CD84+ / mertk+ myeloid derived suppressor cells and methods of use for transplantation tolerance

Abstract

The invention is directed to the compositions comprising myeloid derived suppressor cells (MDSCs); methods of collecting, purifying, and / or expanding the MDSCs; and methods of use in subjects to induce transplantation tolerance.

Description

[0001] CD84+ / MERTK+ MYELOID DERIVED SUPPRESSOR CELLS AND METHODS OF USE FOR TRANSPLANTATION TOLERANCE

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 677,562, filed July 31, 2024, which is hereby incorporated by reference herein in its entirety.

[0004] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0005] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “206085-0185- OOWO_SequenceListing.xml” having a creation date of July 30, 2025, and having a size of 2,595 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

[0006] BACKGROUND OF THE INVENTION

[0007] Transplantation tolerance reduces the morbidity and complexities of conventional pharmacologic immunosuppression (Kawai T, et al., N Engl J Med, 2008, 358( 1 ):353— 361 ; Scalea JR, et al., Am J Transplant, 2014, 14(9):2001-2010). However, the availability of compositions and methods to induce tolerance after transplantation remain limited.

[0008] Thus, there remains a need in the art for improved compositions and methods for inducing tolerance. The present invention satisfies this unmet need.

[0009] SUMMARY OF THE INVENTION

[0010] In some embodiments, the present invention provides a composition for adoptive cell transfer to induce transplantation tolerance comprising one or more myeloid derived suppressor cells (MDSCs) selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC. In some embodiments, the composition comprises a purified population of one or more MDSC selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC. In some embodiments, the CD84+ MerTK+ MDSCs are generated by expanding a population of MDSCs with GCSF.

[0011] In some embodiments, the present invention provides a method of treating a subject receiving a transplant or who has received a transplant, comprising administering a composition of the present invention. In some embodiments, the subject is receiving, or has received, a heart transplant.

[0012] In some embodiments, the present invention provides a method of treating a hyperimmune subject comprising administering a composition of the present invention.

[0013] In some embodiments, the present invention provides a method of suppressing T cell proliferation in a subject comprising administering a composition of the present invention.

[0014] In some embodiments, the present invention provides a method of reducing the dose of an immunosuppressive drug administered to a subject comprising administering a composition of the present invention.

[0015] In some embodiments, the present invention provides a method of stimulating the generation of Treg cells in a subject comprising administering a composition of the present invention.

[0016] In some embodiments, the present invention provides a method comprising collecting myeloid derived suppressor cells (MDSC) from a subject for adoptive cell transfer to induce transplantation tolerance. In some embodiments, the MDSC is one or more MDSC selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC. In some embodiments, the MDSC are splenic MDSC, bone marrow MDSC, or a combination thereof. In some embodiments, the method further comprises purifying the MDSCs for one or more selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC. In some embodiments, the purifying comprises fluorescence-activated cell sorting. In some embodiments, the method further comprises stimulating the MDSC with GCSF, growth factors, or a combination thereof. In some embodiments, the method further comprises freezing the MDSC.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. Figure 1, comprising Figure 1 A through Figure ID, depicts representative experimental results of in vitro expansion of T cells and MDSCs. Figure 1A depicts representative images of immune cell clustering and proliferation analyze under the Leica microscope. Original magnification, X10. 72 h CD4 T cells culture Non- stimulation (1) and stimulation and expansion (2) by CD3 and CD28 monoclonal antibody. Figure IB depicts a graph illustrating MDSC phenotype. Figure 1C depicts representative bar graphs quantifying markers impacted by granulocyte colony stimulating factor (GCSF) such as CD45, Gr-1, CD1 lb, Ly-6G, Ly-6C, F4 / 80, CXCR2, CD84. Figure ID depicts representative bar graphs quantifying biomarkers of MDSCs including CD115, PD-L1, TGF-|3, CCR2, Rae-ly, CD45R / B220, Stat3 and MerTK, as stratified by WT and MerTK KO.

[0019] Figure 2, comprising Figure 2A and Figure 2B, depicts representative experimental results demonstrating of T cell proliferation. Figure 2A depicts representative fluorescence-activated cell sorting plots from T cell proliferation assay with co-culture of MDSCs. CD1 lb+Gr-l+ MDSCs were sorted from mice no treated and treated with GMSF by FACS® Aria II cytometer (BD) and co-cultured with cell trace violet (CTV)-labeled CD4 T cells sorted from spleen and BM of naive mice. Representative histogram images of flow cytometric analysis for total MDSCs. Figure 2B depicts representative bar graphs demonstrating that GCSF expanded MDSCs suppress T cell proliferation in vitro.

[0020] Figure 3, comprising Figure 3A and Figure 3B, depicts representative experimental results demonstrating that MerTK KO MDSC suppression of T cell proliferation is impaired. Figure 3A depicts representative fluorescence-activated cell sorting plots from coculture of T cells with MerTK KO MDSCs which were expanded by GCSF. CD1 lb+Gr-l+ MDSCs were sorted from mice treated with GMSF by FACS® Aria II cytometer (BD) and cocultured with cell trace violet (CTV)-labeled CD4 T cells sorted from spleen and BM of naive C57BL / 6J and MerTK KO mice. Representative histogram images of flow cytometric analysis for total MDSCs. Figure 3B depicts a representative bar graph demonstrating that MerTK KO reduced T cell suppressive capacity of MDSCs in co-culture.

[0021] Figure 4, comprising Figure 4A through Figure 4C, depicts representative experimental results demonstrating that CD84 KO MDSC suppression of T cell proliferation is impaired. Figure 4A depicts representative fluorescence-activated cell sorting gating for CD84+ / KO MDSCs. Figure 4B depicts a representative bar graph quantifying the relationship between CD84 and MerTK among MDSCs expanded by GCSF. CD84 expression is down- regulated in MerTK KO mice. It was found that the MerTK KO MDSCs also fail to express CD84, which is interesting as MerTK KO MDSCs are ineffective in controlling T cell responses to transplantation. Figure 4C depicts a representative bar graph demonstrating that CD84 KO reduces MDSC suppressive capacity of T cell proliferation in vitro.

[0022] Figure 5 depicts a representative bar graph demonstrating that blocking CD84 impairs MDSC function among MDSCs expanded by GCSF. Dose-dependent impairment of MDSC function with anti-CD84 blocking monoclonal antibody.

[0023] Figure 6 depicts a representative bar graph quantifying the results of experiments of MerTK KO mice MDSCs with GCSF and with blocking CD84 antibody. IxlO6MDSC cells were suspended in the presence of 2.5, 10 and 20 pg anti-CD84 antibody and co-culture with CD4 T cells 72 hrs.

[0024] Figure 7 depicts a representative bar graph quantifying the results of experiments turning off MDSCs’ T cell suppressive capacity. Elimination of MerTK and CD84 among GCSF expanded MDSCs nearly completely restores T cell proliferation in vitro, demonstrating near complete impairment of GCSF expanded MDSCs.

[0025] Figure 8, comprising Figure 8A and Figure 8B, depicts representative experimental results relating to MerTK+CD84+ MDSCs. Figure 8A depicts a representative bar graph illustrating how common double positive MerTK+CD84+ MDSCs among MDSCs expanded by GCSF are. Figure 8B depicts a representative bar graph of CD84 and MerTK double positive (DP) sorting spleen and bone marrow MDSCs co-culture with CD4 T cells.

[0026] Figure 9 depicts a representative bar graph demonstrating that CD84 KO does not alter MerTK expression.

[0027] Figure 10 depicts representative images demonstrating the use of CD3 / CD28 to activate and expand T cells. 48 h after the CD4 T cell stimulation and expansion by CD3 and CD28 monoclonal antibody.

[0028] Figure 11 depicts representative results demonstrating immunosuppression of MDSCs potentiated by GMSF. CD1 lb+Gr-l+ MDSCs were sorted from mice not treated and treated with GMSF by FACS® Aria II cytometer (BD) and co-cultured with cell trace violet (CTV)-labeled CD4 T cells sorted from spleen and BM of naive mice. Representative histogram images of flow cytometric analysis for total MDSCs. Figure 12 depicts representative results demonstrating immunosuppression of MDSCs potentiated by GMSF. CD1 lb+Gr-l+ MDSCs were sorted from mice treated with GMSF by FACS® Aria II cytometer (BD) and co-cultured with cell trace violet (CTV)-labeled CD4 T cells sorted from spleen and BM of naive C57BL / 6J and MerTK KO mice. Representative histogram images of flow cytometric analysis for total MDSCs.

[0029] Figure 13 depicts quantification analyses of cell trace violet (CTV) intensity dilution in CTV-labeled CD4 T cells co-cultured with total, PMN-, and M-MDSCs.

[0030] Figure 14 depicts representative results demonstrating MDSCs from MerTK- / - mice have reduced inhibition of cell proliferation.

[0031] Figure 15 depicts representative results demonstrating GCSF + / - impacted study markers: CD84 and MerTK.

[0032] Figure 16 depicts representative results from experiments with WT with and without GCSF and with and without blocking CD84 antibody. IxlO6MDSC cells were suspended in the presence of 2.5, 10 and 20pg anti-CD84 antibody and co-culture with CD4 T cells.

