:200 CD36-APC Mouse Biolegend 102611 1:100 CD5-PE/Cy5 Mouse Biolegend 100609 1:100 CD20-APC Mouse Biolegend 150411 1:100 CD365 (Tim-1)-PE Mouse Biolegend 119505 1:100 CD1d-APC Mouse Biolegend 123521 1:100 CD9-Alexa Fluor 647 Mouse Biolegend 124809 1:100 CD3-Brilliant Violet 421 Mouse Biolegend 100228 1:100 CD43-PE Mouse Biolegend 121207 1:100 CD38-AF647 Mouse Biolegend 102716 1:100 F4/80-PE/Cy5 Mouse Biolegend 123112 1:100 NK1.1-APC Mouse Biolegend 108709 1:100 CD40-PE Mouse Biolegend 102805 1:100 CD86-PE Mouse Biolegend 105007 1:100 Ly6C-APC Mouse Biolegend 105007 1:100 TCR--FITC Mouse eBioscience 11-5961-82 1:100 CD21-eFluor 450 Mouse eBioscience 48-0212-82 1:100 CD23-PE/Cy7 Mouse eBioscience 25-0232-82 1:100 CD93-PE Mouse eBioscience 12-5892-82 1:100 CD80-PE/Cy5 Mouse eBioscience 15-0801-82 1:100 lgD-FITC Mouse eBioscience 11-5993-85 1:100 lgM-APC Mouse eBioscience 17-5790-82 1:100 CD4-FITC Mouse eBioscience 11-0041-85 1:100 Foxp3-PE Mouse eBioscience 12-5773-80 1:100 CD209b (SIGN-R1)-APC Mouse eBioscience 17-2093-80 1:100 CD25-APC Mouse Caltag RM6005 1:100 CD4-APC/Cy7 Human Biolegend 317417 1:100 CD19-PerCP/Cy5.5 Human Biolegend 302229 1:100 CD20-AF700 Human Biolegend 302322 1:100 CD24-PE/Cy7 Human Biolegend 311119 1:100 lgD-Brilliant Violet 421 Human Biolegend 348225 1:100 CD25-PE Human Biolegend 356103 1:100 CD3 Human Biolegend 317303 1:100 CD28 Human Biolegend 302913 1:100 .sup.1Antibody specificity and fluorochrome .sup.2Species specificity of the antibody .sup.3BD Bioscicences, San Diego, CA; Biolegend, San Diego, CA; eBioscience, San Diego, CA; Caltag Laboratories, Burlingame, CA .sup.4Catalog number of the antibody .sup.5Dilution used of the stock vial of antibody
(112) Flow cytometry and FACS purification. Single cell suspensions from mouse and human spleens or human peripheral blood were obtained, counted and 0.2-110.sup.6 cells were cell surface stained with specific antibodies. Frozen human splenocytes were thawed and washed twice prior to staining. Intracellular Foxp3, IgD and IgM staining was performed using the anti-mouse/rat Foxp3-PE staining kit from eBioscience using anti-Foxp3, -IgD or -IgM in the final step (San Diego, CA) per manufacturer instructions. Cells were acquired on a LSRII flow cytometer (BD Biosciences, San Diego, CA) and data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). Fluorochrome labeled and/or EGFP expressing cells were sorted using a FACSAria cell-sorter (BD Biosciences, San Diego, CA).
(113) Histolological analysis. Frozen section (6 M) of spleens from B10.PL mice treated with 250 g anti-CD20 antibody (18B12IgG1) or isotype control (2B8msIgG1) were fixed with acetone for 10 minutes. The fixed splenic sections then were stained with anti-mouse IgM-PE-Dazzle 594, IgD-Alex Fluor 488, anti-mouse CD3-Brilliant Violet 421 and anti-mouse CD209b (SIGN-R1)-APC and images were captured with Olympus VS120 fluorescent microscope at 80 magnification.
(114) EAE induction. EAE was induced by the i.v. adoptive transfer of 110.sup.6 MBP-specific encephalitogenic T cells activated in vitro with MBP Ac.sub.1-11 peptide into sublethally irradiated (360-380 rad) mice as described.sup.2,35. Clinical symptoms of EAE were scored daily as follows: 0, no disease; 1, limp tail; 1.5, hind limb ataxia; 2, hind limb paresis; 2.5, partial hind limb paralysis; 3, total hind limb paralysis; 4, hind and fore limb paralysis; and 5, death.
