An immunosuppressive population of cells and uses thereof

Abstract

An immunosuppressive population of cells comprising ex vivo expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells).

Description

AN IMMUNOSUPPRESSIVE POPULATION OF CELLS AND USES THEREOF Related applications

[0001] This application claims priority from Australian Provisional Patent Application No. 202590246 filed on 30 January 2025, the entire content of which is incorporated by reference.Field

[0002] The present disclosure relates generally to an immunosuppressive population of cells comprising ex vivo expanded CD45+ cells, wherein the expanded CD45+ cells comprise a subpopulation of T cells (CD3+ T cells) that are enriched for T helper cells (CD4+ T cells), and pharmaceutical compositions comprising the same. In some embodiments, the present disclosure relates to methods for the treatment of an inflammatory disease or condition comprising administration of the immunosuppressive population of immune cells or the pharmaceutical compositions disclosed herein, and methods of adoptive cell therapy for the treatment of an inflammatory disease or condition.Background

[0003] One of the key features of aging is the gradual decline of the immune system, which is recognised to play a key role in maintaining human health, e.g., by protecting against infections by upregulating the inflammatory response. A normal inflammatory response is characterized by a temporal upregulation of inflammatory activity when an invading pathogen has been encountered. However, chronically activated, low-grade, non-infective systemic inflammation is often present in aged individuals. Such chronic and dysregulated inflammation can lead to hypertension, depression, hyperglycaemia, dyslipidaemia, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, chronic kidney disease, various types of cancer, neurodegenerative disease, osteoporosis, and autoimmune diseases.

[0004] Inflammatory diseases and conditions affecting all age groups are associated with a loss of immunological tolerance to self-antigens, partially owing to unusual responses by regulatory T cells (Tregs). Moreover, for some diseases and conditions, such as autoimmunity, autoantibodies produced by B cells are a large contributor to pathogenesis.Due to the multi-immunogenic nature of the pathophysiology of inflammatory diseases and conditions, there are limited options. For example, monoclonal antibodies targeting CD20 and CD 19 have been used for the treatment of B cell-derived neurological autoimmune diseases such as multiple sclerosis. However, many patients are resistant to B cell-targeting monoclonal antibodies.

[0005] Adoptive cell therapies have emerged as promising cancer treatments, with remarkable clinical outcome when immunogenic cell populations are generated and administered, e.g., in patients with B-cell acute lymphoblastic leukemia (Kochenderfer et al., 2010, Blood, 116: 4099-4102). Following this initial success, the application of adoptive cell therapies to other indications has also been considered, including for the treatment of conditions where immunosuppression is required. For example, there has been considerable efforts made to harness Tregs for the treatment of inflammatory diseases or conditions, without effective therapeutic results. Similarly, mesenchymal stem cells (MSC) and chimeric antigen receptor (CAR) T cells have also been investigated for use in the treatment of inflammatory diseases or conditions, but a lack of therapeutic efficacy combined with adverse side-effects have limited the use of such adoptive cell therapies across broader indications that require immunosuppression.

[0006] Accordingly, there remains an urgent need to develop alternative adoptive cell therapies for the treatment of inflammatory diseases or conditions, including those associated with aging.Summary

[0007] The present disclosure is predicated, in part, on the surprising finding that an immunosuppressive population of cells can be expanded ex vivo under conditions that favour the enrichment of helper T cells within a subpopulation of CD45+ T cells (CD3+ T cells). Unlike previous methods for the manufacture of immune cell populations for use in adoptive cell therapies, e.g., cascade primed (CAPRI) cells, which favour the generation of immunogenic population of cells, the immunosuppressive population of cells disclosed herein exhibit robust anti-inflammatory effects, which are useful for the treatment of inflammatory diseases and conditions, including those associated with aging. It follows, therefore, that the immunosuppressive population of cells of the present disclosure are particularly beneficial in the preparation of adoptive cell therapies for the treatment ofinflammatory diseases and conditions.

[0008] Accordingly, in one aspect, the present disclosure provides an immunosuppressive population of cells comprising ex vivo expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells).

[0009] In another aspect, there is provided pharmaceutical composition comprising the immunosuppressive population of cells disclosed herein.

[0010] In another aspect, there is provided a method for the treatment of an inflammatory disease or condition in a subject in need thereof, the method comprising administering an effective amount of the immunosuppressive population of cells, or the pharmaceutical composition disclosed herein to the subject.

[0011] In another aspect, there is provided a use of the immunosuppressive population of cells, or the pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment of an inflammatory disease or condition.

[0012] In another aspect, there is provided the immunosuppressive population of cells, or the pharmaceutical composition disclosed herein for use in a method for the treatment of an inflammatory disease or condition in a subject in need thereof.

[0013] In another aspect, there is provided method for the ex vivo manufacture of an immunosuppressive population of cells, the method comprising:(a) providing a population of isolated immune cells;(b) separating the population of immune cells into a first population of immune cells and a second population of immune cells;(c) contacting the first population of immune cells with an activating agent to provide an activated population of immune cells,(d) contacting the activated population of immune cells with a composition comprising:(i) interleukin-2 (IL-2); and(ii) interleukin-34 (IL-34);(e) following step (d), combining the activated population of immune cells with the second population of immune cells;(f) expanding the combined populations of immune cells of step (e) in the presence a composition comprising:(i) IL-2; and(ii) IL-34,for a time and under conditions suitable to generate an immunosuppressive population of cells, wherein the immunosuppressive population of cells comprising expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells).

[0014] In another aspect, there is provided an immunosuppressive population of cells manufactured by the method disclosed herein.

[0015] In another aspect, there is provided a composition for the for the ex vivo manufacture of an immunogenic population of cells, the composition comprising:(a) IL-2; and(b) IL-34.

[0016] In another aspect, there is provided a kit for the ex vivo manufacture of an immunogenic population of cells, the kit comprising:(a) IL-2; and(b) IL-34.

[0017] In another aspect, there is provided a method for the treatment of an inflammatory disease or condition in a subject in need thereof, the method comprising:(a) providing a population of immune cells obtained from the subject;(b) separating the population of immune cells into a first population of immune cells and a second population of immune cells;(c) contacting the first population of immune cells with an activating agent to provide an activated population of immune cells,(d) contacting the activated population of immune cells with a composition comprising:(i) IL-2; and(ii) IL-34,(e) combining the activated population of immune cells of step (d) with the second population of immune cells.(f) expanding the combined populations of immune cells of step (e) in the presence of a composition comprising:(i) IL-2; and(i) IL-34,for a time and under conditions suitable to generate an immunosuppressive population of cells, wherein the immunosuppressive population of cells comprising expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells); and(g) administering an effective amount of the immunosuppressive population of cells to the subject.Brief Description of the Drawings

[0018] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the accompanying drawings.

[0019] Figure 1 shows the CD45+ immune effector cell subpopulations of an immunosuppressive population of cells useful for the treatment of inflammatory diseases or conditions. A graphical representation of cell type (x-axis) and proportion in a total CD45+ cell population (%; y-axis). Mean values shown with error bars representative of standard deviation (SD), n = 5.

[0020] Figure 2 shows that the immunosuppressive population of cells has a higher proportion of Th17 T Cells (CD3+, CD161+ T cells) when compared to an immunogenic(i.e., anti-cancer) population of cells. (A-B) A graphical representation of the flow cytometry gating strategy for (A) an immunogenic population of cells; and (B) the immunosuppressive population of cells using CD3 (y-axis) and CD161 (x-axis) to identify Th17 T cells (CD3+, CD161+ T cells). (C) A graphical representation of the proportion of Th17 cells within the CD45+ cell population (Th17 (%CD45); y-axis) within the immunogenic and immunosuppressive populations of cells (x-axis). Error bars representative of SD, n = 5. ****, p <0.0001.

[0021] Figure 3 shows the immunosuppressive population of cells comprise a subpopulation of T cells (CD3+ T cells) that are enriched for T helper cells (CD4+ T cells). (A-B) A graphical representation of the flow cytometry gating strategy for (A) an immunogenic population of cells; and (B) the immunosuppressive population of cells using CD4 (y-axis) and CD8 (x-axis).

[0022] Figure 4 shows that the immunosuppressive population of cells has a reduced proportion of levels of T-regulatory cells (Tregs; CD3+, CD25+ T cells) when compared to an immunogenic population of cells. (A-B) A graphical representation of the flow cytometry gating strategy for (A) an immunogenic population of cells; and (B) the immunosuppressive population of cells using CD3 (y-axis) and CD25 (x-axis) to identify Tregs (CD3+, CD25+ T cells). (C) A graphical representation of the proportion of Tregs within CD45+ cell populations (Tregs (%CD45) y-axis) in immunogenic and immunosuppressive populations of cells (x-axis). Error bars representative of SD, n = 5. **** p <0.0001.[0023 J Figure 5 shows that the immunosuppressive population of cells is characterized by immunoregulatory T cell subpopulations. A graphical representation of the ratio of CD4 / CD8 cells (CD4 / CD8; y-axis) in immunogenic and immunosuppressive populations of cells (x-axis). Error bars representative of SD, n = 5. ****, p <0.0001.

[0024] Figure 6 shows that the immunosuppressive population of cells significantly reduces secretion of pro-inflammatory cytokines relative to an immunogenic population of cells. (A-L) A series of graphical representations of the level of (A) IFN-y; (B) TNF-a; (C) MIP-la, (D) IP-10, (E) IL-17, (F) IL-31, (G) IL-IRa, (H) RANTES, (I) MIP-ip, (J) TNF-P, (K) IL-8, and (L) IL- 18 (pg / mL; y-axis) in supernatant samples collected following the in vitro culture of immunogenic and immunosuppressive populations of cells (x-axis). Meanvalues of biological replicates shown with error bars representative of SD, n = 5. ** p <0.01, *** p <0.001, and **** p <0.0001.

[0025] Figure 7 shows the CD45+ immune effector cell subpopulations of an immunosuppressive population of cells preparing using the alternative expansion method useful for the treatment of inflammatory diseases or conditions. A graphical representation of cell type (x-axis) and proportion in a total CD45+ cell population (%; y-axis). Mean values shown with error bars representative of standard deviation (SD), n = 5.

