Rebalancing the immune system through depletion of CCR5-expressing myeloid-biased hematopoietic stem cells

Abstract

The present disclosure provides a clinically applicable method to re-balance production of hematopoietic cells in a mammal, including without limitation an aged mammal, by disrupting the CCL5-CCR5 axis.

Description

REBALANCING THE IMMUNE SYSTEM THROUGH DEPLETION OF CCR5-EXPRESSINGMYELOID-BIASED HEMATOPOIETIC STEM CELLSGOVERNMENT SUPPORT RESEARCH

[0001] This invention was made with Government support under contracts CA220434 and DK1 15600 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0002] Hematopoietic stem cells (HSCs) are cells that, in an adult system, mainly reside in the bone marrow niche, and give rise to the mammalian blood system. HSCs are functionally defined through their self-renewal and multilineage differentiation capabilities, and are on top of a hierarchy of progenitor cells that are further restricted in differentiation capabilities but maintain the blood system by giving rise to short lived mature blood cells (MBCs). Previous research has identified cell surface markers, unique in their presence and levels to isolate HSCs and progenitors. Analysis of phenotypic markers have yielded more knowledge regarding fate and function of HSCs under different conditions, including in aging systems.

[0003] Rossi et al. (2024) has shown that HSCs of old mice have a myeloid bias in differentiation. Upon transplanting HSC from 21 - to 24- month-old donors and 3-month-old donors, it was observed that recipient mice transplanted with cells from old donors predominantly showed myeloid lineage reconstitution. They further showed myeloid bias in aged HSCs by observing that HSC aging is accompanied by down- regulation of genes mediating lymphoid specification and function, and up-regulation of genes mediating myeloid specification and function.

[0004] To address pathologies associated with myeloid bias, therapeutic methods are disclosed herein to restore HSCs to a balanced production of lymphoid and myeloid lineage cells.SUMMARY OF THE INVENTION

[0005] It is shown herein that disrupting the CCL5-CCR5 axis in an individual mammal, e.g. an aged mammal, can increase the production of differentiated immune cells from the pool of balanced hematopoietic stem cells (bal-HSC), and decrease production of differentiated immune cells from the pool of myeloid-biased hematopoietic stem cells (my-HSC), thereby rebalancing the hematopoietic system. The mammal may be a human.

[0006] Compositions and methods are provided herein for rebalancing the immune system of a mammalian subject by disruption of the CCL5-CCR5 axis. In some embodiments, an antagonist agent of CCR5 or CCL5 is administered to the individual. In some embodiments, the agent inhibits the interaction between CCL5 and CCR5, for example by binding to CCL5 and / or CCR5. In some embodiments, CCR5 expressing HSC are depleted in vivo or in vitro. The result of thisrebalancing can be a relative enhancement of circulating lymphocyte populations, including lymphocyte progenitor populations, and decreased myeloid cell populations. The rebalanced immune system can have an improved capacity to respond to novel infections, including vaccinations, and has reduced inflammaging properties.

[0007] Conditions that can be treated with the methods include, for example, clonal hematopoiesis of indeterminate potential (CHIP), myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), including pre-malignant AML, atherosclerosis, inflammatory and fibrotic conditions, pathogenic infections, e.g. influenza, Covid- 19, etc., inadequate response to vaccination, prevention or treatment of liquid and solid cancers, immune recovery after cytotoxic agents, and the like.

[0008] In an embodiment, an individual is treated with one or a cocktail of antagonist agents specific for one or both of CCR5 and CCL5. In some embodiments, an agent specific binds to CCR5 and blocks an interaction of CCR5 with CCL5. In some embodiments an agent specifically binds to and inhibits activity of CCR5. In some embodiments, an agent specific binds to CCR5 and blocks an interaction of CCL5 with CCR5. In some embodiments an agent specifically binds to and inhibits activity of CCL5. Agents of interest include drugs approved for human clinical use. In some embodiments the antagonist agent is a monoclonal antibody, e.g. Leronlimab, HGS004, or HGS101 , which specifically binds to CCR5, etc. In some embodiments, the antagonist agent is Leronlimab or a variant thereof. In some embodiments, the antagonist agent is HGS004 or a variant thereof. In some embodiments, the antagonist is HGS101 or a variant thereof. In some embodiments the antagonist is a small molecule, e.g. Maraviroc, Aplaviroc, Vicriviroc, etc. In some embodiments, cells of interest, e.g. HSC, are modified to decrease expression of CCR5, e.g. by genome editing, RNAi, etc.