(115) Sorting and adoptive transfer of B cells. Total splenic B cells from 8-10 wk old mice were purified by negative selection using magnetic cell sorting (STEMCELL Technologies, Vancouver, BC, Canada) followed by FACS purification of FO (B220.sup.+IgM.sup.+ CD21.sup.intCD23.sup.+ CD93.sup.) or MZ (B220.sup.+IgM.sup.hiCD21.sup.hiCD23.sup. CD93.sup.) from anti-CD20-depleted mice or FO (IgD.sup.hi), MZ, BD.sub.L (B220.sup.+IgM.sup.+ CD21.sup.intCD23.sup.+ CD93.sup. IgD.sup.low/), or T2 (B220.sup.+IgM.sup.hiCD21.sup.low/ CD23.sup.+ CD93.sup.+) from WT or Tnfsf18.sup./ mice. B cell purities were 99% as determined by flow cytometry. For some experiments FACS purified Bells were incubated with anti-mouse GITRL (10 g/ml) at 4 C. for 60 min for GITRL blocking. B cells were washed in PBS and 5-2010.sup.6 cells were i.v. injected into each recipient mouse. EAE or CHS challenge was induced three days later or splenic CD4.sup.+Foxp3.sup.+ cells were enumerated on day ten. For CHS, B cells were transferred one day after sensitization. We have found that BD.sub.L functional activity is equivalent on both the B10.PL and C57BL/6 backgrounds, and thus both strains were utilized in the studies described.
(116) Induction of CHS. C57BL/6 mice were sensitized by epicutaneous application of 100 L 3% (wt/vol) oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one) (Sigma-Aldrich, St. Louis, MO) in an acetone:ethanol mixture (1:3 v/v) to shaved abdominal skin. After 4 days, ear thickness was measured using a micrometer. Mice were then challenged by application of 10 L 1% (wt/vol) oxazolone in an acetone:olive oil mixture (1:1 v/v) to both sides of the ears and ear thickness was measured every 24 hr for five days.
(117) RNA sequencing and PC analysis. Total RNA was isolated from sorted B cells using RNAqueous-4PCR kit (Ambion, Austin, Tx). RNA sequencing was performed by PerkinElmer (Waltham, MA) using the TruSeq stranded mRNA kit (Illumina, San Diego, CA) on an Illumina HiSeq2500, running HiSeq Control Software V2.2.68 & Real-time Analysis (RTA) software V1.18.66.3. Basecall files (*.bcl) generated by the Illumina instruments were de-multiplexed and converted to fastq.gz format using bcl2Fastq v1.8.2, one file per direction, 2 files per sample. For samples that were sequenced on multiple lanes and/or multiple flow cells, resulting fastq.gz files were concatenated into single file sets. The raw fastq files were then mapped to the whole mouse genome build mm9 using Tophat v2.1.0 with parameters --no-novel-juncs--no-coverage-search. Cufflinks was used to estimate the transcript abundance in Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). The average FPKM was calculated for each sample and log 2FPKM was used for PC analysis. Differential expression analysis was conducted with Cuffdiff.sup.62 to calculate the most differentially expressed significant genes based on a Benjamini-Hochberg adjusted p-value<0.05 an absolute fold change of 2 or greater. All significantly differentially expressed genes (log 2 fold change<1 or >1; adjusted p-value<0.05) were ordered by descending fold change for gene set overrepresentation analysis. This gene list was submitted to g:Profiler version r1741_e90_eg37.sup.63 as an ordered query to identify overrepresented terms and pathways within the Gene Ontology.sup.64,65 biological process and KEGG.sup.66,67 databases.
(118) B cell development assays. C57BL/6 mice were sublethally irradiated (500 rad) and on days 11, 13, 15, 17, 19, 21 and 24 the absolute number of splenic T2, FO and BD.sub.L was determined by flow cytometry. C57BL/6 mice were lethally irradiated (950 rad) and transplanted with 510.sup.6 total BM cells from C57BL/6J CD45.1 mice. C57BL/6J CD45.1 mice were sublethally irradiated (500 rad) and 15 days later splenic T2 B cells were FACS purified and labeled with carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA) prior to adoptive transfer (1.2510.sup.6) into C57BL/6J CD45.2 recipient mice. Donor (CD45.1.sup.+CFSE.sup.+) T2, FO and BD.sub.L were quantitated 24 and 48 hr post-transfer. C57BL/6J mice were sublethally irradiated (500 rad) and on days 11 post-irradiation, they were adoptively transferred CFSE labeled FO or BD.sub.L (1.510.sup.6) from C57BL/6J CD45.1 donor mice. Donor (CD45.1.sup.+CFSE) FO and BD.sub.L were quantitated 24 and 48 hr post-transfer.