[0033] Figure 17 depicts representative results from experiments with MerTK KO with and without GCSF and with and without blocking CD84 antibody. IxlO6MDSC cells were suspended in the presence of 0.25, lOand 20pg anti-CD84 antibody and co-culture with CD4 T cells.

[0034] Figure 18 depicts representative phenotypical expression of myeloid-derived suppressor cells (MDSCs).

[0035] Figure 19 depicts representative results demonstrating GCSF impacted study markers: CD115, TGF-0, Rae-ly, B220, CXCR2, F4 / 80, PD-L1, STAT3, CD84 and MerTK.

[0036] Figure 20, comprising Figure 20A through Figure 20E, depicts representative results demonstrating the generation of IL-4 / IL-34 / IL-10 fusion protein and the molecular and functional characterization of Fusion Protein IL-4 / IL-34 / IL10 fusion protein. Figure 20A depicts a schematic overview of the I1-4 / IL-34 / IL10 fusion protein and its amino acid sequence. The linker sequence is underlined. Figure 20B depicts the purification of IL-4 / IL-34 / IL10 fusion protein by size exclusion chromatography HiLoad® 16 / 600 Superdex®200 equilibrated in endotoxin-free Dulbecco’s phosphate buffered saline buffer. IL-4 / IL-34 / IL10 fusion protein was eluted between 70-90mL. The molecular weight of IL-4 / IL-34 / IL10 was estimated based on the molecular mass standard calibration performed using Thyroglobulin, bovine (670 kDa, 56.09 mL), y-globulin, bovine (158 kDa, 72.89 mL), ovalbumin, chicken (44 kDa, 87.00 mL), and myoglobin, horse (17 kDa, 97.73 mL) indicated with arrows. Figure 20C and Figure 20D depicts representative results demonstrating the purity and quality of purified protein assessed by SDS- PAGE and western blots analyses using monoclonal mouse IgGl Clone#127107 (R&D Systems; MAB2172) antibody for hIL-10 detection, respectively. Figure 20E depicts an optinal cartoon representation.

[0037] Figure 21, comprising Figure 21 A and Figure 2 IB, depicts representative results demonstrating IL-4 / IL-34 / IL-10 fusion protein prolonged cardiac allograft survival. Figure 21A depicts representative results from experiments where 5 mg / Kg of I1-4 / IL-34 / IL10 fusion protein was intraperitonially injected on day 0 for single dose group (FP1) or on day 0, 3, and 6 for multiple dose group (FP3) post-transplantation. Figure 2 IB depicts representative results demonstrating graft survival analyzed by abdominal palpation. Survival curves show significant difference between control (No treatment) and FP-treated groups (FP1 or FP3) by log-rank (Mantel-Cox) test, *p < 0.05, Mean survival time (MST, days).

[0038] Figure 22, comprising Figure 22A through Figure 22C, depicts representative results demonstrating the effect of IL-4 / IL-34 / IL-10 FP on T cell proliferation. Figure 22A depicts the gating strategy for proliferated CD8+ / CD4+ cells. Figure 22B depicts representative histogram plots showing representative flow cytometric data of proliferating cells in CD4+ or CD8+ cells. Figure 22C depicts representative bar charts showing the frequency of proliferated cells of CD4+ or CD8+ cell. The differences between seven groups were tested by ANOVA and after post Tukey test. T cells were stimulated with CD3 & CD28 antibodies during incubation for 3 days before flow cytometry assay.

[0039] Figure 23, comprising Figure 23A through Figure 23D, depicts representative results demonstrating IL-4 / IL-34 / IL-10 fusion protein promotes in vitro MDSCs expansion. Bone marrow cells were obtained cultured in the presence / absence of IL-4 / IL-34 / IL-10 fusion protein (20 ng / ml). Following incubation for 6 days, cells were collected and analyzed for total cells (Figure 23 A), total MDSCs (Figure 23B), PMN-MDSCs (Figure 23C), and M-MDSCs (Figure 23D). The representative histogram images and quantitative analysis of percentage of MDSC subsets induced 6 days after incubation. Data were analyzed as mean value ± SD and student t-test was used to assess the result significance. *p < 0.05, ***p < 0.001, compared with the control group, ns, not significant.

[0040] Figure 24 depicts representative results demonstrating MDSCs’ ability to suppress T cell proliferation is impaired with Tacrolimus and MMF. The decree of impairment appears to be greater with tacrolimus.

[0041] Figure 25 depicts representative results demonstrating why transplant induced / expanded (Tx-MDSC) alone are insufficient for alloregulation compared to GCSF- treated MDSC.

[0042] Figure 26 depicts representative results demonstrating why GCSF-MDSCs are special. MDSC biomarker expression was significantly affected by GCSF. GCSF-MDSCs expressed higher levels of CD84, CCR2, IL-34, and Stat3 but lower levels of ADAMI 0 and AhR.

[0043] Figure 27 depicts representative results demonstrating immunosuppression of MDSCs potentiated by G-CSF. spleen and bone marrow CD84 and MerTK double positive CD1 lb+Gr-l+ MDSCs were sorted from mice treated with G-CSF by FACS Aria II cytometer (BD) and co-cultured with cell trace violet (CTV)-labeled CD4 T cells purified from spleen of naive C57BL / 6J mice.

[0044] Figure 28 depicts representative results demonstrating immunosuppression of MDSCs potentiated by G-CSF. spleen and bone marrow CD84 and MerTK double negative CD1 lb+Gr-l+ MDSCs were sorted from mice treated with G-CSF by FACS Aria II cytometer (BD) and co-cultured with cell trace violet (CTV)-labeled CD4 T cells purified from spleen of naive Balb / c mice.

[0045] Figure 29 depicts representative results demonstrating immunosuppression of MDSCs potentiated by G-CSF. spleen and bone marrow CD84 and MerTK double positive and double negative CD1 lb+Gr-l+ MDSCs were sorted from mice treated with G-CSF by FACS Aria II cytometer (BD) and co-cultured with cell trace violet (CTV)-labeled CD4 T cells purified from spleen of naive Balb / c mice.

[0046] Figure 30 depicts representative results demonstrating the effect of IL-34 on proliferation and differentiation of bone marrow cells in vitro.

[0047] DETAILED DESCRIPTION The invention is based, in part, on the finding that a specific population of myeloid derived suppressor cells (MDSCs) induces transplantation tolerance. Thus, the invention is based on a previously unknown requirement for MDSCs (e.g. CD84+ MDSCs, MerTK+ MDSCs, CD84+ MerTK+ MDSCs, etc.) in promoting transplantation tolerance.

[0048] Therefore, in various embodiments, the invention provides compositions comprising MDSCs. In some embodiments, the MDSCs are CD84+ MDSCs. In some embodiments, the MDSCs are MerTK+ MDSCs. In some embodiments, the MDSCs are CD84+ MerTK+ MDSCs. In one embodiment, the composition comprises a substantially purified population of CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs.

[0049] In other embodiments, the invention provides methods for collecting, purifying, and / or expanding MDSCs. In some embodiments, the invention provides methods for collecting, purifying, and / or expanding CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs. In some embodiments, the invention provides methods for inducing transplant tolerance in a subject comprising administering to the subject a composition comprising MDSCs or a substantially purified population of MDSCs. In other embodiments, the invention provides methods for inducing transplant tolerance in a subject comprising administering to the subject a composition comprising CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs or a substantially purified population of CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs.

[0050] In some embodiments, the present invention relates to “off the shelf’ MDSCs that can be used in subjects receiving transplants and / or hyperimmune subjects. In certain instances, the compositions and methods described herein allow for the subject receiving the transplant and / or the hyperimmune subject to receive a reduced dose of immunosuppressive drug therapy as compared to what would be needed without the administration of a presently described CD84+ MerTK+ MDSCs.

[0051] In certain embodiments, the MDSC cells (e.g. CD84+ MDSCs, MerTK+ MDSCs, CD84+ MerTK+ MDSCs, etc.) of the present invention control T cell responses in a transplant subject. In some embodiments, MDSCs are extracted and purified from a subject that will receive a transplant in the future. These cells are expanded in vitro using GCSF and growth factors and targeted for CD84+ and / or MerTK+ expression. In some embodiments, single positive and / or dual positive cells are separated using fluorescence-activated cell sorting and again expanded using GCSF. In certain embodiments, MDSCs that have been expanded are frozen for future use. The compositions and methods described herein can be used as a method of controlling alloreactivity, stimulating Treg generation in vivo, and reducing the dose of immunosuppressive drug therapy.

[0052] Definitions

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0054] As used herein, each of the following terms has the meaning associated with it in this section.

[0055] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0056] “About” as used herein when referring to a measurable value, for example numerical values and / or ranges, such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For example, “about 40 [units]” may mean within ± 25% of 40 (e.g., from 30 to 50), within ± 20%, ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, less than ± 1%, or any other value or range of values therein or therebelow. Furthermore, the phrases “less than about [a value]” or “greater than about [a value]” should be understood in view of the definition of the term “about” provided herein.