[0026] Figure 8 shows that the immunosuppressive population of cells has elevated levels of CD4+ cells and increased CD4 / CD8 ratio relative to the immunogenic population of cells. (A-B) A graphical representation of the flow cytometry gating strategy for (A) an immunogenic population of cells; and (B) the immunosuppressive population of cells using CD4 (y-axis) and CD8 (x-axis) to identify CD4+ cells. (C) A graphical representation of the ratio of CD4 / CD8 cells (CD4 / CD8; y-axis) in immunogenic and immunosuppressive populations of cells (x-axis). Error bars representative of SD, n = 5. *, p <0.05.

[0027] Figure 9 shows that the alternative expansion method elevates the proportion of Tregs in the immunosuppressive population of cells relative to the immunogenic population of cells. (A-B) A graphical representation of the flow cytometry gating strategy for (A) an immunogenic population of cells; and (B) the immunosuppressive population of cells using CD3 (y-axis) and CD25 (x-axis) to identify Tregs. (C) A graphical representation of Tregs as a proportion of the total CD3+ cell population (%; y-axis) in the immunogenic or immunosuppressive population of cells (x-axis). Mean values shown with error bars representative of standard deviation (SD), n = 5.<0.0001.

[0028] Figure 10 shows that the immunogenic population of cells potently suppresses the immunogenic activity and viability of human PBMCs relative to the immunogenic population of cells. (A) A graphical representation of the flow cytometry histogram of 7AAD fluorescence of human PBMCs untreated (NT) or treated with either the immunogenic population of cells (anti-cancer) or immunosuppressive population of cells (IPC) for 3 days. (B) A graphical representation of the percentage viability (y-axis; % 7AAD-) of live (7AAD negative) and dead (7AAD positive) PBMCs and time (x-axis; day). Mean values shown with error bars representative of standard deviation (SD), n = 3. **, p <0.01.Detailed Description

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.

[0030] The articles "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells (e.g., a population of cells); reference to "an agent" includes a single agent, as well as two or more agents; and so forth.[003.1] In the context of this specification, the term “about” is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 40%-60%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.

[0032] Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.[0033J The term “optionally” is used herein to mean that the subsequent described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiment in which the event or circumstance occurs as well as embodiments in which it does not.[0034J As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.Immunosuppressive populations of cells

[0035] In an aspect disclosed herein, there is provided an immunosuppressive population of cells comprising ex vivo expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells)

[0036] The term "immunosuppressive" as used herein refers to a composition, e.g., a population of cells, pharmaceutical compositions, etc., which is capable of suppressing an immune response.

[0037] The term "population of cells" refers to heterogeneous cell populations that comprise subpopulations of immune effector cells, e.g., T cells, monocytes, natural killer (NK) cells and / or B cells. It is also contemplated herein the heterogeneous cell populations comprise the progeny of a single parental cell. Due to natural, accidental or deliberate mutation, the progeny cells may not necessarily be identical in morphology or in genome to the original parental cell.

[0038] The terms "expanded", "expansion", and the like as used herein refer to the propagation of cell populations ex vivo or in vitro to reach a desired number or proportion of cells, and subpopulations thereof.

[0039] The term "enriched" as used herein refers to the selective expansion and / or propagation of specific cell types, relative to other cell types in the population. In some embodiments, enrichment of a specific cell type may be evidenced as an increase in the proportion of specific cell types, as described elsewhere herein. For example, in the context of the immunosuppressive population of cells, and the methods disclosed herein, "enrichment" of T helper cells occurs relative to other CD3+ T cells, for example, relative to cytotoxic T cells (CD8+ T cells), natural killer (NK) T cells, Thl7 cells, Tregs, or combinations thereof.

[0040] The term " CD45+ cells" as used herein refers to nuclear hematopoietic cells that express the CD45 transmembrane protein.

[0041] The term " T cell" as used herein refers to CD3+ T cells, and subpopulations thereof. Suitable T cells would be known to persons skilled in the art, illustrative examples of which include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, activated T lymphocytes or tumor infiltrating lymphocytes (TILs). Illustrative subpopulations of T cells encompassed by particular embodiments include but are not limited to heterogeneous subpopulations of CD4+ and CD8+ T cells across naive, central memory and effector linages, T helper cells (CD3+, CD4+T cells), cytotoxic T cells (CD3+, CD8+T cells), natural killer T (NKT) cells (CD3+, CD56+ T cells), T regulatory cells (Tregs; CD3+, CD4+, CD25+, FoxP3+, CD127- T cells), Thl7 cells (CD3+, CD161+ T cells), or any other subset of T cells.

[0042] As disclosed elsewhere herein, the immunogenic population of cells comprising ex vivo expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells). T helper cells are associated with immunoregulation in controlling excessive immune reactions and inflammation.

[0043] The term " T helper cell" refers to CD3+, CD4+ T cells.

[0044] In an embodiment, the subpopulation of T cells (CD3+ T cells) comprises at least about 35% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells) (e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells)).

[0045] In an embodiment, the subpopulation of T cells (CD3+ T cells) comprises at from about 35% to about 50% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells), including all integer values within this range.

[0046] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises:(a) cytotoxic T cells (CD3+, CD8+ T cells);(b) natural killer T (NKT) cells (CD3+, CD56+ T cells);(c) T regulatory cells (Tregs) (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); (d) Thl7 cells (CD3+, CD161+ T cells); and(e) any combination of (a)-(d).

[0047] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells).

[0048] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells).

[0049] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD4+, CD25+, FoxP3+, CD 127- T cells).

[0050] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Thl7 cells (CD3+, CD161+ T cells).

[0051] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and NKT cells (CD3+, CD56+ T cells).

[0052] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127-T cells).

[0053] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0054] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells) and Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells).

[0055] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0056] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0057] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells) and Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells).

[0058] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0059] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells), Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0060] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells), Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0061] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), and wherein the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells), i.e., the CD4 / CD8 ratio, is at least about 0.7 (e.g., at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0).

[0062] In an embodiment, the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is from about 0.7 to about 1.5, including all integer values within this range.

[0063] In an embodiment, the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is from about 0.7 to about 5.0, including all integer values within this range.

[0064] In an embodiment, the expanded CD45+ cells comprise at least about 5% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells (e.g., at least about 5%, 6%, 7%, 8%, 9%, 10%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells).

[0065] In an embodiment, the expanded CD45+ cells comprise at least about 10% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells (e.g., at least about 10%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells).

[0066] In an embodiment, the expanded CD45+ cells comprise from about 5% to about 30% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0067] In an embodiment, the expanded CD45+ cells comprise from about 10% to about 30% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0068] In an embodiment, the expanded CD45+ cells comprise no more than about 30% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells (e.g., about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells).

[0069] In an embodiment, the expanded CD45+ cells comprise from about 10% to about 25% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0070] In an embodiment, the expanded CD45+ cells comprise at least about 15% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells (e.g., about 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells).

[0071] In an embodiment, the expanded CD45+ cells comprise from about 15% to about 90% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0072] In an embodiment, the expanded CD45+ cells further comprise one or more or all of the subpopulations of immune effector cells selected from the group consisting of:(a) monocytes (CD 14+ cell s);(b) natural killer (NK) cells (CD3-, CD56+ cells);(c) B cells (CD 19+ cells);(d) dendritic cells (CD1 lc+ cells); and(d) any combination of (a)-(d).

[0073] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of monocytes (CD 14+ cells).

[0074] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of NK cells (CD3-, CD56+ cells).

[0075] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of B cells (CD 19+ cells).

[0076] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of dendritic cells (CD1 lc+ cells).

[0077] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and NK cells (CD3-, CD56+ cells).

[0078] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and B cells (CD19+ cells).

[0079] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and dendritic cells (CD1 lc+ cells).

[0080] In an embodiment, the expanded CD45+ cells further comprise subpopulations of NK cells (CD3-, CD56+ cells) and B cells (CD19+ cells).

[0081] In an embodiment, the expanded CD45+ cells further comprise subpopulations of NK cells (CD3-, CD56+ cells) and dendritic cells (CD1 lc+ cells).

[0082] In an embodiment, the expanded CD45+ cells further comprise subpopulations of B cells (CD19+ cells) and dendritic cells (CD1 lc+ cells).

[0083] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD 14+ cells), NK cells (CD3-, CD 56+ cells) and B cells (CD 19+ cells).

[0084] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), NK cells (CD3-, CD56+ cells) and dendritic cells (CD1 lc+).

[0085] In an embodiment, the expanded CD45+ cells further comprise subpopulations ofNK cells (CD3-, CD56+ cells), B cells (CD19+ cells) and dendritic cells (CDllc+).

[0086] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), B cells (CD19+ cells) and dendritic cells (CD1 lc+).

[0087] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), NK cells (CD3-, CD56+ cells), B cells (CD19+ cells) and dendritic cells (CD1 lc+).

[0088] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0089] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the expanded CD45+ cells comprise from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) relative to the total population of expanded CD45+ cells, wherein the subpopulation of T cells (CD3+ T cells) further comprises: (i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0090] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) from about 15% to about 35% cytotoxic T cells (CD3+, CD8+ T cells) relative to the total population of expanded CD45+ cells;(ii) from about 0.1% to about 10% NKT cells (CD3+, CD56+ T cells) relative to the total population of expanded CD45+ cells;(iii) from about 40% to about 70% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells; and (iv) from about 5% to about 25% Thl7 cells (CD3+, CD161+ T cells) of the total population of expanded CD45+ cells,(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0091] In an embodiment, the expanded CD45+ cells comprise from about 80% to about 99% CD3+ T cells relative to the total population of expanded CD45+ cells.

[0092] In an embodiment, the expanded CD45+ cells comprise from about 0.05% to about 0.5% of monocytes (CD14+ cells) relative to the total population of expanded CD45+ cells.

[0093] In an embodiment, the expanded CD45+ cells comprise from about 0.1% to about 10% of NK cells (CD3-, CD56+ cells) relative to the total population of expanded CD45+ cells.

[0094] In an embodiment, the expanded CD45+ cells comprise from about 0.5% to about 5% of B cells (CD 19+ cells) relative to the total population of expanded CD45+ cells.