[0009] In some embodiments, the CCL5-CCR5 axis is disrupted by in vivo or in vitro selective immunodepletion of CCR5+HSC. The methods comprise contacting a population of cells, e.g. cells in bone marrow, comprising HSC with an effective dose of one or more agents that specifically bind to CCR5. In some embodiments the agent is an antibody. In some embodiments the anti-CCR5 antibody is conjugated to a toxin.

[0010] In some embodiments, methods of selective immunodepletion comprise administering an effective dose of an agent specific for CD117 in combination with a CCR5 selective agent(s). In some embodiments, methods of selective immunodepletion comprise administering an effective dose of an agent that blocks CD47 interaction with SIRPa, in combination with the CCR5 selective agent(s). In some embodiment, methods of selective immunodepletion comprise administering an effective dose of an agent specific for CD117, and an agent that blocks CD47 interaction with SIRPa, in combination with the CCR5 selective agent(s). In some embodiments one or all of the agents (CCR5 selective agent, CD1 17 specific agent, agent that blocks CD47 interaction with SIRPa) is an antibody. In other embodiments, an antibody specific for CCR5 iscombined with one or more antibodies specific for an additional my-HSC selective marker, e.g. CD150, etc. In some embodiments, the additional my-HSC selective marker is selected from the group consisting of: CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1 . In some embodiments, the additional my-HSC selective marker is selected from the group consisting of: CD150 (Slamfl ), CD62p, and NEO1 .

[0011] In some embodiments the antibody specific for any of CCR5, CCL5, CD1 17, CD47, etc. is a humanized monoclonal antibody. An antibody may comprise an Fc region sequence. In some embodiments, a single dose, or multiple doses, of the antibody is administered in vivo. In some embodiments the dose of antibody is delivered by intravenous infusion. The effective dose of the antibody may be up to about 50 mg / kg, up to about 25 mg / kg, up to about 10 mg / kg; up to about 5 mg / kg; up to about 1 mg / kg; up to about 0.1 mg / kg. In some embodiments an antibody dose is from about 0.1 mg / kg to about 25 mg / kg, from about 0.5 mg / kg to about 15 mg / kg, from about 1 to about 5 mg / kg. The antibody is optionally conjugated to a cytotoxic agent.

[0012] In other embodiments, a population of cells comprising HSC is depleted of CCR5+ cells in vitro by, e.g. flow cytometry, magnetic immunodepletion, and the like.

[0013] In certain embodiments the subject being treated is an aged, or elderly, mammal. The rate of aging is species specific, where a human may be aged at about 50 years; and a rodent at about 2 years. In general terms, a natural progressive decline in body systems starts in early adulthood, but it becomes most evident several decades later. One arbitrary way to define elderly more precisely in humans is to say that it begins at conventional retirement age, around about 60, around about 65 years of age. Another definition sets parameters for aging coincident with the loss of reproductive ability, which is around about age 45, more usually around about 50 in humans, but will, however, vary with the individual.

[0014] In some embodiments an individual diagnosed with CHIP, or a myelodysplastic condition, e.g. pre-malignant AML, MDS, and the like is treated with the methods disclosed herein to rebalance the immune system and to shift production of differentiated immune cells from balanced hematopoietic stem cells (bal-HSC).

[0015] Disrupting the CCL5-CCR5 axis may provide for an enrichment of bal-HSC to my-HSC of at least 1 .5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more. For example, the number of CCR5+HSC may be reduced relative to the number of CCR5 HSC by least 1 .5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5- fold, at least 10-fold, at least 15-fold, or more. After a period of time sufficient for rebalancing, e.g. after about 1 week, after about 2 weeks, after about 3 weeks, the ratio of the number of lymphoid progenitors in bone marrow, e.g. common lymphoid progenitors, to the number of myeloid progenitors, e.g. common myeloid progenitors, may be increased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more. The number of circulating naive T cells relative to the total circulating lymphocytepopulation may be increased at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more. The basal circulating level of ‘inflammaging’ markers, e.g. IL-1 a, CXCL5, IL1 RL1 , IL-23, IL-1 b, CXCL2, IL-31 , IL-5, GM-CSF, may be decreased at least at least 1.5-fold, 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15- fold, or more upon treatment with the methods of the invention.