[0057] As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils, and granulocytes.

[0058] As used herein, the term “immune response” includes T cell mediated and / or B cell mediated immune responses that are influenced by modulation of T cell co-stimulation. The term immune response further includes immune responses that are indirectly effected by T cell activation such as antibody production (humoral responses) and the activation of cytokine responsive cells such as macrophages.

[0059] As used herein, the term “T cell immune response” refers to activation of antigen specific T cells as measured by proliferation or expression of molecules on the cell surface or secretion of proteins such as cytokines.

[0060] As used herein, the term “T cell” refers to a lymphocyte (e.g., white blood cell) that functions in cell-mediated immunity. In some embodiments, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes. As is known in the art, T cells typically do not present antigens, and rely on other lymphocytes (e.g., natural killer cells and B cells) to aid in antigen presentation. Types of T cells include, but are not limited to, T helper cells (TH cells), Memory T cells (Tcm, Tern, or Temra), Regulatory T cells (Treg), Cytotoxic T cells (CTLs), Natural killer T cells (NK cells), gamma delta T cells, and Mucosal associated invariant T cells (MAIT).

[0061] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0062] “Allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0063] “Xenogeneic” refers to a graft derived from an animal of a different species.

[0064] The phrase “disease associated with expression of expression of alloresponsive cells” as used herein includes, but is not limited to, a disease associated with expression of alloresponsive cells or condition associated with cells which express alloresponse including, e.g., an allograft rejection, an immune rejection, a chronic allogeneic rejection, an engraftment rejection, a transplant rejection, an inflammation, an inflammation caused by ischemia / reperfusion, an infection, an immune response to an allograft, and any combination thereof.

[0065] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR complex, BCR complex, etc.) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR complex and / or BCR complex, etc. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-p, and / or reorganization of cytoskeletal structures, and the like.

[0066] A “stimulatory molecule,” as the term is used herein, means a molecule expressed by a cell that provide the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR and / or BCR complex in a stimulatory way for at least some aspect of the cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR and / or BCR complex with a human leukocyte antigen (HLA) I, HLA II, major histocompatibility complex (MHC) I, or MHC II molecule loaded with peptide, and which leads to mediation of a cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d. In a specific CAR of the invention, the cytoplasmic signaling sequence derived from CD3-zeta is derived from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0067] An “antigen presenting cell” or “APC” as used herein, means an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays foreign antigens complexed with HLA I, HLA II, MHC I, or MHC II complexes on their surfaces. For example, T cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0068] A “costimulatory molecule” refers to the cognate binding partner on a cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include, but are not limited to an HLA class I molecule, HLA class II molecule, MHC class I molecule, MHC class II molecule, MHC class III molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD3, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18) and 4-1BB (CD137). “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.

[0069] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0070] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0071] A disease or disorder is “alleviated” if the severity of at least one sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0072] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0073] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0074] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0075] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0076] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In some embodiments, the cells are not cultured in vitro.

[0077] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human, mouse).

[0078] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be constmed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0079] Compositions

[0080] In various embodiments, the present invention provides compositions comprising myeloid derived suppressor cells (MDSCs). In some embodiments, the MDSC is positive for one or more surface markers selected from the group consisting of: Grl, CD1 lb, F4 / 80, CXCR2, CD84, and MerTK. In some embodiments, the MDSC is a CD84+ MDSC. In some embodiments, the MDSC is a MerTK+ MDSC. In some embodiments, the MDSC is a CD84+ MerTK+ MDSC. In some embodiments, the MDSC is a mononuclear MDSC. In some embodiments, the MDSC is a polymorphonuclear MDSC.

[0081] In some embodiments, the composition comprises mononuclear cells. In some embodiments, the mononuclear cell comprises one or more of Grl, CD1 lb, CD84, and MerTK. In some embodiments, the mononuclear cell is a Grl+ mononuclear cell. In some embodiments, the mononuclear cell is a CD1 lb+ mononuclear cell. In some embodiments, the mononuclear cell is a Grl+ CD1 lb+ mononuclear cell. In some embodiments, the mononuclear cell is a CD84+ mononuclear cell. In some embodiments, the mononuclear cell is a MerTK+ mononuclear cell. In some embodiments, the mononuclear cell is a Grl+ CD1 lb+ CD84+ mononuclear cell. In some embodiments, the mononuclear cell is a Grl+ CD1 lb+ MerTK+ mononuclear cell. In some embodiments, the mononuclear cell is a Grl+ CD1 lb+ CD84+ MerTK+ mononuclear cell.

[0082] In some embodiments, the composition comprises MDSCs isolated from a tissue. In some embodiments, the composition comprises MDSCs isolated from spleen tissue. In some embodiments, the composition comprises MDSCs isolated from bone marrow. In some embodiments, the composition comprises MDSCs isolated from spleen tissue, MDSCs isolated from bone marrow, or a combination thereof. In some embodiments, the composition comprises MDSCs collected from a single subject. In some embodiments, the composition comprises MDSCs collected from at least two different subjects.

[0083] In some embodiments, the MDSCs are generated by expansion of a MDSC population. Mediators of MDSC expansion are known in the art and include, but are not limited to, GCSF; GM-CSF; IL-2, IL-4; IL-6; IL-34; IL-10; a fusion protein comprising IL-4, IL-34, and IL- 10, etc. In some embodiments, the MDSC population is expanded by contacting the MDSC population with GCSF; GM-CSF; IL-2, IL-4; IL-6; IL-34; IL-10; a fusion protein comprising IL- 4, IL-34, and IL-10; or a combination thereof. In some embodiments, the fusion protein comprising IL-4, IL-34, and IL-10 comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. In some embodiments, the fusion protein comprising IL-4, IL-34, and IL-10 comprises SEQ ID NO:1.

[0084] In some embodiments, the composition comprises a purified population of MDSCs. In some embodiments, the composition comprises a purified population of CD84+ MDSCs. In some embodiments, the composition comprises a purified population of MerTK+ MDSCs. In some embodiments, the composition comprises a purified population of CD84+ MerTK+ MDSCs.

[0085] In some embodiments, the purified population of CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs is generated by isolating CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs cells from an expanded population of MDSCs. In some embodiments, the expanded population of MDSCs is generated by contacting a MDSC population with GCSF; GM-CSF; IL-2, IL-4; IL-6; IL-34; IL-10; a fusion protein comprising IL- 4, IL-34, and IL-10; or a combination thereof. In certain embodiments, the purified population of CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs is generated by isolating CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs from an expanded population and by further expanding the isolated CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs by contacting the isolated CD84+ MDSCs, MerTK+ MDSCs, and / or CD84+ MerTK+ MDSCs with GCSF; GM-CSF; IL-2, IL-4; IL-6; IL-34; IL-10; a fusion protein comprising IL-4, IL-34, and IL-10; or a combination thereof.

[0086] Methods of Collecting

[0087] In various embodiments, the present invention provides methods of collecting MDSCs of the invention. In some embodiments, the MDSCs are collected from a tissue. In some embodiments, the MDSCs are collected from spleen tissue. In some embodiments, the MDSCs are collected from bone marrow. In some embodiments, MDSCs from different tissues are combined. In some embodiments, MDSCs collected from spleen tissue, bone marrow, another tissue, or a combination thereof are combined. In some embodiments, MDSCs collected from spleen tissue and from bone marrow are combined. In some embodiments, MDSCs are collected from a single subject. In some embodiments, MDSCs collected from at least two different subjects are combined.

[0088] In some embodiments, the method of collecting MSDCs further comprises a purification step. In some embodiments, the purification step comprises flow cytometry. In some embodiments, the purification step comprises fluorescence-activated cell sorting. In some embodiments, samples are sorted for cells positive for at least one marker selected from the group consisting of: Grl, CD1 lb, F4 / 80, CXCR2, CD84, and MerTK. In some embodiments, samples are sorted for cells positive for CD84 and / or MerTK.

[0089] In some embodiments, the MDSCs are further expanded after purification. In some embodiments, the MDSCs are expanded with cytokines. In some embodiments, the MDSCs are expanded with growth factors. Mediators of inflammation, such as cytokines and growth factors, that expand MDSCs are well known in the art and include, but are not limited to, GCSF; GM-CSF; IL-2, IL-4; IL-6; IL-34; IL-10; etc. In some embodiments, the MDSCs are expanded with GCSF; GM-CSF; IL-2, IL-4; IL-6; IL-34; IL-10; a fusion protein comprising IL- 4, IL-34, and IL-10; or a combination thereof. In some embodiments, the MDSCs are expanded with GCSF and at least one additional growth factor. In some embodiments, the MDSCs are expanded with GCSF and at least one additional cytokine. In some embodiments, the MDSCs are expanded with GCSF, at least one additional growth factor, and at least one additional cytokine.

[0090] In certain aspects, the MDSCs are frozen prior to use. For example, in certain embodiments, expanded MDSCs, purified MDSCs, and / or expanded, purified, and expanded MDSCs are frozen for until they are thawed for administration to the subject.