[0095] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0096] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the expanded CD45+ cells comprise from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) relative to the total population of expanded CD45+ cells, wherein the subpopulation of T cells (CD3+ T cells) further comprises: (i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0097] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) from about 15% to about 35% cytotoxic T cells (CD3+, CD8+ T cells) relative to the total population of expanded CD45+ cells;(ii) from about 0.1% to about 10% NKT cells (CD3+, CD56+ T cells) relative to the total population of expanded CD45+ cells;(iii) from about 40% to about 70% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells; and (iv) from about 5% to about 25% Thl7 cells (CD3+, CD161+ T cells) of the total population of expanded CD45+ cells,(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0098] In an embodiment, the expanded CD45+ cells comprise from about 80% to about 99% CD3+ T cells relative to the total population of expanded CD45+ cells.

[0099] In an embodiment, the expanded CD45+ cells comprise from about 0.05% to about 0.5% of monocytes (CD14+ cells) relative to the total population of expanded CD45+ cells.

[0100] In an embodiment, the expanded CD45+ cells comprise from about 0.1% to about 10% of NK cells (CD3-, CD56+ cells) relative to the total population of expanded CD45+ cells.

[0101] In an embodiment, the expanded CD45+ cells comprise from about 0.5% to about 5% of B cells (CD 19+ cells) relative to the total population of expanded CD45+ cells.

[0102] In an embodiment, the expanded CD45+ cells comprise from about 30% to about 40% of dendritic cells (CDllc+ cells) relative to the total population of expanded CD45+ cells.

[0103] In an embodiment, the immunosuppressive population of cells comprises, consists or consists essentially of the CD45+ subpopulations of Table 2 and the CD3+ T cell subpopulations of Table 3 or 4.Pharmaceutical compositions

[0104] In another aspect disclosed herein, there is provided a pharmaceutical composition comprising the immunosuppressive population of cells disclosed herein.

[0105] The term "pharmaceutical composition" as used herein refers to a composition that is in a form that allows the biological activity of the active ingredient (e.g., an immunogenic population of cells as disclosed herein) to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the pharmaceutical composition is to be administered.

[0106] In an embodiment, the pharmaceutical composition comprises the immunosuppressive population of cells disclosed herein in sufficient number to administer a dosage of from about 104to about 109cells / kg body weight per dose, including all integer values within this range. Accordingly, the pharmaceutical composition may comprise the immunosuppressive population of cells in sufficient number to administer a dosage of 104, 105, 106, 107, 108or 109cells / kg body weight per dose.

[0107] In an embodiment, the pharmaceutical composition comprises the immunosuppressive population of cells disclosed herein in sufficient number to administer a dosage of from about 2 * 107to about 3 * 108cells per dose, including all integer values within this range.

[0108] In an embodiment, the pharmaceutical composition comprises the immunosuppressive population of cells disclosed herein in a sufficient number to administer a dosage of from about 0.5 x io8to about 2.0 x io8cells per dose, including all integer values within this range (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 x io8cells per dose).

[0109] In some embodiments, periodic re-administration of the pharmaceutical composition may be required to achieve a desirable therapeutic effect. The exact amounts and rates of administration of the pharmaceutical composition will depend on a number of factors, examples of which are described elsewhere herein, such as the subject’s age, bodyweight, general health, sex and dietary requirements, as well as any drugs or agents used in combination or coincidental with the administration of the composition. Where multiple divided doses are required, these may be administered hourly, daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

[0110] In an embodiment, the pharmaceutical composition is to be administered from about 1 day to about 7 days per week, including all integer values within this range (e.g., 1, 2, 3, 4, 5, 6 or 7 days per week).

[0111] In an embodiment, the pharmaceutical composition is to be administered about 5 days per week.

[0112] In an embodiment, the pharmaceutical composition is to be administered about 2 days per week.

[0113] In an embodiment, the pharmaceutical composition is to be administered about 1 day per week.

[0114] In an embodiment, the pharmaceutical composition is for parenteral administration. In another embodiment, the pharmaceutical composition is for intravenous administration.

[0115] In an embodiment, the pharmaceutical composition is to be administered as an intravenous infusion.

[0116] In an embodiment, the pharmaceutical composition is to be administered as a continuous infusion.

[0117] In an embodiment, the pharmaceutical composition is to be administered as an intravenous bolus infusion.

[0118] In an embodiment, the pharmaceutical composition is to be administered as an intravenous bolus infusion over a period of from about 1 minute to about 10 minutes, including all integer values within this range (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes).

[0119] The pharmaceutical compositions disclosed herein may be prepared according to conventional methods well known in the pharmaceutical industries, such as thosedescribed in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA), comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0120] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal and anti-bacterial agents, isotonic and absorption agents, and the like.

[0121] It is further contemplated herein that the pharmaceutical composition may be coadministered with one or more other agents suitable for the treatment or amelioration of symptoms associated with inflammatory diseases or conditions, illustrative examples of which include glucocorticoids, cyclooxygenase (COX) inhibitors, antimetabolites (e.g., fluorouracil, methotrexate), vitamins (e.g., vitamin A, vitamin D3), minerals (e.g., calcium, iron, magnesium, zinc), aminosalicylates (e.g., sulfasalazine, mesalazine), immunosuppressants (e.g., cyclosporin A, tacrolimus, pimecrolimus), cyclostatics (e.g., 6-thioguanine, azathioprine, hydroxyurea, methotrexate, fumaric acid esters, mycophenolic acid), antibacterial chemotherapies (e.g., metronidazole, ornidazole, ofloxacin, ciprofloxacin), non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics.

[0122] Such combinations may be administered simultaneous with the pharmaceutical composition or concurrently with the pharmaceutical composition.Methods for the ex vivo manufacture of an immunosuppressive population of cells

[0123] In another aspect disclosed herein, there is provided a method for the ex vivo manufacture of an immunogenic population of cells, the method comprising:(a) providing a population of immune cells;(b) separating the population of immune cells into a first population of immune cells and a second population of immune cells;(c) contacting the first population of immune cells to an activating agent to provide an activated population of immune cells;(d) contacting the activated population of immune cells to a composition comprising:(i) interleukin-2 (IL-2); and(ii) interleukin-34 (IL-34);(e) following step (d), combining the activated population of immune cells with the second population of immune cells;(f) expanding the combined populations of immune cells of step (e) in the presence of a composition comprising:(i) IL-2; and(ii) IL-34,for a time and under conditions suitable to generate an immunosuppressive population of cells, wherein the immunosuppressive population of cells comprising expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells).

[0124] The immune cells contemplated herein may be isolated from any species, particularly a vertebrate, and even more particularly a mammal. Suitable vertebrates that fall within the scope of the disclosure include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fciscicularis. and / or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.), and fish. In a preferred embodiment, the immune cells are isolated from a human.

[0125] In an embodiment, the population of immune cells is isolated from a blood sample.100126] In an embodiment, the population of immune cells is isolated from peripheral blood mononuclear cells (PBMCs).

[0127] The term “isolated” as used herein refers to an immune cell (e.g., a CD45+ cell), which is substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment, e.g., whole blood. Methods for the isolation of immune cells from whole blood would be known to persons skilled in the art, illustrative examples of which include the isolation of immune cells from whole blood using the Ficoll-Paque method and density gradient centrifugation.

[0128] The term "activation" as used herein refers to a process of stimulating populations of immune cells comprising a subpopulation of T cells in preparation for expansion ex vivo. Suitable methods for the activation of populations of immune cells would be known to persons skilled in the art, illustrative examples of which include polyclonal stimulation by antibodies (e.g., anti-CD28, anti-CD3 antibodies), soluble activation proteins, lipid microbubbles, dissolvable microspheres and linked antibodies.

[0129] As detailed elsewhere herein, the term “expansion” as used herein refers to the propagation of cell populations ex vivo or in vitro to reach a desired number or proportion of cells, and subpopulations thereof. Persons skilled in the art will appreciate that the term “expansion” includes standard cell biology techniques such as maintaining the cell populations in a suitable cell culture medium at a suitable temperature and humidity. Accordingly, in an embodiment, the expansion of the combined populations of immune cells in step (f) comprises one or more routine cell culture medium changes.

[0130] Persons skilled in the art will appreciate that the timing of such cell culture medium changes will be based upon the condition of the medium and concentrations of waste, e.g., reflected by color changes in the medium. In an embodiment, the cell culture medium is changed at least every second day. In another embodiment, the cell culture medium is changed at least every third day. In another embodiment, the cell culture medium is changed at least every fourth day. In another embodiment, the cell culture medium is changed every fifth day.

[0131] In an embodiment, the cell culture medium change further comprises conducting a cell count.

[0132] In an embodiment, the cell culture medium change further comprises standardizing the concentration of cells.

[0133] In an embodiment, the cell culture medium change further comprises standardizing the concentration of cells to at least about 1.0 × 106cells mL.

[0134] In an embodiment, the first population of immune cells is activated by contacting the first population of immune cells with an activating agent selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing. In accordance with this embodiment, therefore, the first population of immune cells is activated by polyclonal stimulation by antibodies.

[0135] In an embodiment, the first population of immune cells is activated with a monoclonal anti-CD3 antibody.

[0136] In an embodiment the monoclonal anti-CD3 antibody is coated on the surface of the culture vessel (e.g., flask, dish, etc.).

[0137] In an embodiment, the first population of immune cells is activated with a monoclonal anti-CD28 antibody.

[0138] In an embodiment, the first population of immune cells is activated with about 0.1 pg / mL to about 2.0 pg / mL of the monoclonal anti-CD28 antibody (e.g., about 0.1, 0.2, 0.3 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg / mL of the anti-CD28 monoclonal antibody).

[0139] In an embodiment, the first population of immune cells is activated with a monoclonal anti-CD3 antibody and a monoclonal anti-CD28 antibody.

[0140] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific antibodies e.g., bispecific antibodies), and single variable domain antibodies so long as they exhibit the desired biological activity. The term “antibody” includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (which may be abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CLI). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed herein, the FRs of an antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Included within the scope of the term “antibody” is an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called a, 5, a, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0141] In an embodiment, the composition of step (d) comprises from about 10 ng / mL to about 40 ng / mL IL-2 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng / mL IL-2).

[0142] In an embodiment, the composition of step (d) comprises about 25 ng / mL IL-2.

[0143] In an embodiment, the composition of step (d) comprises from about 5 ng / mL to about 20 ng / mL IL-34 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-34).

[0144] In an embodiment, the composition of step (d) comprises about 12.5 ng / mL IL-34.

[0145] In an embodiment, the composition of step (f) comprises from about 10 ng / mL to about 40 ng / mL IL-2 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng / mL IL-2).