[0016] In some embodiments, the balance of immune cells, e.g. the relative number of one or more of naive T cells, exhausted T cells, age-associated B cells” (ABC), myeloid progenitors and lymphoid progenitors is determined before disrupting the CCL5-CCR5 axis. In some embodiments, the balance of immune cells, e.g. the relative number of one or more of naive T cells, exhausted T cells, ABC, myeloid progenitors and lymphoid progenitors is determined before disruption, where an improvement in the desired balance of lymphoid to myeloid cells is associated with successful disruption. In some embodiments, the improvement is an increase in the number of naive T cells relative to the total circulating lymphocyte population. In some embodiments, the improvement is an increase in the number of lymphoid progenitors compared to the number of myeloid progenitors (e.g., as measured in the bone marrow of the subject). In some embodiments, the improvement is an increase in the number of bal-HSC (e.g., CCR5 HSC) relative to my-HSC (e.g., CCR5 HSC).

[0017] Disrupting the CCL5-CCR5 axis may provide for an improved immune response, e.g. response to viral infection; response to bacterial infection; response to pre-malignant or malignant tumor; response to vaccination; generation of antibodies in response to an immunogen; and the like, relative to the individual’s response prior to treatment with the methods of the disclosure. For example and without limitation, an antigen-specific CD8+ T cell response can be increased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more in a rebalanced individual. An antigen-specific antibody response can be increased at least 1 .5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more in a rebalanced individual. The severity of infection or tumor burden may be reduced, e.g. a decrease in hospitalization, infected cells, mortality, tumor burden, metastases, cancer relapse and the like.

[0018] In some embodiments a method is provided for preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective dose of an agent as disclosed herein, that disrupts the CCR5-CCL5 axis, wherein the administering results in a reduction of myeloid-biased hematopoietic stem cells (my-HSC) relative to balanced hematopoietic stem cells (bal-HSC), thereby preventing or treating the disease or disorder. In some embodiments, the subject is at risk of infection. In some embodiments, the subject has received or will receive a vaccine for preventing the infection. In some embodiments, the administering results in an improved immune response to the vaccine. In some embodiments, the subject has or is at risk of developing cancer or a hematological malignancy. In someembodiments, the administering results in an improved anti-cancer immune response. In some embodiments, the subject has or is at risk of developing clonal hematopoiesis of indeterminate potential (CHIP). In some embodiments, the administering results in reduced clonal expansion of HSCs. In some embodiments, the subject has or is at risk of developing a chronic inflammatory disorder. In some embodiments, the administering results in a reduction in at least one inflammaging marker, optionally wherein the inflammaging marker is basal circulating level of a cytokine selected from the group consisting of IL-1 a, CXCL5, IL1 RL1 , IL-23, IL-1 b, CXCL2, IL- 31 , IL-5, GM-CSF, and a combination thereof. In any such embodiments the subject may be human. In any such embodiments the subject may be aged. Following the administering step, there may be an enrichment of bal-HSC to my-HSC of at least 1.5-fold to at least 15-fold. Following the administering step, there may be an enrichment of CCR5 HSC to CCR5+HSC of at least 1.5-fold to at least 15-fold. Following the administering step, the ratio of the number of lymphoid progenitors in bone marrow of the subject to the number of myeloid progenitors in bone marrow of the subject may be increased. Following the administering step the number of circulating naive T cells relative to the total circulating lymphocyte population may be increased.BRIEF DESCRIPTION OF THE FIGURES

[0019] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to- scale.

[0020] FIG 1A-1C. A) Gating strategy for CCR5+ cells in mouse and human HSCs, as well as multipotent progenitor subsets a and b. B) CCR5 expression in mouse HSPCs and mouse HSCs increases with age. CCL5 protein levels in whole mice bone marrow increases with age. (Young mice: <4 months, Old mice: >18 months old) C) CCR5+ HSCs increase with age in human samples.