(119) Isolation of human splenocytes and peripheral blood mononuclear cells. Human splenic tissue was obtained from the Wisconsin Donor Network and Tissue Bank, Versiti Wisconsin (Milwaukee, WI) through written informed consent and approved by the Institutional Review Board (IRB) of Versiti Wisconsin as non-human subject research. Portions of human splenic tissue were digested with collagenase D (1 mg/mL) (Roche) for 1 hr followed by dissociation using gentleMACS C tubes in combination with the gentleMACS Dissociator (Miltenyi Biotec, San Diego, CA).sup.68,69. Red blood cells were lysed with RBC lysis buffer (eBioscience) and lymphocytes were further purified using 60% percoll (Sigma, St. Louis, MO). Cells were washed and stored frozen in liquid nitrogen. Peripheral blood lymphocytes were isolated from buffy coats obtained from Versiti Wisconsin from healthy donors in a non-human subject research manner. Buffy coats were diluted 2 with PBS and lymphocytes were separated using a 60% percoll gradient.
(120) In vitro co-culture of human B cells and Treg. B cells, APC (CD19.sup. CD3.sup.) and CD4.sup.+ CD25.sup.hi Tregs were FACS purified from human splenocytes or peripheral blood from the same donor. The sorted Tregs were labeled with 3 M CFSE and cultured alone (0.510.sup.5) or with sorted B cells (110.sup.5) in the presence of anti-CD3 (OKT3) (2 g/ml) and irradiated (3000 rad) APC or in the presence of plate-bound anti-CD3 (10 mg/ml) and anti-CD28 (10 mg/ml). After 96 hr the cells were stained with CD4, CD25 and DAPI (Thermo Fisher Scientific, Waltham, MA) and proliferation of Treg were determined by dye dilution by flow cytometry.sup.3.
(121) Statistical analysis. Data were analyzed using GraphPad prism (San Diego, CA) and were presented as meanSEM. Statistical significance was determined using the unpaired t-test. p-values<0.05 were considered significant.
REFERENCES
(122) 1 Wolf, S. D., Dittel, B. N., Hardardottir, F. & Janeway, C. A., Jr. Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice. J. Exp. Med. 184, 2271-2278 (1996). 2 Mann, M. K., Maresz, K., Shriver, L. P., Tan, Y. & Dittel, B. N. B cell regulation of CD4+CD25+T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J. Immunol. 178, 3447-3456 (2007). 3 Ray, A., Basu, S., Williams, C. B., Salzman, N. H. & Dittel, B. N. A novel IL-10-independent regulatory role for B cells in suppressing autoimmunity by maintenance of regulatory T cells via GITR ligand. J. Immunol. 188, 3188-3198 (2012). 4 Fillatreau, S., Sweenie, C. H., McGeachy, M. J., Gray, D. & Anderton, S. M. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3, 944-950 (2002). 5. Tedder, T. F. B10 cells: a functionally defined regulatory B cell subset. J. Immunol. 194, 1395-1401 (2015). 6 Ray, A. & Dittel, B. N. Mechanisms of Regulatory B cell Function in Autoimmune and Inflammatory Diseases beyond IL-10. J. Clin. Med. 6, doi:10.3390/jcm6010012 (2017). 7 Ray, A., Mann, M. K., Basu, S. & Dittel, B. N. A case for regulatory B cells in controlling the severity of autoimmune-mediated inflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. J. Neuroimmunol. 230, 1-9 (2010). 8 Mauri, C. & Menon, M. Human regulatory B cells in health and disease: therapeutic potential. J. Clin. Invest. 127, 772-779 (2017). 9 Rudensky, A. Y. Regulatory T cells and Foxp3i. Immunol. Rev. 241, 260-268 (2011). 10 Liao, G. et al. GITR engagement preferentially enhances proliferation of functionally competent CD4+CD25+FoxP3+ regulatory T cells. Int. Immunol. 22, 259-270 (2010). 11 Ronchetti, S. et al. Glucocorticoid-induced tumour necrosis factor receptor-related protein: a key marker of functional regulatory T cells. J. Immunol. Res. 2015, 171520, doi:10.1155/2015/171520 (2015). 12 Zhang, A. H., Skupsky, J. & Scott, D. W. Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice. Blood 117, 2223-2226 (2011). 13 Allman, D. et al. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167, 6834-6840 (2001). 