[0146] In an embodiment, the composition of step (f) comprises about 25 ng / mL IL-2.

[0147] In an embodiment, the composition of step (f) comprises from about 5 ng / mL to about 20 ng / mL IL-34 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-34).

[0148] In an embodiment, the composition of step (f) comprises about 12.5 ng / mL IL-34.

[0149] In an embodiment, the method further comprising expanding the combined populations of immune cells in step (f) in the presence of an activating agent.

[0150] In an embodiment, the activating agent is selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing.

[0151] In an embodiment, the combined populations of immune cells in step (f) are expanded in the presence of from about 0.1 pg / mL to about 2.0 pg / mL of the activating agent (e.g., about 0.1, 0.2, 0.3 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg / mL of the activating agent).

[0152] In an embodiment, the combined populations of immune cells in step (f) are expanded in the presence of from about 0.1 pg / mL to about 2.0 pg / mL of an anti-CD28 monoclonal antibody (e.g., about 0.1, 0.2, 0.3 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg / mL of the anti-CD28 monoclonal antibody).

[0153] In an embodiment, the combined populations of immune cells in step (f) are expanded in the presence of about 0.5 pg / mL or about 1.0 pg / mL of an anti-CD28 monoclonal antibody.

[0154] In an embodiment, the combined populations of immune cells in step (f) are expanded in the presence of a monoclonal anti-CD3 antibody.

[0155] In an embodiment the monoclonal anti-CD3 antibody is coated on the surface of the culture vessel (e.g., flask, dish, etc.).

[0156] In an embodiment, the combined populations of immune cells in step (f) are expanded in the presence of a monoclonal anti-CD3 antibody and a monoclonal anti-CD28 antibody.

[0157] In an embodiment, the activated population of immune cells comprises activated T cells (CD3+ T cells).

[0158] In an embodiment, the second population of immune cells comprises naive T cells (e.g., CD25-, CD45RA+, CD45RO- and / or CD127+).

[0159] In an embodiment, the ratio of the activated population of immune cells and the second population of immune cells combined in step (e) is about 1 (i.e., about 1:1 activated population of immune cells: second population of immune cells).

[0160] In an embodiment, the combined populations of immune cells of step (e) are expanded for at least about 24 hours (e.g., at least about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, or 480 hours).

[0161] In an embodiment, the combined populations of immune cells of step (e) are expanded for from about 240 hours to about 360 hours, including all integer values within this range.

[0162] In an embodiment, the combined populations of immune cells of step (e) are expanded for about 288 hours.

[0163] In an embodiment, the immunosuppressive population of cells is characterized by one or more or all of the features disclosed elsewhere herein.

[0164] Accordingly, in an embodiment, the subpopulation of T cells (CD3+ T cells) comprises at least about 35% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells) (e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells)).

[0165] In an embodiment, the subpopulation of T cells (CD3+ T cells) comprises at from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells), including all integer values within this range.

[0166] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises:(a) cytotoxic T cells (CD3+, CD8+ T cells);(b) NKT cells (CD3+, CD56+ T cells);(c) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells);(d) Thl7 cells (CD3+, CD161+ T cells); and(e) any combination of (a)-(d).

[0167] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells).

[0168] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells).

[0169] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD25+, FoxP3+, CD127- T cells).

[0170] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Thl7 cells (CD3+, CD161+ T cells).

[0171] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and NKT cells (CD3+, CD56+ T cells).

[0172] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and Tregs (CD3+, CD25+, FoxP3+, CD127- T cells).

[0173] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0174] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells) and Tregs (CD3+, CD25+, FoxP3+, CD 127- T cells).

[0175] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0176] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0177] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells) and Tregs (CD3+, CD25+, FoxP3+, CD127- T cells).

[0178] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0179] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells), Tregs (CD3+, CD25+, FoxP3+, CD 127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0180] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells), Tregs (CD3+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0181] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), and wherein the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells, i.e., the CD4 / CD8 ratio, (CD3+, CD8+ T cells) is at least about 0.7 (e.g., at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0).

[0182] In an embodiment, the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is from about 0.7 to about 1.5, including all integer values within this range.[00183J In an embodiment, the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is from about 0.7 to about 5.0, including all integer values within this range.

[0184] In an embodiment, the expanded CD45+ cells comprise at least about 5% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells (e.g., at least about 5%, 6%, 7%, 8%, 9%, 10%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells).

[0185] In an embodiment, the expanded CD45+ cells comprise at least about 10% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells (e.g., at least about 10%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells).

[0186] In an embodiment, the expanded CD45+ cells comprise from about 5% to about 30% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0187] In an embodiment, the expanded CD45+ cells comprise from about 10% to about 30% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0188] In an embodiment, the expanded CD45+ cells comprise no more than about 30% Tregs (CD3+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells (e.g., about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% Tregs (CD3+, CD25+ T cells) of the total population of the expanded CD45+ cells).100189] In an embodiment, the expanded CD45+ cells comprise from about 10% to about 25% Tregs (CD3+, CD25+, FoxP3+, CD 127- T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0190] In an embodiment, the expanded CD45+ cells comprise at least about 15% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells (e.g., about 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells).

[0191] In an embodiment, the expanded CD45+ cells comprise from about 15% to about 90% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0192] In an embodiment, the expanded CD45+ cells further comprise one or more or all of the subpopulations of immune effector cells selected from the group consisting of:(a) monocytes (CD 14+ cell s);(b) natural killer (NK) cells (CD3-, CD56+ cells);(c) B cells (CD 19+ cells);(d) dendritic cells (CD1 lc+ cells); and(d) any combination of (a)-(d).

[0193] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of monocytes (CD 14+ cells).

[0194] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of NK cells (CD3-, CD56+ cells).

[0195] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of B cells (CD 19+ cells).

[0196] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of dendritic cells (CD1 lc+ cells).

[0197] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and NK cells (CD3-, CD56+ cells).

[0198] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and B cells (CD19+ cells).

[0199] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and dendritic cells (CD1 lc+ cells).

[0200] In an embodiment, the expanded CD45+ cells further comprise subpopulations of NK cells (CD3-, CD56+ cells) and B cells (CD19+ cells).

[0201] In an embodiment, the expanded CD45+ cells further comprise subpopulations of NK cells (CD3-, CD56+ cells) and dendritic cells (CD1 lc+ cells).

[0202] In an embodiment, the expanded CD45+ cells further comprise subpopulations of B cells (CD 19+ cells) and dendritic cells (CD1 lc+ cells).

[0203] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD 14+ cells), NK cells (CD3-, CD 56+ cells) and B cells (CD 19+ cells).

[0204] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), NK cells (CD3-, CD56+ cells) and dendritic cells (CD1 lc+).

[0205] In an embodiment, the expanded CD45+ cells further comprise subpopulations ofNK cells (CD3-, CD56+ cells), B cells (CD19+ cells) and dendritic cells (CDllc+).

[0206] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), B cells (CD19+ cells) and dendritic cells (CD1 lc+).

[0207] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), NK cells (CD3-, CD56+ cells), B cells (CD19+ cells) and dendritic cells (CD1 lc+).

[0208] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0209] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the expanded CD45+ cells comprise from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) relative to the total population of expanded CD45+ cells, wherein the subpopulation of T cells (CD3+ T cells) further comprises: (i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0210] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) from about 15% to about 35% cytotoxic T cells (CD3+, CD8+ T cells) relative to the total population of expanded CD45+ cells;(ii) from about 0.1% to about 10% NKT cells (CD3+, CD56+ T cells) relative to the total population of expanded CD45+ cells;(iii) from about 40% to about 70% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells; and (iv) from about 5% to about 25% Thl7 cells (CD3+, CD161+ T cells) of the total population of expanded CD45+ cells,(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0211] In an embodiment, the expanded CD45+ cells comprise from about 80% to about 99% CD3+ T cells relative to the total population of expanded CD45+ cells.

[0212] In an embodiment, the expanded CD45+ cells comprise from about 0.05% to about 0.5% of monocytes (CD14+ cells) relative to the total population of expanded CD45+ cells.

[0213] In an embodiment, the expanded CD45+ cells comprise from about 0.1% to about 10% of NK cells (CD3-, CD56+ cells) relative to the total population of expanded CD45+ cells.

[0214] In an embodiment, the expanded CD45+ cells comprise from about 0.5% to about 5% of B cells (CD 19+ cells) relative to the total population of expanded CD45+ cells.

[0215] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).100216] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the expanded CD45+ cells comprise from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) relative to the total population of expanded CD45+ cells, wherein the subpopulation of T cells (CD3+ T cells) further comprises: (i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).100217] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) from about 15% to about 35% cytotoxic T cells (CD3+, CD8+ T cells) relative to the total population of expanded CD45+ cells;(ii) from about 0.1% to about 10% NKT cells (CD3+, CD56+ T cells) relative to the total population of expanded CD45+ cells;(iii) from about 40% to about 70% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells; and (iv) from about 5% to about 25% Thl7 cells (CD3+, CD161+ T cells) of the total population of expanded CD45+ cells,(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0218] In an embodiment, the expanded CD45+ cells comprise from about 80% to about 99% CD3+ T cells relative to the total population of expanded CD45+ cells.

[0219] In an embodiment, the expanded CD45+ cells comprise from about 0.05% to about 0.5% of monocytes (CD14+ cells) relative to the total population of expanded CD45+ cells.

[0220] In an embodiment, the expanded CD45+ cells comprise from about 0.1% to about 10% of NK cells (CD3-, CD56+ cells) relative to the total population of expanded CD45+ cells.

[0221] In an embodiment, the expanded CD45+ cells comprise from about 0.5% to about 5% of B cells (CD 19+ cells) relative to the total population of expanded CD45+ cells.

[0222] In an embodiment, the expanded CD45+ cells comprise from about 30% to about 40% of dendritic cells (CDllc+ cells) relative to the total population of expanded CD45+ cells.

[0223] In an embodiment, the immunosuppressive population of cells comprises, consists or consists essentially of the CD45+ subpopulations of Table 2 and the CD3+ T cell subpopulations of Table 3 or 4.

[0224] In an embodiment, the method further comprises freezing the immunosuppressive population of cells.

[0225] In an aspect disclosed herein, there is provided an immunosuppressive population of cells manufactured by the method described herein.Compositions and kits for the ex vivo manufacture of an immunosuppressive population of immune cells

[0226] In an aspect disclosed herein, there is provided a composition for the ex vivo manufacture of an immunosuppressive population of cells, the composition comprising:(a) IL-2; and(b) IL-34.