[0021] FIG. 2A-2C. A. CCR5 expression is higher in My-bi (CD150high ) HSCs. B) CCR5+ HSCs co-express My-bi markers CD41 , CD62p and Neogeninl and long-term HSC marker Hoxb5, more than CCR5- HSCs. C) Following myeloablative stress with 5FU challenge, CD150high HSPCs increase in the bone marrow of old and young mice. Consequently, CCR5 expression on bone marrow resident HSPCs increases in young mice, indicating selective expansion in response to myeloablative stress.

[0022] FIG. 3A-3B. A) CCR5+ HSPCs from old donor mouse give rise to higher frequency of myeloid cells in young recipient mice throughout primary transplant. Mean CCR5+ donor derived (CD45.2+) myeloid cells stay consistently higher than CCR5- donor derived myeloid cells insecondary transplant, although the difference in frequencies is not significant. At the end of the secondary transplant, recipient mice that had CCR5- HSPCs transplanted have higher mean frequency of donor derived lymphoid cells (determined by the mean frequency of CD4+ CD19+ and NK1 .1 + cells). Additionally, recipient mice that received CCR5+ HSPCs have a higher frequency of my-HSCs, as determined by CD15Ohl0hsubset of HSPCs in their bone marrow after serial transplantation. B) CCR5+ HSPCs from young donor mouse give rise to higher frequency of myeloid cells in old recipient mice.

[0023] FIG. 4. CCR5+ human HSPCs give rise to higher frequency of donor derived (human CD45+) myeloid cells in young recipient mice throughout primary transplant. Recipient mice that received CCR5+ HSPCs have a higher frequency of my-HSCs, as determined by CD41 + HSCs in their bone marrow after serial transplantation.

[0024] FIG. 5A-5C. A) Gating strategy for naive T cells and mature B cells. B) 5-day long treatment with CCR5 antagonist Maraviroc (100mg / kg) causes significant increase of naive T cells and mature B cells in peripheral blood of old mice compared to old untreated mice. C) 1- month long treatment with CCL5 mAb (2.5mg / kg), every week, causes significant increase of mature B cells, and increases frequency of common lymphoid progenitors in the bone marrow of old mice compared to old untreated mice.

[0025] FIG. 6. Old mice treated with 2.5 mg / kg every 5 days for 1 month, then exposed to a sublethal dose of 0.5ug / g LPS to induce acute inflammatory challenge, have lower frequency of Cd1 1 b+ myeloid cells, and higher frequency of CD4+ T cells and B cells in their peripheral blood.

[0026] FIGS. 7A-7D. Bulk RNA sequencing shows differential gene expression between CCR5+ and CCR5- KLS cells from young mice and between CCR5+ and CCR5- KLS cells from old mice, heatmaps are visualizing genes with p-values < 0.0001. Genes involved in myeloid cell differentiation, Tyrobp, Dapk2 and Hmgn2, and genes overexpressed in myeloid malignancies including acute myeloid leukemia and myelodysplastic syndromes, Srgn, Cstb, Spp1 and Lef1 , were upregulated in young CCR5+ KLS compared to young CCR5- KLS. Additionally, Aif1 , involved in myeloid differentiation, and CCR2, shown to be associated with myeloid bias, were upregulated in old CCR5+ KLS compared to old CCR5- KLS. A. Genes upregulated in old CCR5- cells (left 3 columns) and downregulated in old CCR5+ cells (right 3 columns) B. Genes downregulated in old CCR5- cells (left 3 columns) and upregulated in old CCR5+ cells (right 3 columns) C. Genes upregulated in young CCR5- cells (left 3 columns) and downregulated in young CCR5+ cells (right 3 columns). D. Genes downregulated in young CCR5- cells (left 3 columns) and upregulated in young CCR5+ cells (right 3 columns).