14 Basu, S., Ray, A. & Dittel, B. N. Cannabinoid receptor 2 is critical for the homing and retention of marginal zone B lineage cells and for efficient T-independent immune responses. J. Immunol. 187, 5720-5732 (2011). Seidel-Guyenot, W., Alt, R., Perschon, S., Knop, J. & Steinbrink, K. B cells are not required for T cell priming in low zone tolerance to contact allergens and contact hypersensitivity. Eur. J. Immunol. 34, 3082-3090 (2004). 16 Saito, T. et al. Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development. Immunity 18, 675-685 (2003). 17 Allman, D. & Pillai, S. Peripheral B cell subsets. Curr. Opin. Immunol. 20, 149-157 (2008). 18 Meyer-Bahlburg, A., Andrews, S. F., Yu, K. O., Porcelli, S. A. & Rawlings, D. J. Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation. J. Exp. Med 205, 155-168 (2008). 19 Barr, T. A. et al. B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. J. Exp. Med 209, 1001-1010 (2012). 20 Loder, F. et al. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med 190, 75-89 (1999). 21 Srivastava, B., Quinn, W. J., 3rd, Hazard, K., Erikson, J. & Allman, D. Characterization of marginal zone B cell precursors. J. Exp. Med 202, 1225-1234 (2005). 22 Mayer, R. J. et al. CD23 shedding: requirements for substrate recognition and inhibition by dipeptide hydroxamic acids. Inflamm. Res. 51, 85-90 (2002). 23 Woodland, R. T. & Schmidt, M. R. Homeostatic proliferation of B cells. Semin. Immunol. 17, 209-217 (2005). 24 Katz, S. I., Parker, D. & Turk, J. L. B-cell suppression of delayed hypersensitivity reactions. Nature 251, 550-551 (1974). Gorczynski, R. M. Immunity to murine sarcoma virus-inducted tumors. II. Suppression of T cell-mediated immunity by cells from progressor animals. J. Immunol. 112, 1826-1838 (1974). 26 Russell, A. S., Liburd, E. M. & Diener, E. In vitro suppression of cell mediated autoimmunity in NZB mice. Nature 249, 43-45 (1974). 27 Mizoguchi, E., Mizoguchi, A., Preffer, F. I. & Bhan, A. K. Regulatory role of mature B cells in a murine model of inflammatory bowel disease. Int. Immunol. 12, 597-605 (2000). 28 Mizoguchi, A., Mizoguchi, E., Takedatsu, H., Blumberg, R. S. & Bhan, A. K. Chronic intestinal inflammatory condition generates IL-10-producing regulatory B cell subset characterized by CD1d upregulation. Immunity 16, 219-230 (2002). 29 Mauri, C. & Bosma, A. Immune regulatory function of B cells. Annu. Rev. Immunol. 30, 221-241 (2012). 30 Rosser, E. C. & Mauri, C. Regulatory B cells: origin, phenotype, and function. Immunity 42, 607-612 (2015). 31 Heine, G. et al. Autocrine IL-10 promotes human B-cell differentiation into IgM- or IgG-secreting plasmablasts. Eur. J. Immunol. 44, 1615-1621 (2014). 32 Barr, T. A., Brown, S., Ryan, G., Zhao, J. & Gray, D. TLR-mediated stimulation of APC: Distinct cytokine responses of B cells and dendritic cells. Eur. J. Immunol. 37, 3040-3053 (2007). 33 Lino, A. C. et al. LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells. Immunity 49, 120-133 e129 (2018). 34 Blair, P. A. et al. CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity 32, 129-140 (2010). 35 Dittel, B. N., Merchant, R. M. & Janeway, C. A., Jr. Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis. J. Immunol. 162, 6392-6400 (1999). 36 Pescovitz, M. D. Rituximab, an anti-cd20 monoclonal antibody: history and mechanism of action. Am. J. Transplant 6, 859-866 (2006). 37 Clark, E. A. & Ledbetter, J. A. How does B cell depletion therapy work, and how can it be improved?Ann. Rheum. Dis. 64 Suppl 4, iv77-80, doi:10.1136/ard.2005.042507 (2005). 38 Weiner, G. J. Rituximab: mechanism of action. Semin. Hematol. 47, 115-123 (2010). 39 Bruhns, P. & Jonsson, F. Mouse and human FcR effector functions. Immunol. Rev. 268, 25-51 (2015). Geisberger, R., Lamers, M. & Achatz, G. The riddle of the dual expression of IgM and IgD. Immunology 118, 429-437 (2006). 