[0227] In an embodiment, the composition comprises from about 10 ng / mL to about 40 ng / mL IL-2 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng / mL IL-2).

[0228] In an embodiment, the composition comprises about 25 ng / mL IL-2.

[0229] In an embodiment, the composition comprises from about 5 ng / mL to about 20 ng / mL IL-34 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-34).

[0230] In an embodiment, the composition comprises about 12.5 ng / mL IL-34.

[0231] Persons skilled in the art would appreciate that the compositions disclosed herein may be provided in concentrated forms. Such concentrated compositions may be diluted in the culture medium upon contact with the cells, or diluted prior to use. Accordingly, in an embodiment, the composition is provided as a xlO, *20, *30, *40, *50, *60, *70, *80, *90, or x 100 concentrate composition.

[0232] In an embodiment the composition is provided as a xioo concentrate composition.

[0233] In an embodiment, the composition comprises from about 1000 ng / mL to about 4000 ng / mL IL-2 (e.g., about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 ng / mL IL-2).

[0234] In an embodiment, the composition comprises about 2500 ng / mL IL-2.

[0235] In an embodiment, the composition from about 500 ng / mL to about 2000 ng / mL IL-34 (e.g., about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ng / mL IL- 15).

[0236] In an embodiment, the composition comprises about 1250 ng / mL IL-34.

[0237] In another aspect disclosed herein, there is provided a kit for the ex vivo manufacture of an immunosuppressive population of cells, the kit comprising:(a) IL-2; and(b) IL-34.

[0238] In an embodiment, the kit comprises the IL-2 at a concentration of from about 10 ng / mL to about 40 ng / mL (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng / mL).

[0239] In an embodiment, the kit comprises the IL-2 at a concentration of about 25 ng / mL.

[0240] In an embodiment, the kit comprises the IL-34 at a concentration of from about 5 ng / mL to about 20 ng / mL (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL).

[0241] In an embodiment, the kit comprises the IL-34 at a concentration of about 12.5 ng / mL.

[0242] Persons skilled in the art would appreciate that the cytokines disclosed herein may be provided in concentrated forms. Such concentrates may be diluted in the culture medium upon contact with the cells, or diluted prior to use. In accordance with this embodiment, the kit may further comprise instructions for use for the ex vivo manufacture of an immunosuppressive population of cells, inclusive of methods to dilute the components of the kit prior to, or during use.

[0243] In an embodiment, the components of the kit are provided as a xlO, x20, x30, x40, x50, x60, x70, x80, x90, or xl00 concentrate.

[0244] In an embodiment, the components of the kit are provided as a x 100 concentrate.100245] In an embodiment, the kit comprises the IL-2 at a concentration of from about 1000 ng / mL to about 4000 ng / mL (e.g., about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 ng / mL).

[0246] In an embodiment, the kit comprises the IL-2 at a concentration of about 2500 ng / mL.

[0247] In an embodiment, the kit comprises the IL-34 at a concentration of from about 500 ng / mL to about 2000 ng / mL (e.g., about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 ng / mL).

[0248] In an embodiment, the kit comprises the IL-34 at a concentration of about 1250 ng / mL.

[0249] In an embodiment, the kit further comprises an activating agent.

[0250] In an embodiment, the activating agent is selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing.Methods for the treatment of an inflammatory disease or condition and associated therapeutic uses

[0251] It is further contemplated that the immunosuppressive population of cells disclosed herein, and pharmaceutical compositions comprising the same may be adapted for the treatment of an inflammatory disease or condition. Accordingly, in an aspect, the present disclosure provides a method of treating an inflammatory disease or condition in a subject in need thereof, the method comprising administering an effective amount of the immunosuppressive population of cells, or the pharmaceutical composition disclosed herein to the subject.

[0252] In another aspect, the present disclosure provides the use of the immunosuppressive population of cells, or the pharmaceutical composition disclosed herein in the manufacture of a medicament for the treatment of an inflammatory disease or condition.

[0253] In yet another aspect, the present disclosure provides the immunosuppressive population of cells, or the pharmaceutical composition disclosed herein for use in the treatment of an inflammatory disease or condition.

[0254] The therapeutic regimen for the treatment of an inflammatory disease or condition can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type of inflammatory disease or condition in addition to the age, weight and general health of the subject. Another determinative factor may be the risk of developing recurrent or chronic disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent or chronic disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent or chronic disease.

[0255] The term “inflammatory disease or condition” as used herein means any refers to diseases or conditions characterized by inflammation caused by a noxious stimulus, e.g., damage, autoimmunity, or infection. The clinical features of an inflammatory disease or condition will be dependent on the noxious stimulus (or stimuli), but typically include heat, pain, redness, and swelling of the affected organ or tissue. The inflammatory disease or condition may be acute or chronic.

[0256] In an embodiment, the inflammatory disease or condition is associated with an increase or upregulation in the level of an inflammatory cytokine selected from the group consisting of IFN-γ, TNF-a, MIP-la, IP-10, IL-17, IL-31, IL-IRa, RANTES, MIP-ip, TNF-P, IL-8, IL- 18, and combinations of the foregoing.

[0257] In an embodiment, the inflammatory disease or condition is selected from the group consisting of hypertension, depression, hyperglycemia, dyslipidemia, diabetes, nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, chronic kidney disease, cancer, neurodegenerative disease, osteoporosis, auto-immune disease, arthritis, lupus, coeliac disease, multiple sclerosis, and aging-related diseases or conditions.

[0258] In an embodiment, the inflammatory disease or condition is auto-immune disease or aging-related diseases or conditions.100259] The term “subject” as used herein refers to any mammal, including livestock and other farm animals (e.g., cattle, goats, sheep, horses, pigs and chickens), performance animals (e.g., racehorses), companion animals (e.g., cats and dogs), laboratory test animals and humans. In an embodiment, the subject is a human. In an embodiment, the subject is an adult. In another embodiment, the subject is a child.100260] In an embodiment, the immunosuppressive population of cells is autologous to the subject.

[0261] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later to be administered into the subject in accordance with the methods disclosed herein. Accordingly, in certain embodiments, immune cells isolated from the subject may be contacted with the compositions disclosed herein and / or expanded ex vivo by the methods disclosed herein, before being reinfused back into the subject in accordance with the methods of treatment disclosed herein.

[0262] In another embodiment, the immunosuppressive population of cells is allogenic to the subject.

[0263] The term "allogenic" as used herein refers to any material derived from a different animal of the same species as the subject to whom the material is administered.

[0264] As used herein, the term “effective amount” typically refers to an amount of the immunosuppressive population of cells, or pharmaceutical composition disclosed herein that is sufficient to affect one or more beneficial or desired therapeutic outcomes (e.g., antiinflammatory effect, reduction in pro-inflammatory cytokines, etc.). Said beneficial or desired therapeutic outcomes may be measured using clinical techniques known in the art, illustrative examples of which include the measurement of CD45+ cell subpopulations (e.g., T helper cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), Tregs (CD4+, CD25+, FoxP3+, CD127-), B cells (CD19+), NK cells (CD56+, CD3-), NKT cells (CD3+, CD56+), conventional DCs (CD3-, CD56-, CDlc+, CD303-), plasmacytoid DCs (CD3-, CD56-, CDlc-, CD303+) and myeloid derived suppressor cells (CDllb+, HLA-DRlow / -, CD33+), and quantification of blood markers and / or cytokine concentrations (e.g., IL-lb, IL-6, TNF-a, IL-2, IL-8, IL- 10, IFN-y, macrophage inflammatory protein 1 (MIP-1), monocyte chemoattractant protein 1 (MCP-1), IL-12, IL-4, IL-5, IL-13, TGF-P, immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin E (IgE), complement component 3 (C3), complement component 4 (C4), rheumatoid factor (RF), antinuclear antibody (ANA), extractable nuclear antigen (ENA), anti-double stranded DNA (anti-dsDNA), IL-17A, transaminase, etc.).

[0265] An “effective amount” can be provided in one or more administrations. The exact amount required may vary depending on factors such as the nature and severity of the inflammatory disease or condition to be treated, and the age and general health of the subject.

[0266] In an embodiment, the method comprises administering the immunosuppressive population of cells disclosed herein in sufficient number to administer a dosage of from about 2 x 107to about 3 x 108cells per dose, including all integer values within this range.

[0267] In an embodiment, the method comprises administering the immunosuppressive population of cells disclosed herein in a sufficient number to administer a dosage of from about 0.5 x io8to about 2.0 x io8cells per dose, including all integer values within this range (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 x io8cells per dose).

[0268] In an embodiment, the method comprises administration of the immunosuppressive population of cells from about 1 day to about 7 days per week, including all integer values within this range (e.g., 1, 2, 3, 4, 5, 6 or 7 days per week).

[0269] In an embodiment, the method comprises administration of the immunosuppressive population of cells about 5 days per week.

[0270] In an embodiment, the method comprises administration of the immunosuppressive population of cells about 2 days per week.

[0271] In an embodiment, the method comprises administration of the immunosuppressive population of cells about 1 day per week.

[0272] In an embodiment, the method comprises parenteral administration of the immunosuppressive population of cells.

[0273] In an embodiment, the method comprises intravenous administration of the immunosuppressive population of cells.

[0274] In an embodiment, the method comprises intravenous infusion of the immunosuppressive population of cells.

[0275] In an embodiment, the method comprises intravenous continuous infusion of the immunosuppressive population of cells.

[0276] In an embodiment, the method comprises intravenous bolus infusion of the immunosuppressive population of cells.

[0277] In an embodiment, the intravenous bolus infusion is to be administered over a period of from about 1 minute to about 10 minutes, including all integer values within this range (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes).

[0278] The terms “treat”, "treating", “treatment” and the like are used interchangeably herein to mean relieving, reducing, alleviating, ameliorating or otherwise inhibiting the severity and / or progression of an inflammatory disease or condition, or a symptom thereof, in a subject. It is to be understood that the terms “treat”, "treating", “treatment” and the like, as used herein, do not imply that a subject is treated until clinical symptoms of the inflammatory disease or condition have been eliminated or are no longer evident. Said treatment may also reduce the severity of the inflammatory disease or condition by preventing progression or alleviating the symptoms associated with the inflammatory disease or condition.