[0027] FIGS. 8A-8G. Characterization of CCR5+HSPCs in young and old bone marrow. (A) Representative flow cytometry plots showing the gating strategy used to identify and analyze CCR5 expression in KLS cells, pHSCs, and progenitors (MPPa, MPPb, MPPc, CLPs) of young and old mice. (B) Bar graphs showing the frequency of CCR5+ KLS cells, pHSCs, andprogenitors (n = 5 young mice, 6 old mice). (C) Bar graphs showing the concentration (pg / ml) of CCL5 in whole bone marrow samples of young and old mice (n = 3 young and 3 old mice). (D) Bar graphs showing the frequency of CD62p7CD41+ / CD150h'9h / Neogenin-1+cells in CCR5+and CCR5 pHSCs from old mice (n = 6 old mice). (E) GSEA of CCR5+versus CCR5 KLS cells from old mice for myeloid differentiation signatures. (F) GSEA of CCR5+versus CCR5 KLS cells from old mice for T cell differentiation signatures. (G) GSEA of CCR5+versus CCR5 KLS cells from old mice for B cell differentiation signatures. NES, normalized enrichment score; FDR, false discovery rate; pval, p-value. (n = 3 old mice)

[0028] FIG. 9A-9E. Cell cycle dynamics of CCR5+and CCR5 HSCs. (A) Representative flow cytometry plots, from Hoxb5-tri-mCherry old mice, showing the gating strategy used to identify CCR5+and Hoxb5+LT-HSCs. (B) Bar graphs showing the frequency of CCR5+and Hoxb5+LT- HSCs from young and old mice. (C) Representative flow cytometry plots, from old mice, showing the gating strategy used to identify CCR5+and CCR5 LT-HSCs in stages GO, G1 , G2 / S of cell cycle. (D) Bar graph showing the frequency of CCR5+and CCR5 LT-HSCs from young and old mice in different cell cycle stages, according to flow cytometry analysis (n = 4 young mice, 5 old mice). (E) Bar graphs showing the frequency of CCR5+pHSCs and CCR5+LT-HSCs in treated or untreated young and old mice, post-5 days of the 5-FU challenge (n = 4 young and old untreated, 5 young and old 5-FU treated mice).

[0029] FIGS. 10A-10H. CCR5+HSCs show myeloid bias in serial transplantation assays. (A) Schematic diagram depicting the protocol for the serial transplantation of CCR5+and CCR5 KLS cells from the bone marrow of CD45.2+old donors into lethally irradiated CD45.1+young recipients, Peripheral blood is evaluated every 4 weeks for 16 weeks following each transplant, and bone marrow is evaluated on the 16thweek of the secondary transplant. (B) Percent of myeloid cells (CD1 1 b+granulocytes and monocytes) among donor-derived CD45.2+cells at weeks 4, 8, 12 and 16 post primary transplant. (C) Percent of lymphoid cells (B220+B cells, CD8+T cells, NK1 ,1+NK cells) among donor-derived CD45.2+cells at weeks 4, 8, 12, 16 post primary transplant, (n of CCR5+recipients for every week, without outliers and without recipients with no donor chimerism, = 5, 4, 4, 4 and of CCR5 recipients = 14, 13, 12, 10). (D) Percent of donor- derived CD45.2+cells among all peripheral blood cells at week 4, 8, 12, 16 post primary transplant, (n of CCR5+recipients for every week, without outliers = 6, 5, 5, 5, of CCR5 recipients = 14, 15, 13, 13) (E,F) Same as in B-C but analyzing peripheral blood in secondary recipients transplanted with 5x105whole bone marrow cells from primary hosts post primary transplant, (n of CCR5+recipients for every week, without outliers and without recipients with no donor chimerism = 8, 5, 6, 5 and of CCR5 recipients = 11 , 5, 6, 8) (G) Same as in D but for secondary recipients (n of CCR5+recipients for every week, without outliers = 9, 6, 7, 5 and of CCR5 recipients = 1 1 , 8, 10, 8) (H) Bone marrow analysis post-secondary transplantation. Bar graphsshow frequency of CCR5+ donor derived KLS cells, GMPs / CMPs, MkPs, MEPs, and CLPs (n for CCR5+ recipients = 10, n for CCR5- recipients = 8).