[0091] Modified MDSCs

[0092] In some embodiments, the composition of the present invention comprises modified MDSC. In some embodiments, the MDSCs are modified in vivo. In some embodiments, the MDSCs are modified ex vivo. The modified MDSC may be modified from MDSCs or modified from a purified population of MDSCs.

[0093] Expression of human leukocyte antigen (HLA) molecules is a well-known barrier to organ transplantation. Because the protein carries so many allelic differences, meticulous matching of the donor HLA class I alleles to the recipient to thwart organ rejection is necessary even in combination with immunosuppression drugs administered to the recipient. Another approach to tackle transplant rejection is not only to target host T cells through immunosuppressive drugs but also to remove donor HLA class I structures from the donor organ. Therefore, the MDSC of the present invention may be modified to be stripped of HLA molecules to generate MDSCs that do not stimulate an immune response. Methods of stripping HLA molecules are well known in the art and include, but are not limited to, treatment by papain or another cleavage agent. Therefore, in some embodiments, the modified MDSC comprises a MDSC lacking HLA molecules. In some embodiments, the HLA molecule is an HLA I molecule, an HLA II molecule, or a combination thereof. In some embodiments, the MDSC lacking HLA molecules comprises a MDSC or a purified population of MDSC stripped of HLA. In some embodiments, the MDSC or purified population of MDSC is contacted with a cleavage agent. In some embodiments, the cleavage agent is papain. In some embodiments, the MDSC or purified population of MDSC is contacted with a cleavage agent ex vivo. In some embodiments, the MDSC or purified population of MDSC is contacted with a cleavage agent for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 9 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 21 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or at least 5 years. In some embodiments, the MDSC is contacted with a cleavage agent at about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C. The MDSC may be analyzed to confirm removal of HLA molecules through techniques known in the art including, but not limited to, flow cytometry, ELISA, immunofluorescent staining, western blot, etc.

[0094] In some embodiments, the modified MDSC comprises CD84, MerTK, or a combination thereof. In some embodiments, the modified MDSC is modified from a MDSC or a purified population of MDSC that lack CD84 (i.e. CD84 negative or CD84- MDSCs). In some embodiments, the modified MDSC is modified from a MDSC or a purified population of MDSC that lack MerTK (i.e. MerTK negative or MerTK- MDSCs). In some embodiments, the modified MDSC is modified from a MDSC or a purified population of MDSC that lack CD84 and MerTK (i.e. CD84 MerTK double negative or CD84- MerTK- MDSCs).

[0095] In some embodiments, the modified MDSC comprises CD84 and is modified from a CD84- MDSC, a MerTK- MDSC, and / or a CD84- MerTK- MDSC. In some embodiments, the modified MDSC comprises MerTK and is modified from a CD84- MDSC, a MerTK- MDSC, and / or a CD84- MerTK- MDSC. In some embodiments, the modified MDSC comprises CD84 and MerTK and is modified from a CD84- MDSC, a MerTK- MDSC, and / or a CD84- MerTK- MDSC.

[0096] Methods of modifying MDSCs are well-known in the arts. Such methods include, but are not limited to, microinjection, electroporation, sonoporation, nanoscale injection devices, protein transduction domains (PTDs), cell-penetrating peptides (CPPs), chemical modifications, liposomes, inorganic nanoparticles, viral carriers, endocytosis, biolistics, lipofection, calcium phosphate, transduction, transfection, infection, etc. Such methods and elements to modify MDSCs are further described in detail below.

[0097] In some embodiments, the CD84- MDSC, MerTK- MDSC, and / or CD84- MerTK- MDSC is contacted with GCSF; GM-CSF; IL-2; IL-4; IL-6; IL-34; IL-10; a fusion protein comprising IL-4, IL-34, and IL- 10; or a combination thereof.

[0098] Methods of Modifying MDSCs

[0099] In various embodiments, the present invention provides methods of modifying MDSCs of the present invention. The MDSCs of the invention may be modified ex vivo and / or in vivo. With respect to ex vivo immunization, a method of modifying the MDSCs comprises at least one of the following in vitro prior to administering a cell into a subject: i) expanding the MDSC, ii) introducing an activator of CD84, MerTK, or combination thereof to the MDSC, iii) purifying the MDSC, or iv) cry opreserving the MDSC.

[0100] Ex vivo procedures are well known in the art. Briefly, cells are isolated from a subject (e.g., a human) and modified. The modified MDSC can be administered to a subject to provide a therapeutic benefit. The subject may be a human and the modified MDSC can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0101] In various embodiments, the method comprises contacting the MDSC with an activator. In various embodiments, the activator is an activator of CD84, MerTK, or a combination thereof. In some embodiments, the activator is an activator of CD84, MerTK, or a combination thereof in MDSCs. It will be understood by one of skill in the art that an increase in activity of CD84, MerTK, or a combination thereof can similarly be accomplished by increasing the levels of CD84, MerTK, or a combination thereof. In some embodiments, the method comprises increasing the levels of CD84, MerTK, or a combination thereof. In some embodiments, the method comprises increasing the levels of CD84, MerTK, or a combination thereof in the MDSC. In one embodiment, the method comprises contacting the MDSC with an activator, wherein the activator increases the amount of CD84 polypeptide, the amount of CD84 mRNA, the amount of CD84 activity, the amount of MerTK polypeptide, the amount of MerTK mRNA, the amount of MerTK activity, or a combination thereof.

[0102] It is understood by one skilled in the art, that an increase in the level CD84, MerTK, or a combination thereof encompasses the increase of CD84 protein expression, MerTK protein expression, or a combination thereof. Thus, increasing the level of CD84, MerTK, or a combination thereof includes, but is not limited to, increasing the protein levels of CD84, MerTK, or a combination thereof; and increasing transcription, translation, or both, of a nucleic acid encoding CD84, MerTK, or a combination thereof.

[0103] Thus, the present invention relates to methods of modifying MDSCs comprising contacting the MDSC with at least one of a CD84 polypeptide, a MerTK polypeptide, a recombinant CD84 polypeptide, a recombinant MerTK polypeptide, an active CD84 polypeptide fragment, an active MerTK polypeptide fragment, an activator of CD84, or an activator of MerTK.

[0104] Activation of CD84, MerTK, or a combination thereof can be assessed using a wide variety of methods, including those disclosed herein, as well as methods well-known in the art or to be developed in the future. That is, the routineer would appreciate, based upon the disclosure provided herein, that increasing the level or activity of CD84, MerTK, or a combination thereof can be readily assessed using methods that assess the level of a nucleic acid encoding one or more of CD84 and MerTK (e.g., mRNA) and / or the level of one or more of CD84 and MerTK polypeptide in a biological sample obtained from a subject.

[0105] An activator of CD84, MerTK, or a combination thereof can include, but should not be construed as being limited to, a chemical compound, a protein, a peptidomemetic, an antibody, a nucleic acid molecule. One of skill in the art would readily appreciate, based on the disclosure provided herein, that an activator of CD84, MerTK, or a combination thereof encompasses a chemical compound that increases the level, enzymatic activity, or the like of CD84, MerTK, or a combination thereof. In some embodiments, the enzymatic activity is one or more of increased MDSC activity, suppression of T cell activity, suppression of T cell proliferation, and induction of transplantation tolerance. Additionally, an activator of CD84, MerTK, or a combination thereof encompasses a chemically modified compound, and derivatives, as is well known to one of skill in the chemical arts.

[0106] It will be understood by one skilled in the art, based upon the disclosure provided herein, that modifying MDSCs to comprise an increase in the level of CD84, MerTK, or a combination thereof encompasses the increase in CD84 expression, MerTK expression, or a combination thereof, including transcription, translation, or both. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that an increase in the level of CD84, MerTK, or a combination thereof includes an increase in CD84 activity, MerTK activity, or a combination thereof (e.g., enzymatic activity, receptor binding activity, etc.). Thus, increasing the level or activity of CD84, MerTK, or a combination thereof includes, but is not limited to, increasing the amount of CD84 polypeptide, MerTK polypeptide, or a combination thereof, increasing transcription, translation, or both, of a nucleic acid encoding CD84, MerTK, or a combination thereof; and it also includes increasing any activity of a CD84 polypeptide, MerTK polypeptide, or a combination thereof as well. The method of modifying MDSCs can selectively activate CD84, MerTK, or a combination thereof. Thus, the present invention relates to modifying MDSCs by contacting the MDSC with a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, an activator of CD84 expression or activity, MerTK polypeptide, a recombinant MerTK polypeptide, an active MerTK polypeptide fragment, an activator of MerTK expression or activity, or a combination thereof.

[0107] Further, one of skill in the art would, when equipped with this disclosure and the methods exemplified herein, appreciate that an activator of CD84, MerTK, or a combination thereof includes such activators as discovered in the future, as can be identified by well-known criteria in the art of pharmacology, such as the physiological results of activation of CD84, MerTK, or a combination thereof as described in detail herein and / or as known in the art. Therefore, the present invention is not limited in any way to any particular activator of CD84, MerTK, or a combination thereof as exemplified or disclosed herein; rather, the invention encompasses those activators that would be understood by the routineer to be useful as are known in the art and as are discovered in the future.