[0279] In another aspect disclosed herein, there is provided a method for the treatment of an inflammatory disease or condition in a subject in need thereof, the method comprising:(a) providing a population of immune cells obtained from the subject;(b) separating the population of immune cells into a first population of immune cells and a second population of immune cells;(c) contacting the first population of immune cells to an activating agent to provide an activated population of immune cells;(d) contacting the activated population of immune cells to a composition comprising:(i) IL-2; and(ii) IL-34,(e) combining the activated population of immune cells of step (d) with the second population of immune cells.(f) expanding the combined populations of immune cells of step (e) in the presence of a composition comprising:(i) IL-2; and(i) IL-34,for a time and under conditions suitable to generate an immunosuppressive population of cells, wherein the immunosuppressive population of cells comprising expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells); and(g) administering an effective amount of the immunosuppressive population of cells to the subject.[00280J In an embodiment, the population of immune cells is isolated from a blood sample.

[0281] In an embodiment, the population of immune cells is isolated from a blood sample obtained from the subject prior to the administration of an immunodepleting therapy (e.g., methotrexate, mycophenolate mofetil, azathiprine)

[0282] In an embodiment, the population of immune cells is isolated from peripheral blood mononuclear cells (PBMCs).

[0283] In an embodiment, the first population of immune cells is activated by contacting the first population of immune cells with an activating agent selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing, in accordance with this embodiment, therefore, the first population of immune cells is activated by polyclonal stimulation by antibodies.

[0284] In an embodiment, the first population of immune cells is activated with a monoclonal anti-CD3 antibody.

[0285] In an embodiment the monoclonal anti-CD3 antibody is coated on the surface of the culture vessel (e.g., flask, dish, etc.).

[0286] In an embodiment, the first population of immune cells is activated with a monoclonal anti-CD28 antibody.[00287J In an embodiment, the first population of immune cells is activated with about 0.1 pg / mL to about 2.0 pg / mL of the monoclonal anti-CD28 antibody (e.g., about 0.1, 0.2, 0.3 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg / mL of the anti-CD28 monoclonal antibody).

[0288] In an embodiment, the first population of immune cells is activated with a monoclonal anti-CD3 antibody and a monoclonal anti-CD28 antibody.

[0289] In an embodiment, the composition of step (d) comprises from about 10 ng / mL to about 40 ng / mL IL-2 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng / mL IL-2).

[0290] In an embodiment, the composition of step (d) comprises about 25 ng / mL IL-2.

[0291] In an embodiment, the composition of step (d) comprises from about 5 ng / mL to about 20 ng / mL IL-34 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-34).

[0292] In an embodiment, the composition of step (d) comprises about 12.5 ng / mL IL-34.

[0293] In an embodiment, the composition of step (f) comprises from about 10 ng / mL to about 40 ng / mL IL-2 (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 ng / mL IL-2).

[0294] In an embodiment, the composition of step (f) comprises about 25 ng / mL IL-2.

[0295] In an embodiment, the composition of step (f) comprises from about 5 ng / mL to about 20 ng / mL IL-34 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL IL-34).

[0296] In an embodiment, the composition of step (f) comprises about 12.5 ng / mL IL-34.

[0297] In an embodiment, the activated population of immune cells comprises activated T cells (CD3+ T cells).

[0298] In an embodiment, the second population of immune cells comprises naive T cells (e.g., CD45RA+, CCR7+).

[0299] In an embodiment, the ratio of the activated population of immune cells and the second population of immune cells combined in step (e) is 1 (i.e., about 1:1 activated population of immune cells: second population of immune cells).

[0300] In an embodiment, the immunosuppressive population of cells is characterized by one or more or all of the features disclosed elsewhere herein.

[0301] Accordingly, in an embodiment, the subpopulation of T cells (CD3+ T cells) comprises at least about 35% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells) (e.g., about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells)).

[0302] In an embodiment, the subpopulation of T cells (CD3+ T cells) comprises at from about 35% to about 50% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells), including all integer values within this range.

[0303] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises:(a) cytotoxic T cells (CD3+, CD8+ T cells);(b) NKT cells (CD3+, CD56+ T cells);(c) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells);(d) Thl7 cells (CD3+, CD161+ T cells); and(e) any combination of (a)-(d).[00304J In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells).

[0305] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells).100306] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD4+, CD25+, FoxP3+, CD 127- T cells).100307] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Thl7 cells (CD3+, CD161+ T cells).

[0308] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and NKT cells (CD3+, CD56+ T cells).

[0309] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127-T cells).

[0310] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0311] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells) and Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells).

[0312] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0313] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0314] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells) and Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells).

[0315] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0316] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises NKT cells (CD3+, CD56+ T cells), Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0317] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), NKT cells (CD3+, CD56+ T cells), Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) and Thl7 cells (CD3+, CD161+ T cells).

[0318] In an embodiment, the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells), and wherein the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells), i.e., the CD4 / CD8 ratio, is at least about 0.7 (e.g., at least about 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0).

[0319] In an embodiment, the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is from about 0.7 to about 1.5, including all integer values within this range.

[0320] In an embodiment, the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is from about 0.7 to about 5.0, including all integer values within this range.

[0321] In an embodiment, the expanded CD45+ cells comprise at least about 5% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells (e.g., at least about 5%, 6%, 7%, 8%, 9%, 10%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells).

[0322] In an embodiment, the expanded CD45+ cells comprise at least about 10% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells (e.g., at least about 10%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells).

[0323] In an embodiment, the expanded CD45+ cells comprise from about 5% to about 30% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0324] In an embodiment, the expanded CD45+ cells comprise from about 10% to about 30% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0325] In an embodiment, the expanded CD45+ cells comprise no more than about 30% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells (e.g., about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% Tregs (CD3+, CD25+ T cells) of the total population of the expanded CD45+ cells).

[0326] In an embodiment, the expanded CD45+ cells comprise from about 10% to about 25% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0327] In an embodiment, the expanded CD45+ cells comprise at least about 15% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells (e.g., about 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%,47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells).

[0328] In an embodiment, the expanded CD45+ cells comprise from about 15% to about 90% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells, including all integer values within this range.

[0329] In an embodiment, the expanded CD45+ cells further comprise one or more or all of the subpopulations of immune effector cells selected from the group consisting of:(a) monocytes (CD 14+ cell s);(b) natural killer (NK) cells (CD3-, CD56+ cells);(c) B cells (CD 19+ cells);(d) dendritic cells (CD1 lc+ cells); and(d) any combination of (a)-(d).

[0330] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of monocytes (CD 14+ cells).

[0331] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of NK cells (CD3-, CD56+ cells).

[0332] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of B cells (CD 19+ cells).

[0333] In an embodiment, the expanded CD45+ cells further comprise a subpopulation of dendritic cells (CD1 lc+ cells).

[0334] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and NK cells (CD3-, CD56+ cells).

[0335] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and B cells (CD19+ cells).

[0336] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells) and dendritic cells (CD1 lc+ cells).

[0337] In an embodiment, the expanded CD45+ cells further comprise subpopulations of NK cells (CD3-, CD56+ cells) and B cells (CD19+ cells).

[0338] In an embodiment, the expanded CD45+ cells further comprise subpopulations of NK cells (CD3-, CD56+ cells) and dendritic cells (CD1 lc+ cells).

[0339] In an embodiment, the expanded CD45+ cells further comprise subpopulations of B cells (CD 19+ cells) and dendritic cells (CD1 lc+ cells).

[0340] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD 14+ cells), NK cells (CD3-, CD 56+ cells) and B cells (CD 19+ cells).

[0341] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), NK cells (CD3-, CD56+ cells) and dendritic cells (CD1 lc+).

[0342] In an embodiment, the expanded CD45+ cells further comprise subpopulations ofNK cells (CD3-, CD56+ cells), B cells (CD19+ cells) and dendritic cells (CDllc+).

[0343] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), B cells (CD19+ cells) and dendritic cells (CD1 lc+).

[0344] In an embodiment, the expanded CD45+ cells further comprise subpopulations of monocytes (CD14+ cells), NK cells (CD3-, CD56+ cells), B cells (CD19+ cells) and dendritic cells (CD1 lc+).

[0345] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0346] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the expanded CD45+ cells comprise from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) relative to the total population of expanded CD45+ cells, wherein the subpopulation of T cells (CD3+ T cells) further comprises: (i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0347] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) from about 15% to about 35% cytotoxic T cells (CD3+, CD8+ T cells) relative to the total population of expanded CD45+ cells;(ii) from about 0.1% to about 10% NKT cells (CD3+, CD56+ T cells) relative to the total population of expanded CD45+ cells;(iii) from about 40% to about 70% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells; and (iv) from about 5% to about 25% Thl7 cells (CD3+, CD161+ T cells) of the total population of expanded CD45+ cells,(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells); and(d) a subpopulation of B cells (CD 19+ cells).

[0348] In an embodiment, the expanded CD45+ cells comprise from about 80% to about 99% CD3+ T cells relative to the total population of expanded CD45+ cells.

[0349] In an embodiment, the expanded CD45+ cells comprise from about 0.05% to about 0.5% of monocytes (CD14+ cells) relative to the total population of expanded CD45+ cells.

[0350] In an embodiment, the expanded CD45+ cells comprise from about 0.1% to about 10% of NK cells (CD3-, CD56+ cells) relative to the total population of expanded CD45+ cells.

[0351] In an embodiment, the expanded CD45+ cells comprise from about 0.5% to about 5% of B cells (CD 19+ cells) relative to the total population of expanded CD45+ cells.

[0352] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0353] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein theexpanded CD45+ cells comprise from about 35% to about 70% T helper cells (CD3+, CD4+ T cells) relative to the total population of expanded CD45+ cells, wherein the subpopulation of T cells (CD3+ T cells) further comprises: (i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD25+, FoxP3+, CD127- T cells); and (iv) Thl7 cells (CD3+, CD161+ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0354] In an embodiment, the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells consisting of:(a) a subpopulation of T cells (CD3+ T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (CD3+ T cells) further comprises:(i) from about 15% to about 35% cytotoxic T cells (CD3+, CD8+ T cells) relative to the total population of expanded CD45+ cells;(ii) from about 0.1% to about 10% NKT cells (CD3+, CD56+ T cells) relative to the total population of expanded CD45+ cells;(iii) from about 40% to about 70% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells; and (iv) from about 5% to about 25% Thl7 cells (CD3+, CD161+ T cells) of the total population of expanded CD45+ cells,(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD1 lc+ cells).