[0030] FIGS. 1 1A-11 E. Effects of anti-CCL5 and maraviroc treatment on hematopoietic output and pHSCs. (A) Schematic diagram depicting the protocol for anti-CCL5 and Maraviroc treatment. Old anti-CCL5 treated and mice received 2.5mg / kg monoclonal CCL5 antibody of rat lgG2A isotype (R&D Systems) and old untreated mice received rat lgG2A isotype control, weekly for 4 weeks. Maraviroc mice received 200mg / kg Maraviroc (MedChem Express) dissolved in 10% DMSO + 40%PEG300 + 5% Tween80 + 45% PBS, 200mg / kg per mice, for 5 consecutive days. DMSO mice received the maraviroc dissolving solution in the same regimen (n of young mice = 5 n of old untreated mice = 4, n of anti-CCL5 treated old mice = 8 and IgG treated old mice = 6, n of maraviroc and vehicle treated old mice = 5). (B) Bar graphs show frequency of naive T and memory T cells in young untreated, old untreated, old anti-CCL5 treated, old Maraviroc treated, and old vehicle treated mice. (C) Bar graphs show frequency of mature B and aged B cells. (D) Bar graph showing frequency of CD150highpHSCs and CLPs. (E) Schematic diagram depicting the protocol for anti-CCL5 treatment and acute inflammatory challenge with LPS (n of old treated and untreated mice = 5). Bar graph shows the frequency of Cd1 1 b+myeloid cells, Cd11 b CD8+T cells and Cd1 1 b B220+B cells from all live cells before LPS challenge and 3, 7, 21 days after LPS challenge.

[0031] FIG. 12. Representative flow cytometry plots, from old mice, showing my-HSC marker (CD62p, CD41 , Neogenin-1 , and CD150high expressions) in CCR5 pHSCs, and CCR5+pHSCs.

[0032] FIGS. 13A-13C. (A) Representative flow cytometry plots showing gating strategy to quantify donor derived lymphoid and myeloid cells in peripheral blood of recipients post serial transplantation. (B) Representative flow cytometry plots showing gating strategy to quantify donor derived bone marrow stem and progenitor populations (CLPs, CMP / GMPs, MkPs, MEPs, and CD15Ohi0hKLS cells). (C) Representative flow cytometry plots showing gating strategy to quantify donor derived KLS cells, and bar graph showing frequency of donor derived KLS cells post 16 weeks of the primary and secondary transplant.

[0033] FIGS. 14A-14C. (A) Representative flow cytometry plots showing gating strategy to quantify frequency of mature B and aged B cells. (B) Representative flow cytometry plots showing gating strategy to quantify frequency of naive T and memory T cells. (C) Bar graph showing frequency of CD150lowpHSCs as calculated by 100% - frequency of CD150hi9hpHSCs.

[0034] FIGS. 15A-B. (A) Gating strategy to quantify CCR5+and CCR5 HSCs in human bone marrow samples. (B) Bar graph shows frequency of CCR5+ HSCs in young and old (from fresh donor) human bone marrow.DETAILED DESCRIPTION OF THE EMBODIMENTSDefinitions

[0035] It is to be understood that this invention is not limited to the particular methodology, products, apparatus and factors described, as such methods, apparatus and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by appended claims.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

[0037] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0038] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” refers to one or mixtures of such candidates, and reference to “a method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0039] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar in magnitude and / or within a similar range to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0041] As used herein, the terms “polypeptide,” “peptide,” and “protein” refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, to include disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0042] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect with a therapeutic agent. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof, e.g., reducing the likelihood that the disease or symptom thereof occurs in the subject, and / or may be therapeutic in terms of completely or partially reducing a symptom, or a partial or complete cure for a disease and / or adverse effect attributable to the disease. "T reatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting or slowing the onset or development of the disease; or (c) relieving the disease, e.g., causing regression of the disease or symptoms associated with the disease. The therapeutic agent may be administered before, during or after the onset of disease. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, may be of particular interest. In some embodiments, treatment is performed prior to complete loss of function in the affected tissues. In some embodiments, the subject’s treatment will be administered during the symptomatic stage of the disease, and in some embodiments, after the symptomatic stage of the disease.

[0043] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to any subject for whom treatment is desired. The subject may be a mammalian subject. Mammalian subjects include, e. g., humans, non-human primates, rodents, (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, for example a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., cats, dogs).

[0044] Hematopoietic stem cells (HSC). HSCs are functionally defined by their unique capacity to self-renew and to differentiate to produce all mature blood cell types. The term “HSC” therefore refers to multipotent cells capable of differentiating into all the cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, dendritic cells; and self-renewal activity, i.e. the ability to divide and generate at least one daughter cell with the identical (e.g., self-renewing) characteristics of the parent cell.