[0108] Further methods of identifying and producing an activator of CD84, MerTK, or a combination thereof are well known to those of ordinary skill in the art, including, but not limited to, obtaining an activator from a naturally occurring source. Alternatively, an activator of CD84, MerTK, or a combination thereof can be synthesized chemically. Further, the routineer would appreciate, based upon the teachings provided herein, that an activator of CD84, MerTK, or a combination thereof can be obtained from a recombinant organism. Compositions and methods for chemically synthesizing activators of CD84, MerTK, or a combination thereof and for obtaining them from natural sources are well known in the art and are described in the art.

[0109] One of skill in the art will appreciate that an activator can be administered as a small molecule chemical, a protein, a nucleic acid construct encoding a protein, or combinations thereof. Numerous vectors and other compositions and methods are well known for administering a protein or a nucleic acid construct encoding a protein to cells or tissues. Therefore, the invention includes a method of modifying an MDSC comprising contacting the MDSC with a protein or a nucleic acid encoding a protein that is an activator of CD84, MerTK, or a combination thereof.

[0110] One of skill in the art will realize that diminishing the amount or activity of a molecule that itself diminishes the amount or activity of CD84, MerTK, or a combination thereof can serve to increase the amount or activity of CD84, MerTK, or a combination thereof. Any inhibitor of a regulator of CD84, MerTK, or a combination thereof is encompassed in the invention. As a non-limiting example, antisense is described as a form of inhibiting a regulator of CD84, MerTK, or a combination thereof in order to increase the amount or activity of CD84, MerTK, or a combination thereof. Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of a mRNA molecule. When present in a cell, antisense oligonucleotides hybridize to an existing mRNA molecule and inhibit translation into a gene product. Inhibiting the expression of a gene using an antisense oligonucleotide is well known in the art (Marcus- Sekura, 1988, Anal. Biochem. 172:289), as are methods of expressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No. 5,190,931). The methods of the invention include the use of antisense oligonucleotide to diminish the amount of a molecule that causes a decrease in the amount or activity CD84, MerTK, or a combination thereof, thereby increasing the amount or activity of CD84, MerTK, or a combination thereof. Contemplated in the present invention are antisense oligonucleotides that are synthesized and provided to the cell by way of methods well known to those of ordinary skill in the art. As an example, an antisense oligonucleotide can be synthesized to be between about 10 and about 100, more preferably between about 15 and about 50 nucleotides long. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides to improve biological activity in comparison to unmodified antisense oligonucleotides (Tullis, 1991, U.S. Pat. No. 5,023,243).

[0111] Similarly, the expression of a gene may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory element of a gene, thereby affecting the transcription of the gene. Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are well known in the art, and include such methods as the yeast two hybrid system (Bartel and Fields, eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary, N.C.).

[0112] Alternatively, inhibition of a gene expressing a protein that diminishes the level or activity of CD84, MerTK, or a combination thereof can be accomplished through the use of a ribozyme. Using ribozymes for inhibiting gene expression is well known to those of skill in the art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267: 17479; Hampel et al., 1989, Biochemistry 28: 4929; Altman et al., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA molecules with the ability to cleave other single-stranded RNA molecules. Ribozymes are known to be sequence specific, and can therefore be modified to recognize a specific nucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing the selective cleavage of specific mRNA molecules. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozyme without undue experimentation, provided with the disclosure and references incorporated herein.

[0113] One of skill in the art will appreciate that a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, a MerTK polypeptide, a recombinant MerTK polypeptide, or an active MerTK polypeptide fragment can be contacted to the MDSC singly or in any combination thereof. Further, a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, a MerTK polypeptide, a recombinant MerTK polypeptide, or an active MerTK polypeptide fragment can be contacted to the MDSC singly or in any combination thereof in a temporal sense, in that they may be administered simultaneously, before, and / or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that a cell contacted with a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, a MerTK polypeptide, a recombinant MerTK polypeptide, or an active MerTK polypeptide fragment can be used to induce transplantation tolerance, and that an activator can be used alone or in any combination with another CD84 polypeptide, recombinant CD84 polypeptide, active CD84 polypeptide fragment, CD84 activator, MerTK polypeptide, recombinant MerTK polypeptide, active MerTK polypeptide fragment, or MerTK activator to effect a therapeutic result.

[0114] One of skill in the art, when armed with the disclosure herein, would appreciate that inducing transplantation tolerance encompasses administering a cell that has been contacted with a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, CD84 activator, a MerTK polypeptide, a recombinant MerTK polypeptide, an active MerTK polypeptide fragment, or MerTK activator as a preventative measure against a loss / lack of transplantation tolerance. As more fully discussed elsewhere herein, methods of increasing the level or activity of CD84, MerTK, or a combination thereof encompass a wide plethora of techniques for increasing not only activity of CD84, MerTK, or a combination thereof, but also for increasing expression of a nucleic acid encoding CD84, MerTK, or a combination thereof. Additionally, as disclosed elsewhere herein, one skilled in the art would understand, once armed with the teaching provided herein, that the present invention encompasses a method of preventing a wide variety of diseases where increased expression and / or activity of CD84, MerTK, or a combination thereof mediates, treats or prevents the disease. Further, the invention encompasses treatment or prevention of such diseases discovered in the future.

[0115] The invention encompasses contacting the MDSC with a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, a CD84 activator, a MerTK polypeptide, a recombinant MerTK polypeptide, an active MerTK polypeptide fragment, a MerTK activator, or a combination thereof to practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and contact the MDSC with the appropriate CD84 polypeptide, recombinant CD84 polypeptide, active CD84 polypeptide fragment, CD84 activator, MerTK polypeptide, recombinant MerTK polypeptide, active MerTK polypeptide fragment, MerTK activator, or a combination thereof to a subject. However, the present invention is not limited to any particular method of contacting or modifying the cell. This is especially true where it would be appreciated by one skilled in the art, equipped with the disclosure provided herein, including the reduction to practice using an art- recognized model of a transplantation tolerance, that methods of contacting the MDSC with a CD84 polypeptide, a recombinant CD84 polypeptide, an active CD84 polypeptide fragment, CD84 activator, a MerTK polypeptide, a recombinant MerTK polypeptide, an active MerTK polypeptide fragment, MerTK activator, or a combination thereof can be determined by one of skill in the pharmacological arts.

[0116] As used herein, the term “pharmaceutically-acceptable carrier” means a chemical composition with which an appropriate CD84 polypeptide, recombinant CD84 polypeptide, active CD84 polypeptide fragment, CD84 activator, MerTK polypeptide, recombinant MerTK polypeptide, active MerTK polypeptide fragment, or MerTK activator may be combined and which, following the combination, can be used to contact the MDSC with the appropriate CD84 polypeptide, recombinant CD84 polypeptide, active CD84 polypeptide fragment, CD84 activator, MerTK polypeptide, recombinant MerTK polypeptide, active MerTK polypeptide fragment, or MerTK activator to a subject.

[0117] Pharmaceutical Compositions

[0118] Pharmaceutical compositions of the present invention may comprise a MDSC as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e g., aluminum hydroxide); and preservatives. Compositions of the present invention are in one embodiment formulated for intravenous administration.

[0119] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0120] When “an immunologically effective amount” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the cells described herein may be administered at a dosage of 10e4 to 10e9 cells / kg body weight, in some instances 10e5 to 10e6 cells / kg body weight, including all integer values within those ranges. The cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0121] In various embodiments, it may be desired to administer activated cells (e.g. MDSCs) to a subject and then subsequently redraw blood (or have an apheresis performed), activate cells therefrom according to the present invention, and reinfuse the patient with these activated and expanded cells. This process can be carried out multiple times every few weeks. In various embodiments, cells can be activated from blood draws of from lOcc to 400cc. In various embodiments, cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or lOOcc. Not to be bound by theory, using this multiple blood draw / multiple reinfusion protocol, may select out certain populations of cells.

[0122] The administration of a subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one embodiment, the cell compositions of the present invention are administered by i.v. injection. The compositions of cells may be injected directly into a transplant site, lymph node, or site of infection.

[0123] In various embodiments of the present invention, cells activated and expanded using the methods described herein, or other methods known in the art where cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In some embodiments, the cells of the invention may be used in a treatment regimen in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. Drugs that inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In one embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In one embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment

[0124] Methods of Use

[0125] In various embodiments, the present invention provides methods of administering a subject with the composition of the invention. In some embodiments, the subject is receiving a transplant or has received a transplant. In some embodiments, the subject is receiving or has received a heart transplant.

[0126] In certain embodiments, the method comprises adoptive cell therapy comprising administering MDSCs to a subject. In some embodiments, the MDSCs are CD84+ MDSCs, MerTK+ MDSCs, CD84+ MerTK+ MDSCs, or a combination thereof. In one embodiment, the method comprises administering a purified population of MDSCs to the subject. In certain aspects, administering MDSCs or purified population of MDSCs induces transplant tolerance in the subject.

[0127] In some embodiments, the present invention provides methods of treating a subject comprising administering a composition of the invention to the subject. In some embodiments, the subject is hyperimmune. In some embodiments, the present invention provides methods of suppressing T cell proliferation and / or activation in a subject comprising administering a composition of the invention to the subject. In some embodiments, the present invention provides methods of stimulating the generation of Treg cells in a subject comprising administering a composition of the invention to the subject.