[0355] In an embodiment, the expanded CD45+ cells comprise from about 80% to about 99% CD3+ T cells relative to the total population of expanded CD45+ cells.

[0356] In an embodiment, the expanded CD45+ cells comprise from about 0.05% to about 0.5% of monocytes (CD14+ cells) relative to the total population of expanded CD45+ cells.

[0357] In an embodiment, the expanded CD45+ cells comprise from about 0.1% to about 10% of NK cells (CD3-, CD56+ cells) relative to the total population of expanded CD45+ cells.

[0358] In an embodiment, the expanded CD45+ cells comprise from about 0.5% to about 5% of B cells (CD 19+ cells) relative to the total population of expanded CD45+ cells.

[0359] In an embodiment, the expanded CD45+ cells comprise from about 30% to about 40% of dendritic cells (CDllc+ cells) relative to the total population of expanded CD45+ cells.

[0360] In an embodiment, the immunosuppressive population of cells comprises, consists or consists essentially of the CD45+ subpopulations of Table 2 and the CD3+ T cell subpopulations of Table 3 or 4.

[0361] In an embodiment, the inflammatory disease or condition is associated with an increase or upregulation in the level of an inflammatory cytokine selected from the group consisting of IFN-γ, TNF-a, MIP-la, IP-10, IL-17, IL-31, IL-IRa, RANTES, MIP-ip, TNF-P, IL-8, IL- 18, and combinations of the foregoing.

[0362] In an embodiment, the inflammatory disease or condition is selected from the group consisting of hypertension, depression, hyperglycemia, dyslipidemia, diabetes, nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, chronic kidney disease, cancer, neurodegenerative disease, osteoporosis, auto-immune disease, arthritis, lupus, coeliac disease, multiple sclerosis, and aging-related diseases or conditions.

[0363] In an embodiment, the inflammatory disease or condition is auto-immune disease or aging-related diseases or conditions.

[0364] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (orinformation derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0365] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the present disclosure without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0366] The present disclosure will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.Example 1Ex vivo generation of an immunosuppressive population of cells

[0367] PBMCs were isolated from peripheral blood by density gradient centrifugation using lymphocyte separation medium (LSM).

[0368] To activate T cells in PBMC bulk cultures, PBMCs were resuspended in X-VIVO 15 media before incubating at 37°C in a humidified atmosphere of 5% CO2 in 25 cm2culture flasks coated with the anti-CD3 monoclonal antibody, OKT3. After 3 hours, a mixture of GMP-grade components (Table 1) were added to the media before incubating for a further 3 hours to generate a population of CD3 -activated PBMCs.

[0369] An equal number of autologous, non-activated PBMCs were then added to the CD3-activated PBMCs and incubated for a further 16 hours. Thereafter, the cells were transferred to 75 cm2culture flasks for expansion in the presence of the mixture of GMP-grade components (Table 1), which was replaced every 4 days. Following a 12 day incubation period, the resulting population of cells was harvested and characterized.

[0370] To increase the proportion of Tregs in the immunogenic cell population, an alternative expansion method was developed, which incorporated the use of both anti-CD3 and anti-CD28 monoclonal antibodies as activating agents, and the expansion of the combined activated / non-activated PBMCs in the presence of these activating agents. Briefly, to activate T cells in PBMC bulk cultures, PBMCs were resuspended in X-VIVO 15 media and anti-CD28 (1 pg / mL) before incubating at 37°C in a humidified atmosphereof 5% CO2 in 25 cm2culture flasks coated with anti-CD3 monoclonal antibody. An equal number of autologous, non-activated PBMCs were added to the CD28 / CD3-activated PBMCs and incubated for a further 48 hours. Thereafter, 1 * 106cells / mL of the combined cells were transferred to 75 cm2culture flasks coated with the anti-CD3 monoclonal antibody for expansion in the presence of the mixture of GMP -grade components (Table 1) and anti-CD28 (0.5 pg / mL), which was replaced every 2-3 days. Upon replacement of the mixture of GMP-grade components, cell counts were performed to ensure that 1 x 106cells / mL were reseeded. Following a 12 day incubation period, the resulting population of cells was harvested and characterized.Example 2Characterization of ex vivo generated immunosuppressive population of cells

[0371] The populations of cells generated in Example 1 was initially assessed for cell growth and viability, without any sign of abnormal cell death or morphology.

[0372] The immune subpopulations of the population of cells were assessed by flow cytometry. As shown in Figure 1, the immunosuppressive population of cells was highly enriched for CD3+ T cells relative to other CD45+ cells. In particular, the CD3+ cell population, comprising CD4+ and CD8+ T cells, represented the majority (-80%) of the CD3+ cell population, bearing in mind that the CD3+ also includes regulatory T cells (Tregs; -22%) and Thl7 cells (-20%). In addition to the CD3+ T cell population, the remaining effector cells comprised in the immunosuppressive population of cells are natural killer T (NKT) cells (-6%), B cells (-1%) and natural killer (NK) cells (<1%).

[0373] To evaluate the functional characteristics of the immunosuppressive population of cells, a comparative analysis was performed between the immunosuppressive population of cells and an immunogenic population of cells, characterized by heterogenous subpopulations of immune effector cells consisting of:(a) cytotoxic T cells (CD3+, CD8+ T cells), wherein the subpopulation of cytotoxic T cells (CD3+, CD8+ T cells) is enriched for memory T cells; (b) neutrophils (CD 15+ cells);(c) monocytes (CD 14+ cells);(d) dendritic cells (CD1 lc+ cells);(e) NK cells (CD3-, CD56+ cells);(f) B cells (CD 19+ cells);(g) T helper cells (CD3+, CD4+ T cells);(h) NKT cells (CD3+, CD56+ T cells);(i) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells); and(j) Thl7 cells (CD3+ CD161+ T cells).

[0374] The immune profile analysis of the immunosuppressive population of cells demonstrated enrichment for Thl7 cells and T helper cells relative to the immunogenic population of cells (Figures 2 and 3), reduced levels of Tregs relative to the immunogenic population of cells (Figure 4). Moreover, analysis of the comparative ratio of CD4+ and CD8+ T cells indicated a significant increase in the CD4 / CD8 ratio in the immunosuppressive population of cells relative to the immunogenic population of cells, which demonstrate the immunoregulatory nature of the immunosuppressive population of cells (Figure 5). Finally, the immunosuppressive population of cells showed a significantly reduced pro-inflammatory cytokine profile (e.g., IFN-y, TNF-a, MIP-la, IP- 10, IL- 17, IL-31, IL-IRa, RANTES, MIP-ip, TNF-P, IL-8, IL-18) relative the immunogenic population of cells (Figure 6).

[0375] Using the alternative expansion method, the analysis showed CD3+ T cells remained enriched as the predominant (-99%) part of the entire CD45+ population (Figure 7). Within the CD45+ population, Tregs (-81%) account the most dominant immune subpopulation cell types followed by CD4+ cells (-64%), dendritic cells (CDllc+; -35%), CD8+ cells (-18%) and Thl7 cells (-9%).

[0376] Taken together, these data demonstrate that the immunosuppressive population of cells generated in Example 1 is functionally characterized by immune cell populations that are known to exert immunosuppressive effects, as such, the immunosuppressive population of cells is particularly useful for therapeutic applications, for example, in the treatment of inflammatory diseases or conditions, including those associated with aging.Example 3Immunosuppressive population of cells have elevated levels of CD4+ cells

[0377] The immune profile analysis of the immunosuppressive population of cells demonstrated elevated levels of CD4+ T cells compared to the immune profile of the immunogenic population of cells (Figure 8). While CD8+ T cells are well -recognized for their cytotoxicity and effector functions playing crucial roles against infection and cancer, CD4+ T cells play an immune regulatory role in controlling excessive immune reactions and inflammation. Therefore, a comparative ratio between CD4+ and CD8+ T cells indicates an overall immune phenotype in terms of immune reactions and regulations. Compared to the immunogenic population of cells, the immune profiles of the immunosuppressive population of cells showed a significantly increased CD4 / CD8 ratio mainly owing to elevated levels of CD4+ T cells (Figure 3). The elevated levels of CD4+ T cells in the immunosuppressive population of cells further demonstrated the immune regulatory nature of these cells.Example 4Immunosuppressive population of cells have elevated levels of regulatory T cells (Tregs)

[0378] The immune profile analysis of the immunosuppressive population of cells showed an elevated level of regulatory T cells (Tregs) compared to the immune profile of the immunogenic population of cells (Figure 9). Tregs, a subset of specialized T cells, play an essential role in inducing and maintaining peripheral immunity. Tregs have been widely implicated as key to preventing excessive immune responses and suppressing autoimmunity. An elevated level of Tregs in the immunosuppressive population of cells relative to the immunogenic population of cells further supports the immunosuppressive characteristics of the immunosuppressive population of cells.Example 5Immunosuppressive population of cells significantly suppress the proliferation of PMBCs

[0379] To further characterize the immunosuppressive function of the immunosuppressive population of cells, the ability of the immunosuppressive population of cells to suppress immune cells was assessed relative to the immunogenic population of cells.Briefly, PMBCs were co-cultured in the presence of either the immunosuppressive or immunogenic population of cells. As shown in Additional Figure 10A, the viability of PBMCs was significantly inhibited by the immunosuppressive population of cells compared to the immunogenic population of cells as indicated by 7AAD+ (dead) cells at day 3 of coculture. The observed cell-mediated suppression was even greater when the viability of PBMCs were assessed at day 6 (Figure 10B).100380] Taken together, these data demonstrate that the immunosuppressive population of cells is capable of specifically and selectively suppressing immunogenic subsets of immune cells in primary human samples. It follows, therefore, that the immunogenic population of cells is capable of treating (or preventing) inflammatory diseases or conditions, including those associated with aging.Table 1. Composition for the ex vivo manufacture of an immunosuppressive population of cellsComponent (GMP-grade) Kit concentration (xlOO)Interleukin-2 (IL-2) 2500 ng / mlInterferon gamma (IFN-y) 5000 ng / mlTable 2. Exemplary immunosuppressive population of cellsCD45+ subpopulation Mean proportion of cells relative to total number of CD45+ cells (%)Expansion method Alternative expansion methodT cells (CD3+) 85.27 98.54Monocytes (CD 14+ cells) 0.12 0.10Natural killer (NK) cells (CD3- 0.71 0.48, CD56+ cells)B cells (CD 19+ cells) 1.06 2.18Dendritic cells (CD1 lc+ cells) 35.23Table 3. T cell subsets of the exemplary immunosuppressive population of cells of Table 2CD3+ T cell subpopulation Mean proportion of cells relative to total number of CD45+ cells (%)Expansion method Alternative expansion method Cytotoxic T cells (CD8+ T 33.56 18.63cells)T helper cells (CD4+ T cells) 42.06 64.78Natural killer T (NKT) cells 6.30 0.48(CD3+, CD56+ T cells)T regulatory cells (Tregs) 22.63 81.13(CD3+, CD4+, CD25+,FoxP3+, CD127- T cells)Thl7 cells (CD161+ T cells) 20.48 9.58Table 4. T cell subsets of an exemplary population of cells produced using the alternative expansion methodCD3+ T cell subpopulation Mean proportion of cells relative to total number of CD3+ cells (%) Cytotoxic T cells (CD8+ T cells) 18.90T helper cells (CD4+ T cells) 65.73Natural killer T (NKT) cells (CD3+, CD56+ T 0.48cells)T regulatory cells (Tregs) (CD3+, CD4+, CD25+, 82.33FoxP3+, CD127- T cells)Thl7 cells (CD161+ T cells) 9.72

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An immunosuppressive population of cells comprising ex vivo expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells).