41 Lykken, J. M. et al. Acute and chronic B cell depletion disrupts CD4+ and CD8+ T cell homeostasis and expansion during acute viral infection in mice. J. Immunol. 193, 746-756 (2014). 42 Kobayashi, M. et al. Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbfbeta for their development. Proc. Natl. Acad. Sci. USA 111, 12151-12156 (2014). 43 Ghosn, E. E. et al. Distinct B-cell lineage commitment distinguishes adult bone marrow hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 109, 5394-5398 (2012). 44 Zhang, X. Regulatory functions of innate-like B cells. Cell. Mol. Immunol. 10, 113-121 (2013). Savage, H. P. & Baumgarth, N. Characteristics of natural antibody-secreting cells. Ann. N.Y. Acad. Sci. 1362, 132-142 (2015). 46 Cunningham, A. F. et al. B1b cells recognize protective antigens after natural infection and vaccination. Front. Immunol. 5, 535, doi:10.3389/fimmu.2014.00535 (2014). 47 Pillai, S., Cariappa, A. & Moran, S. T. Marginal zone B cells. Annu. Rev. Immunol. 23, 161-196 (2005). 48 Zhang, Y., Garcia-Ibanez, L. & Toellner, K. M. Regulation of germinal center B-cell differentiation. Immunol. Rev. 270, 8-19 (2016). 49 Moore, T. C., Gonzaga, L. M., Mather, J. M., Messer, R. J. & Hasenkrug, K. J. B Cell Requirement for Robust Regulatory T Cell Responses to Friend Retrovirus Infection. MBio. 8, doi:10.1128/mBio.01122-17 (2017). 50 Arellano, B., Graber, D. J. & Sentman, C. L. Regulatory T cell-based therapies for autoimmunity. Discov. Med. 22, 73-80 (2016). 51 Takeuchi, Y. & Nishikawa, H. Roles of regulatory T cells in cancer immunity. Int. Immunol. 28, 401-409 (2016). 52 Naismith, R. T. et al. Rituximab add-on therapy for breakthrough relapsing multiple sclerosis: a 52-week phase II trial. Neurology 74, 1860-1867 (2010). 53 Bar-Or, A. et al. Rituximab in relapsing-remitting multiple sclerosis: a 72-week, open-label, phase I trial. Ann. Neurol. 63, 395-400 (2008). 54 Tipton, T. R. et al. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies. Blood 125, 1901-1909 (2015). 55 Beers, S. A. et al. Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection. Blood 115, 5191-5201 (2010). 56 Beers, S. A. et al. Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation. Blood 112, 4170-4177 (2008). 57 Ivanov, A. et al. Monoclonal antibodies directed to CD20 and HLA-DR can elicit homotypic adhesion followed by lysosome-mediated cell death in human lymphoma and leukemia cells. J. Clin. Invest. 119, 2143-2159 (2009). 58 Alduaij, W. et al. Novel type II anti-CD20 monoclonal antibody (GA101) evokes homotypic adhesion and actin-dependent, lysosome-mediated cell death in B-cell malignancies. Blood 117, 4519-4529 (2011). 59 Lim, S. H. et al. Anti-CD20 monoclonal antibodies: historical and future perspectives. Haematologica 95, 135-143 (2010). 60 Bologna, L. et al. Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab. J. Immunol. 186, 3762-3769 (2011). 61 Ray, A. et al. Gut Microbial Dysbiosis Due to Helicobacter Drives an Increase in Marginal Zone B Cells in the Absence of IL-10 Signaling in Macrophages. J. Immunol. 195, 3071-3085 (2015). 62 Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotechnol. 31, 46-53 (2013). 63 Reimand, J. et al. g:Profiler-a web server for functional interpretation of gene lists (2016 update). Nucleic Acids Res. 44, W83-89 (2016). 64 Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25-29 (2000). 65 The Gene Ontology, C. Expansion of the Gene Ontology knowledgebase and resources. Nucleic Acids Res. 45, D331-D338 (2017). 66 Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27-30 (2000). 67 Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. & Morishima, K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45, D353-D361 (2017). 68 Thome, J. J. et al. Spatial map of human T cell compartmentalization and maintenance over decades of life. Cell 159, 814-828 (2014). 69 Meng, W. et al. An atlas of B-cell clonal distribution in the human body. Nat. Biotechnol. 35, 879-884 (2017).