[0128] In some embodiments, the method comprises administering to a subject MDSCs. In some embodiments, the MDSCs comprise MDSCs collected from the subject. In some embodiments, the MDSCs comprise MDSCs collected from a second subject. In some embodiments, the second subject is the same species as the subject being administered the MDSCs. In some embodiments, the second subject is from another species than the subject being administered the MDSCs. In some embodiments, the MDSCs comprise MDSCs collected from the subject and MDSCs collected from at least one other subject. In some embodiments, the MDSCs comprise MDSCs that have been collected from at least two other subjects.

[0129] In various embodiments, the MDSC administered to the patient persist in the patient for at least one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, a month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of a MDSC to the patient

[0130] In some embodiments, the subject is administered MDSCs that have been stored. In some embodiments, the subject is administered MDSCs that have been frozen and thawed.

[0131] Embodiments

[0132] The disclosure includes at least the following numbered embodiments:

[0133] 1. A composition for adoptive cell transfer to induce transplantation tolerance comprising one or more myeloid derived suppressor cells (MDSCs) selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

[0134] 2. The composition of embodiment 1, wherein the composition comprises a purified population of one or more MDSC selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

[0135] 3. The composition of any of embodiments 1-2, wherein the CD84+ MerTK+ MDSCs are generated by expanding a population of MDSCs with one or more selected from the group consisting of: GCSF; GM-CSF; IL-2; IL-4; IL-6; IL-34; IL-10; and a fusion protein comprising IL-4, IL-34, and IL- 10.

[0136] 4. The composition of embodiments 1-3, wherein the MDSC lacks a human leukocyte antigen (HLA) molecule.

[0137] 5. A method of treating a subject receiving a transplant or who has received a transplant, comprising administering a composition of any of embodiments 1-4 to the subject.

[0138] 6. The method of embodiment 4, wherein the subject is receiving, or has received, a heart transplant.

[0139] 7. A method of treating a hyperimmune subject comprising administering a composition of any of embodiments 1-4 to the subject.

[0140] 8. A method of suppressing T cell proliferation in a subject comprising administering a composition of any of embodiments 1-4 to the subject.

[0141] 9. A method of reducing the dose of an immunosuppressive drug administered to a subject comprising administering a composition of any of embodiments 1-4 to the subject.

[0142] 10. A method of stimulating the generation of Treg cells in a subject comprising administering a composition of any of embodiments 1-4 to the subject.

[0143] 11. A method comprising collecting myeloid derived suppressor cells (MDSC) from a subject for adoptive cell transfer to induce transplantation tolerance.

[0144] 12. The method of embodiment 11, wherein the MDSC are one or more MDSC selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

[0145] 13. The method of any of embodiments 11-12, wherein the MDSC are splenic MDSC, bone marrow MDSC, or a combination thereof.

[0146] 14. The method of any of embodiments 11-13, further comprising purifying the MDSCs for one or more selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

[0147] 15. The method of embodiment 13, wherein the purifying comprises fluorescence- activated cell sorting. 16. The method of any of embodiments 11-15, further comprising stimulating the MDSC with GCSF, growth factors, or a combination thereof.

[0148] 17. The method of any of embodiments 11-15, further comprising freezing the MDSC.

[0149] EXPERIMENTAL EXAMPLES

[0150] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0151] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0152] Example 1 : CD84+ / MerTK+ MDSCs for the purposes of controlling alloreactivity

[0153] It was previously shown that adoptive transfers of largely undifferentiated myeloid derived suppressor cells (MDSCs) led to tolerance and that the stimuli for MDSC development was an important indicator for MDSC potency (Lee Y, et al., Cell Immunol, 2021, 364: 104346; Scalea JR, et al., Transplantation, 2018, 102(3):359-367; Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355). Indeed, MDSCs which originate in bone marrow are generated from downstream immature myeloid cells (IMCs) under conditions of acute inflammation. Notably, the type of inflammation to which the MDSCs are exposed informs the MDSC potency (Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355; Gabrilovich D, et al., Nat Rev Immunol, 2009, 9(3): 162-174).

[0154] It was previously observed that MDSCs expanded by GCSF were more effective in controlling T cell responses in vitro and in vivo to heterotopic heart transplants than were MDSCs expanded by tumors (Lee Y, et al., Cell Immunol, 2021, 364: 104346; Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355; Scalea JR, et al., Transplantation, 2018, 102(3):359-367). Interestingly, transplantation alone also stimulates the expansion of MDSCs. These MDSCs, however, were also less effective in controlling anti-donor T cell responses than GCSF expanded MDSCs. What was unclear from these initial studies was which specific MDSC population was required for transplantation tolerance induction. Indeed, the heterogeneity of the MDSC population has impeded the understanding of the “ideal” MDSCs required for the purposes of controlling alloreactivity.

[0155] In phenotypic analyses, it was found that GCSF expanded MDSCs expressed CD84 (Lewinsky H, et al., JCI Insight, 2021, 6(6) :e 141683 ; Veglia F, et al., Nat Rev Immunol, 2021, 21 (8):485— 98). For T cell regulation, CD84+ MDSCs have been shown to be more potent than CD84- MDSCs (Lewinsky H, et al., JCI Insight, 2021, 6(6):el41683). CD84 is a membrane glycoprotein and a member of the signaling lymphocyte activation molecule family (SLAM) (Yan Q, et al., Proc Natl Acad Sci U S A, 2007, 104(26): 10583-10588). CD84 is an adhesion molecule expressed on multiple immune cells, a biomarker in malignancies, and regulates T:B cell interactions as well as T cell cytokine secretion (Cuenca M, et al., Clin Immunol, 2019, 204:43-49). CD84+ MDSCs exhibit T cell suppressive capacity and increased ROS production (Veglia F, et al., Nat Rev Immunol, 2021, 21(8):485-498).

[0156] MerTK is a receptor tyrosine kinase that regulates cell survival, migration, and differentiation as well as efferocytosis (Mehrotra P, et al., Nat Rev Drug Discov, 2022, 21(8):601-620; Holtzhausen A, et al., Cancer Immunol Res, 2019, 7(10): 1672-1686; Li K, et al., Signal Transduct Target Ther, 2021, 6:362). MerTK is among the TAM family of tyrosine kinases and is expressed on antigen presenting cells which contribute to inhibition of inflammatory responses (Peeters MJW, et al., Cancer Immunol Res, 2019, 7(9): 1472-1484). MerTK is expressed on MDSCs and controls T cell activation, and thus may be of benefit in a transplant model. MDSCs expressing MerTK show reduced ischemia reperfusion injury after lung transplantation (Leroy V, et al., bioRxiv, 2024). Further, in a model of allogeneic tolerance using apoptotic splenocytes, MerTK was required for expansion of MDSCs and for suppression of proinflammatory cytokines including IFN-alpha. Conversely, MerTK deficiency resulted in tolerance failure (Zhang L, et al., Am J Transplant, 2019, 19(3):674- 685).

[0157] Here, it was examined whether GCSF expanded MDSCs which express both CD84 and MerTK are more effective than CD84- and MerTK- MDSCs. If so, the potential synergies between the two may then be studied. In this regard, an in-vitro model of T cell suppression was used to determine how CD84 and MerTK contribute to MDSC function. It was found that absence of CD84 and MerTK eliminates T cell suppression among GCSF expanded MDSCs. It was found that CD84+ MerTK+ MDCSs represent approximately 10-15% of GCSF expanded MDSCs and may be responsible for (or at least contribute to) the effects of tolerance observed in heterotopic heart transplant model.

[0158] Based on these results, it appears that naturally occurring CD84+ MerTK+ MDSCs may be augmented to suppress anti-donor T cell responses in transplant recipients. This is important because it could be possible to generate large populations of CD84+ MerTK+ MDSCs for the purposes of inducing transplantation tolerance. By better characterizing the ideal MDSC, innovations such as off the shelf treatments for tolerance induction or immunosuppressive optimization will become a reality.

[0159] The methods are described herein.

[0160] Mice

[0161] Male and female wild-type and 6 to 8- week old C57BL / 6J and BALB / c mice. MerTK knockout (MerTK- / -) mice on C57BL / 6J background were generated and a breeding colony established (Zhang H, et al., JCI Insight, 2023, 8:el 51819). CD84 knockout (CD84- / -) mice were also used. All mice were housed in the pathogen-free facilities and mice were fed on standard laboratory food and given tap water ad libitum with a light-dark cycle of 12 hours.