2. The immunosuppressive population of cells of claim 1, wherein the subpopulation of T cells (CD3+ T cells) comprises at least about 35% T helper cells (CD3+, CD4+ T cells) of the total population of T cells (CD3+ T cells).

3. The immunosuppressive population of cells of claim 2, wherein the subpopulation of T cells (CD3-J- T cells) comprises from about 35% to about 70% T helper cells (CD4+ T cells) of the total population of T cells (CD3+ T cells).

4. The immunosuppressive population of cells of any one of claims 1 to 3, wherein the subpopulation of T cells (CD3+ T cells) further comprises:(a) cytotoxic T cells (CD3+, CD8+ T cells);(b) natural killer T (NKT) cells (CD3+, CD56+ T cells);(c) T regulatory’ cells (Tregs) (CD3+, CD4+, CD25+, FoxP3+, CD 127- T cells); (d) Th 17 cells (CD3+, CD161 + T cells); and(e) any combination of (a)-(d).

5. The immunosuppressive population of cells of claim 4, wherein the subpopulation of T cells (CD3+ T cells) further comprises cytotoxic T cells (CD3+, CD8+ T cells).

6. The immunosuppressive population of cells of claim 5, wherein the ratio of T helper cells (CD3+, CD4+ T cells) to cytotoxic T cells (CD3+, CD8+ T cells) is at least about 0.7.

7. The immunosuppressive population of cells of claim 6, wherein the ratio of T helper cells (CD3+;CD4+ T cells) to cytotoxic T cells (CD3+;CD8+ T cells) is from about 0.7 to about 5.0.

8. The immunosuppressive population of cells of any one of claims 4 to 7, wherein the subpopulation of T cells (CD3+ T cells) further comprises Thl7 cells (CD3+, CD161+ T cells).

9. The mmunosuppressive population of cells of claim 8, wherein the expanded CD45+ cells comprise at least about 5% Thl7 cells (CD3+, CD161+ T cells) of the total population of the expanded CD45+ cells.

10. The immunosuppressive population of cells of claim 9, wherein the expanded CD45+ cells comprise from about 5% to about 30% Thl7 cells (CD3+, CD 161+ T cells) of the total population of the expanded CD45+ cells.

11. The immunosuppressive population of cells of any one of claims 4 to 10, wherein the subpopulation of T cells (CD3+ T cells) further comprises Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells).

12. The immunosuppressive population of cells of claim 11, wherein the expanded CD45+ cells comprise at least about 15% Tregs (CD.3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of the expanded CD45+ cells.

13. The immunosuppressive population of cells of claim 12, wherein the expanded CD45+ cells comprise from about 15% to about 90% Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells) of the total population of expanded CD45+ cells.

14. The immunosuppressive population of cells of any one of claims 1 to 13, wherein the expanded CD45+ cells further comprise one or more or all of the subpopulations of cells selected from the group consisting of:(a) monocytes (CD 14+ cells);(b) natural killer (NK) cells (CD3-, CD56+ cells);(c) B cells (CD 19+ cells),(d) dendritic cells (CD I lc+ cells); and(d) any combination of (a)-(d).

15. The immunosuppressive population of cells of any one of claims 1 to 14, wherein the expanded CD45+ cells consist of:(a) a subpopulation of T cells (('1)3 T cells), wherein the subpopulation of T cells is enriched for T helper cells (CD3+, CD4+ T cells), wherein the subpopulation of T cells (('1)3 T cells) further comprises:(i) cytotoxic T cells (CD3+, CD8+ T cells);(ii) NKT cells (CD3+, CD56+ T cells);(iii) Tregs (CD3+, CD4+, CD25+, FoxP3+, CD127- T cells);(iv) Thl7 cells (CD3+, ('1)161 ■ T cells),(b) a subpopulation of monocytes (CD14+ cells);(c) a subpopulation of NK cells (CD3-, CD56+ cells);(d) a subpopulation of B cells (CD 19+ cells); and(e) a subpopulation of dendritic cells (CD11 c+).

16. A pharmaceutical composition comprising the immunosuppressive population of cells of any one of claims 1 to 15.

17. A method for the treatment of an inflammatory disease or condition in a subject in need thereof, the method comprising administering an effective amount of the immunosuppressive population of cells of any one of claims 1 to 15, or the pharmaceutical composition of claim 16 to the subject.

18. Use of the immunosuppressive population of cells of any one of claims 1 to 15, or the pharmaceutical composition of claim 16, in the manufacture of a medicament of a treatment for an inflammatory’ disease or condition.

19. The immunosuppressive population of cells of any one of claims 1 to 15, or the pharmaceutical composition of claim 16 for use in a method for the treatment of an inflammatory' disease or condition in a subject in need thereof.

20. The method of claim 17, the use of claim 18, or the immunosuppressive population of cells for use in accordance with claim 19, wherein the inflammatory disease or conditionis selected from the group consisting of hypertension, depression, hyperglycaemia, dyslipidaemia, diabetes, non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, chronic kidney disease, cancer, neurodegenerative disease, osteoporosis, auto-immune disease, arthritis, lupus, coeliac disease, multiple sclerosis, and aging-related diseases or conditions.

21. A method for the ex vivo manufacture of an immunosuppressive population of cells, the method comprising:(a) providing a population of immune cells;(b) separating the population of immune cells into a first population of immune cells and a second population of immune cells;(c) contacting the first population of immune cells to an activating agent to provide an activated population of immune cells;(d) contacting the activated population of immune cells to a composition comprising:(i) interleukin-2 (IL -2); and(ii) interleukin-34 (IL-34);(e) following step (d), combining the activated population of immune cells with the second population of immune cells;(f) expanding the combined populations of immune cells of step (e) in the presence of a composition comprising:(i) IL-2; and(ii) IL-34,for a time and under conditions suitable to generate an immunosuppressive population of cells, wherein the immunosuppressive population of cells comprising expanded CD45+ cells, wherein the expanded CD45+ cells comprise heterogenou s subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3-r T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3+, CD4+ T cells).

22. The method of claim 21, wherein the population of immune cells has been isolated from a blood sample.

23. The method of cia m 22, wherein the population of immune cells are isolated peripheral blood mononuclear cells (PBMCs).

24. The method of any one of claims 21 to 23, wherein the ratio of the activated population immune cells, and the second population of non-activated immune cells combined is step (e) is 1.

25. The method of any one of claims 21 to 24, wherein the activating agent is selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing.

26. The methods of any one of claims 21 to 25, wherein the activated population of immune cells comprises activated T cells (CD3+ cells).

27. The method of any one of claims 21 to 26, wherein the second population of immune cells comprises naive T cells (CD3+, CD45RA+, CCR7+ T cells).

28. The method of any one of claims 21 to 27, further comprising expanding the combined populations of immune cells in step (f) in the presence of an activating agent.

29. The method of claim 28, wherein the activating agent is selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing.

30. The method of any one of claims 21 to 29, wherein the immunosuppressive population of cells is characterized by one or more or all of the features of any one of claims 2 to 15.

31. An immunosuppressive population of cells manufactured by the method of any one of claims 21 to 30.

32. A composition for the ex vivo manufacture of an immunosuppressive population of cells comprising the following:(a) IL-2; and(b) IL-34.

33. A kit for the ex vivo manufacture of an immunosuppressive population of cells, the kit comprising:(a) IL-2; and,(b) IL-34.

34. The kit of claim 33, further comprising an activating agent.

35. The kit of claim 34, wherein the activating agent is selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoi ng.

36. A method for the treatment of an inflammatory disease or condition in a subject in need thereof, the method comprising:(a) providing a population of immune cells obtained from the subject;(b) separating the population of immune cells into a first population of immune cells and a second population of immune cells;(c) contacting the first population of immune cells to an activating agent to provide an activated population of immune cells;(d) contacting the activated population of immune cells to a composition comprising:(i) IL-2; and(ii) IL-34,(e) combining the activated population of immune cells of step (d) with the second population of immune cells.(f) expanding the combined populations of immune cells of step (e) in the presence of a composition comprising:(i) IL-2; and(i) IL-34,for a time and under conditions suitable to generate an immunosuppressive population of cells, wherein the immunosuppressive population of cells comprising expanded CD45+ cells, wherein the expanded CD45+ ceils comprise heterogenous subpopulations of immune effector cells, wherein the subpopulations of immune effector cells include a subpopulation of T cells (CD3+ T cells), and wherein the subpopulation of T cells (CD3+ T cells) is enriched for T helper cells (CD3-J-, CD4+ T cells); and(g) administering an effective amount of the immunosuppressive population of cells to the subject.

37. The method of claim 36, further comprising expanding the combined populations of immune cells in step (f) in the presence of an activating agent.

38. The method of claim 37, wherein the activating agent is selected from the group consisting of a monoclonal anti-CD3 antibody, a monoclonal anti-CD28 antibody, and combinations of the foregoing.

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