[0162] Administration of GCSF and Isolation of CD4+ T cells and MDSCS

[0163] C57BL / 6J or BALB / c or MerTK KO or CD84 KO mice were intraperitoneal injected with 200ng of granulocyte-colony stimulating factor (GMSF) daily for 4 days and spleens or bone marrow were harvested next day after the last dose (Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355). Splenic and bone marrow were isolated and purified using the EasySep Mouse CD4+ T cell isolation kit or mouse T cell isolation kit, Purity ranged between 95 to 99%, and determined by flow cytometry. MDSC cell were isolated using the EasySep Mouse MDSC (CD1 lb+ Grl+) isolation kit (Stemcell Technologies, Vancouver, BC, Canada), Purity ranged from 98 to 99% (Beliakova-Bethell N, et al., Cytometry A, 2014, 85(2):94-104). Antibodies and reagents

[0164] CellTrace™ Violet Cell Proliferation Kit, CellTrace™ CFSE Cell Proliferation Kit, anti-mCD3 (17A2) and anti-mCD28 (37.51), ). Anti-mCD25- PE / Cy7 (PC61.5), anti- mF4 / 80- PerCP / Cy5.5 (BM8), Anti-mCD8a- PE (53-6.7), anti-mCD8a- AF700 (53-67), anti- mCD4-PE / Cy5 (RM-4-5) were purchased from invitrogen (Thermo Fisher, Hillsboro, OR). Anti- mCD3e (KT3) and anti-mCD28 (37.51) were purchased from BioXCell. Anti-mCD16 / CD32 (2.4G2), anti-mCDl lb- PE (MI / 70), anti-mCD45- FITC (30-F11), anti-mLy-6G and Ly-6c- PerCP / Cy5.5 (2.4G2), anti-mLy-6G / Ly-6C (Gr-l)-Pacific blue (RB6-8C5), anti-mCD4- APC (RM4-5) were purchased from BD Pharmingen (San Diego, CA). Anti-mLy6G- APC / Cy7 (1A8), anti-mLy6C- PerCP (HK 1.4), anti-mCD192(CCR2)- PE (SA203G11), anti-mRAE-ly- AF647 (CXI), purified anti-mouse CD84 antibody (mCD84.7), anti-mCD84- PE (mCD84.7), anti-mCD4-FITC (RM4-5), anti-mCD4-PE / Cy5 (RM4-5), anti-mCDl lb- FITC (MI / 70), anti- mCD274 (PD-L1)- PerCP / Cy5.5 (0F.9G2), anti-mCD45R / B220- AF700 (RA3-6B2), anti- mCD182(CXCR2)- APC (SA044G4), Anti-mCD182(CXCR2)- APC (SA044G4), anti -mouse MERTK (mer) antibody-APC (2B10C42), anti-MerTK- PE (2B10C42), recombinant mGMSF were purchased from BioLegend (San Diego, CA, USA). Anti- mMer- APC (108928), anti- mSTAT3- AF594 (232209), Anti- mTGF-01,2,3- APC (1D11), Anti-mCD115- APC (460615), Recombinant mGM-CSF were purchased from R&D Systems (Minneapolis, MN). CB-1158, hydrochloride (Chemietek, IN, USA), 1400W dihydrochloride (APExBIO technology, Houston, TX, USA).

[0165] Analysis of flow cytometry

[0166] The single cell suspensions from mouse spleen and bone marrow were treated with the anti-mouse CD16 / 32 (Clone 2.4G2 , BD pharmingen) to block FcRIII / II receptors 10 minutes, then stained with antibodies according to the recommended assay procedure, and then washed two times in eBioscience™Flow Cytometry Staining Buffer. Samples were analyzed using a LSR Fortessa Flow Cytometer (BD Biosciences) and data were analyzed with FlowJo (Tree Star).

[0167] In vitro MDSCs suppression assay Suppression assays similar to prior work were leveraged (Lee Y, et al., Cell Immunol, 2021, 364: 104346; Lee Y, et al., Am J Transplant, 2020, 20(8):2343-2355). CD4+ T cells were isolated from the spleens from the C57BL / 6 or BALB / c mice using CD4 T cell isolation kit according to the manufacturer’s procedures. Enriched CD4+ T cells were labeled with 5 pm / L CellTrace™ Violet (CTV) in 1 mL of PBS for 15 minutes at 37°C 5% CO2 incubator. Added an excess of FCS (4 ml) to halt the labeling, and the samples were washed twice with PBS or RPMI 1640 (Gibco, Life Technologies), The CTV-labeled cells were cultured in an anti-CD3 antibody (OKT3, 5pg / ml, Invitrogen or BioxCell )-coated 96-well plate in the presence / absence of Gr-1+ MDSCs obtained from the spleen and bone marrow of WT naive controls at different ratios in the complete T cell culture medium supplemented with anti-CD28 (37.51, 2pg / ml, Invitrogen or BioxCell). A total of 1x106-labeled CD4+ T cells were plated in complete media (RPMI 1640, 10% FBS, 20 units / mL penicillin, 50 mg / mL streptomycin) onto flat-bottomed 96-well plates (Corning, BV) coated with 5 pg / mL anti-CD3 and 2 pg / mL anti- CD28 (BioXCell). MDSCs isolated from WT naive and MerTK KO and CD84 KO mice were added at various ratios. T / MDSC Cells were co-cultured for at 37°C in a 5% CO2 incubator. 3 days after incubation, cells were collected and stained with CD4-Apc, CD25-PE / Cy7 for 30min and the CTV fluorescence intensity was analyzed by flow cytometry. MDSCs suppression was analyzed by assessing T cell proliferation using flow cytometric analysis of CTV dilution.

[0168] The results are described herein:

[0169] In-Vitro Expansion of CD4+ T cells and MDSCs: GCSF Expansion Impacts Expression of Key Tolerance Mediators

[0170] Mouse PBMCs were purified from whole blood. From PMBCs, CD4+ T cells were purified and cultured in vitro with anti-CD3 / CD28 stimulation. Cultures for 72 hours showed lOOOx increase in T cell counts, as well as T cell clustering as shown in Figure 1A. MDSCs were isolated from spleen as well as from bone marrow, and with GCSF expansion, were primarily of the granulocytic (Ly-6GG) phenotype (Figure IB). The M-MDSCs phenotype was more common in bone marrow. Phenotypic analysis of GCSF expanded MDSCs (Figure 1C) showed expression of F4 / 80, CXCR2, as well as CD84. CD84 was expressed among -15% of BM-MDSCs and nearly 40% of splenic MDSCs. GCSF increased expression of MerTK among WT animals. MerTK KO mice expressed no MerTK (control), but also showed lower expression of critical mediators of MDSC-mediated alloregulation including CD115, PD-L1, TGF-B, CCR2, and Stat3 (Figure ID).

[0171] MerTK KO MDSCs show impaired suppression of T cell proliferation

[0172] The effect of eliminating MerTK among previously suppressive MDSCs was determined. T cells expanded by CD3 / CD28 were co-cultured with GCSF and WT MDSCs (Figure 2A). GCSF expansion augmented suppression of T cell proliferation in a dose-dependent fashion. GCSF-expanded MDSCs reduced proliferation 3-fold as shown in Figure 2B. Whereas GCSF-expanded MDSCs from WT mice showed significant reduction in T cell proliferation (Figure 3A), MerTK KO mice demonstrated negligible reduction in T cell proliferation (Figure 3B). These data suggest that MerTK is an important driver of MDSC-mediated T cell suppression.

[0173] CD84 KO MDSCs show impaired suppression of T cell proliferation

[0174] Next, the expression of CD84 among MerTK KO mice and the impact of CD84 on T cell proliferation was examined (Figure 4A). Surprisingly, the MerTK KO animals expressed very little CD84 (Figure 4B). When compared with WT MDSCs, CD84KO mice demonstrated impaired suppression of T cell proliferation in a dose dependent manner, similar to the pattern of effectiveness observed with MerTK KO animals (Figure 4C).

[0175] Anti-CD84 and MerTK KO eliminated the T cells suppressive effects of MDSCs To learn the impact on T cell proliferation among GCSF-expanded MDSCs in the absence of both CD84 and of MerTK, it was determined whether blocking CD84 among MerTK KO animals would further impair T cell proliferation. Different doses of anti-CD84 blocking antibody were tested (Figure 5 and Figure 6). Blocking CD84 with anti-CD84 blocking antibody showed a modest dose-response from 0-20pg in vitro, such that 20 pg of anti-CD84 antibody was most significantly associated with impaired T cell proliferation. The effects of anti-CD84 antibody with MerTK KO were combined to model the effect of eliminating both MerTK as well as CD84 (Figure 7). Combining both MerTK KO and anti-CD84 nearly eliminated all suppression of T cell proliferation. Frequency of CD84+ / MerTK+ MDSCs and control of T cell responses

[0176] Because it appeared that CD84 and MerTK are both important for control of T cell proliferation, it was determined what percent of total MDSCs are double positive for CD84 and MerTK. Indeed, this combination of markers may represent an ideal subset of MDSCs for the purposes of controlling anti-donor T cell responses. Among WT MDSCs expanded by GCSF, it was found that approximately 9% of splenic and as many as 20% of bone marrow MDSCs are both CD84+ and MerTK+. In vitro CD84+ / MerTK+ MDSCs expanded from spleen and bone marrow were quite potent. Indeed, in co-culture, both purified splenic and bone marrow CD84+ / MerTK+ T MDSCs expanded by GCSF near-completely or completely eliminated T cell proliferation at doses of 1 : 1 and 1 :2. Indeed, purified CD84+ / MerTK+ MDSCs are able to control anti-donor T cell responses in vitro.

[0177] Organ transplantation tolerance has been an elusive yet intriguing goal in transplantation for decades. In exciting developments, initial studies led to human trials of bone- marrow transplant induced chimerism and subsequent tolerance of human kidneys (Leventhal JR, et al., Hum Immunol, 2018, 79(4):272-276; Kawai T, et al., N Engl J Med, 2008, 358(1):353-361)

[0178] While the most successful studies of tolerance include bone marrow transplant induction, several animal studies highlight the importance of peripheral mechanisms of tolerance (Leventhal JR, et al., Hum Immunol, 2018, 79(4):272-276; Leventhal J, et al., Sci Transl Med, 2012, 4(124): 124ral28). Such peripheral tolerance models include Tregs, suppressive monocytes, and in the present example, MDSCs. There has been a particular interest in peripheral models, with a focus on MDSCs, as MDSCs are naturally occurring and could be harnessed to the advantage of the transplant recipient as a mechanisms for reducing reliance on pharmacologic immunosuppression, whilst also reducing the requirement for bone marrow transplantation.

[0179] In an early study of bone marrow transplantation mediated tolerance in a heterotopic heart model of mice, MDSCs were surprisingly shown to be required for tolerance induction (Hongo D, et al., Am J Transplant, 2014, 14(11):2467-2477). These MDSCs expressed Argl and PDL1, consistent with mechanistic studies of MDSC mediated T cell control (Scalea JR, et al., Transplantation, 2018, 102(3)359-367; Gabrilovich D, et al., Nat Rev Immunol, 2009, 9(3): 162-174; Hongo D, et al., Am J Transplant, 2014, 14(1 1):2467-2477). Similarly, it was found that depletion of MDSCs using anti-GRl mAb treatment led to loss of chimerism and subsequent loss of tolerance (Hongo D, et al., Am J Transplant, 2014, 14(11):2467-2477).

[0180] Intrigued by prior findings linking MDSCs to tolerance induction and inspired that MDSCs might represent a pathway to tolerance independent of bone marrow transplantation, it was sought to be determined if MDSCs could themselves control anti-donor T cell responses in vitro and in vivo (Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355; Scalea JR, et al., Transplantation, 2018, 102(3):359-367). It was found that, while transplant-induced MDSCs can control T cell responses, the most potent MDSCs were generated by GCSF. In this regard, the efficacy of GCSF-expanded MDSCs to establish tolerance in vivo was tested, and it was found that adoptive transfers of large numbers of GCSF-expanded MDSCs led to tolerance (Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355). Further, elimination of non-MDSC splenocytes failed to induce tolerance, reducing the possibility that there was a donor-specific transfusionlike effect driving alternative pathways of T cell regulation. As shown in cancer models, it was also found that MDSC infusions for transplantation led to downstream Treg expansion (Fujimura T, et al., Oncoimmunology, 2012, 1(9): 1433-1434). Relevant to Treg generation, depletion of MDSCs in the recipient up to 3 weeks after transplantation were observed to eliminate tolerance, but later depletion did not (Lee Y, et al., Am J Transplant, 2020, 20(9):2343-2355). This suggested that perhaps tolerance in the longer term after the MDSC adoptive transfer was mediated not directly by MDSCs, but rather Tregs.

[0181] Those studies, though impactful, failed to show which MDSCs among the many, varied phenotypes were primarily responsible for T cell regulation. To answer this question, the phenotypic analysis was reviewed for important MDSC markers that may indicate potency and relevance in transplantation. These were then cross-referenced with published reports to determine possible targets of highly effective, potentially “ideal” MDSC markers. Like others, CD84 was observed to be expressed among GCSF-expanded MDSCs. Intriguingly, MerTK was seen to be a potent mediator of immune responses, but poorly studied in transplantation (Lewinsky H, et al., JCI Insight, 2021, 6(6):el41683; Yan Q, et al., Proc Natl Acad Sci U S A, 2007, 104(26): 10583-10588; Holtzhausen A, et al., Cancer Immunol Res, 2019, 7(10): 1672- 1686; Peeters MJW, et al., Cancer Immunol Res, 2019, 7(9): 1472-1484; Leroy V, et al., bioRxiv, 2024). In evaluating both CD84 and MerTK, it was found that while treatment / KO individually may reduce T cell proliferation through MDSC impairment, eliminating both CD84 and MerTK appeared to be synergistic and (possibly) point toward an ideal MDSC that when expanded might control T cells and, even induce tolerance. These studies were corroborated by finding herein that co-culture of purified CD84+ / MerTK+ MDSCs expanded by GCSF completely eliminated T cell responses.

[0182] Several findings in this study warrant further discussion. Firstly, it is particularly interesting that CD84 was not expressed among MerTK KO animals. The reason for this is not clear. The CD84 gene is expressed on chromosome 1, in the CD2 super family (Farhangnia P, et al., Front Immunol, 2023, 14: 1174138). In contrast, the MerTK gene is encoded on chromosome 2. MerTK contributes to control of cellular growth and proliferation and perhaps, however, downregulates CD84 which is a known cell survivor receptor (Binsky-Ehrenreich I, et al., Oncogene, 2014, 33(8): 1006-1016). Interestingly, it was found the same expression relationship was not true in reverse. Specifically, as shown in Figure 9, MerTK was indeed expressed among CD84 KO MDSCs. More work will need to be done on this to elucidate the underlying relationship.

[0183] The synergy between CD84 and MerTK is also notable. Recent work showed the CD84+ monocytes were highly potent controllers of T cells in humans. Separately, MerTK is a key regulator of immune responses and is expressed on MDSCs. CD84 not only contributes to T cell regulation, but also controls M-MDSC expansion as well as CD8 exhaustion (Zhou L, et al., Cell Death Dis, 2024, 15:507). The finding that both CD84 and MerTK contribute to MDSC expansion as well as T cell suppression may partly explain the additive or synergistic effects of simultaneous expression..

[0184] The heterogeneity of MDSC populations has been a rate limiting step for studies of MDSCs in transplantation. In some ways, this heterogeneity has been the Achilles heel of MDSC studies. The examples herein may shed light on the MDSC populations most relevant for transplantation.

[0185] In vivo studies are performed using a heterotopic heart transplant model. Transplants using adoptive transfers of purified CD84+ / MerTK+ MDSCs are contemplated. Further, breeding of CD84KO / MerTK KO mice and assessment of the immune function of their MDSCs is contemplated. Interestingly, because both CD84 and MerTK individually help expanded MDSC generation, it is possible that CD84KO / MerTK KO animal may make very few natural MDSCs without GCSF.

[0186] The purification and expansion of CD84+ / MerTK+ MDSCs for the purposes of adoptive transfer for use in larger animals are conducted. Indeed, work shown here leads to the development of an “off-the-shelf’ option for transplant tolerance induction for human transplant recipients. While an off-the-shelf model may seem challenging, antigen specificity / restriction of MDSCs has not yet been encountered, and thus it is conceivable that one could expand, freeze, and infuse off-the-shelf MDSCs at the time of transplant with the goal of inducing tolerance, or minimizing reliance on conventional immunosuppressive drugs.

[0187] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A composition for adoptive cell transfer to induce transplantation tolerance comprising one or more myeloid derived suppressor cells (MDSCs) selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

2. The composition of claim 1, wherein the composition comprises a purified population of one or more MDSC selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

3. The composition of claim 1, wherein the CD84+ MerTK+ MDSCs are generated by expanding a population of MDSCs with one or more selected from the group consisting of: GCSF; GM-CSF; IL-2; IL-4; IL-6; IL-34; IL-10; and a fusion protein comprising IL-4, IL-34, and IL- 10.

4. The composition of claim 1, wherein the MDSC lacks a human leukocyte antigen (HLA) molecule.

5. A method of treating a subject receiving a transplant or who has received a transplant, comprising administering a composition of claim 1 to the subject.

6. The method of claim 5, wherein the subject is receiving, or has received, a heart transplant.

7. A method of treating a hyperimmune subject comprising administering a composition of claim 1 to the subject.

8. A method of suppressing T cell proliferation in a subject comprising administering a composition of claim 1 to the subject.

9. A method of reducing the dose of an immunosuppressive drug administered to a subject comprising administering a composition of claim 1 to the subject.

10. A method of stimulating the generation of Treg cells in a subjectcomprising administering a composition of claim 1 to the subject.

11. A method comprising collecting myeloid derived suppressor cells (MDSC) from a subject for adoptive cell transfer to induce transplantation tolerance.

12. The method of claim 11, wherein the MDSC are one or more MDSC selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

13. The method of claim 11, wherein the MDSC are splenic MDSC, bone marrow MDSC, or a combination thereof.

14. The method of claim 11, further comprising purifying the MDSCs for one or more selected from the group consisting of: CD84+ MDSC, MerTK+ MDSC, and CD84+ MerTK+ MDSC.

15. The method of claim 14, wherein the purifying comprises fluorescence- activated cell sorting.

16. The method of claim 11, further comprising stimulating the MDSC with GCSF, growth factors, or a combination thereof.

17. The method of claim 11, further comprising freezing the MDSC.

Images