[0045] In general, the process of development from pluripotent progenitors to mature cells with specific functions involves the progressive loss of developmental potential to other lineages. The earliest known lymphoid-restricted cell in adult mouse bone marrow is the common lymphocyteprogenitor (CLP), and the earliest known myeloid-restricted cell is the common myeloid progenitor (CMP). A complete description of these cell subsets may be found in Akashi et al. (2000) Nature 404(6774):193, U.S. Pat. No. 7,300,760 (common myeloid progenitor); Kondo et al. (1997) Cell 91 (5):661 -7, U.S. Pat. No. 7,297,329 (common lymphoid progenitor); and is reviewed by Kondo et al. (2003) Annu Rev Immunol. 21 :759-806, each of which is herein specifically incorporated by reference.

[0046] Human HSC have been characterized as, for example, CD34+; CD90 (thy-1 )+; CD59+; CD1 10 (c-mpl)+; c-kit (CD-1 17)+. A human HSC cell may be characterized or selected by the phenotype, for example, of Lin CD34+CD38 CD90+CD45RA . Mouse HSC are, for example, CD90 (thy-1 )10; Sca1+; c-kit (CD-117)+. A mouse HSC cell may be characterized or selected by the phenotype, for example, Lin cKIT+Sca1+Flk2 CD34 CD150+.

[0047] A “lin” or lineage panel may comprise one or more of the markers CD3, CD4, CD8, CD19, CD20, CD56, CD11 b, CD14, and CD15. For example, a lineage panel may comprise antibodies specific for CD3e, CD4, CD8a, B220, CD19, Gr-1 , TER119, and CD1 1 b.

[0048] Reference may be made herein to subpopulations of HSC and cell populations that encompass HSC. The term “KLS” refers to a cell population that encompasses HSC, and which may be characterized in mouse as Lin_cKIT+SCA1+(KLS) cells.

[0049] The term phenotypic HSCs (hereafter referred to as pHSCs) refers to a population that encompasses long term HSCs, short term HSCs, and multipotent progenitors (MPP) cells. This population may be characterized in mouse as Lin c-KIT+SCA1 +CD48-FLK2-CD150+CD34 . The human counterpart may be characterized as Lin CD34+CD38 CD45RA CD90+.

[0050] Long term HSC have been characterized in humans as, for example, Lin CD34+CD38 CD45RA CD49f+CD90+cells. Long term HSC in mouse may also be identified as positive for expression of Hoxb5. Short-term hematopoietic stem cells have been characterized in humans as, for example, CD34+CD38 CD45RA CD49f+CD90+. Multipotent progenitors (MPPs) have been characterized in humans as, for example, CD34+CD38-CD90-CD45RA-.

[0051] my-HSC and bal-HSC. A myeloid-biased HSC (also referred to herein as my-bi) generates differentiated progeny with a greater proportion of myeloid progenitors, relative to a balanced HSC. My-HSCs can be defined by the ratio between lymphoid and myeloid cells in blood that are derived from the my-HSC. Balanced HSCs can give rise to a blood population that is from about 10% to about 20% myeloid cells, with the remainder lymphocytes. The mean lymphoid- to-myeloid cell ratio in normal blood is usually from about 2:1 to 4:1 , and may be around 3:1. My-HSCs may generate a mean lymphoid- to-myeloid cell ratio in the blood of less than about 3:1 , e.g. 4:1 , 5:1 , 6:1 , etc., but greater than 0. My-HSC generate myeloid and lymphoid progeny, but with an altered bias toward myeloid cells.

[0052] Human my-HSC have been described as expressing a set of markers inducing, without limitation CD304, TIE2, ESAM, CD9, CD105, CD166, CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1. These markers may be referred to as my-HSC selective markers. In some embodiments, a my-HSC selected marker comprises one or more of CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1 . In some embodiments a my-HSC selective markers is CD150.

[0053] Markers of mouse my-HSC include, for example, CD150, CD62p, NEO1 , CD38, CD51 (Itgav), CD201 (Procr), CD202b (Tie2), ESAM (Esam), CD105 (Eng), and CD9.

[0054] Myeloid progenitor cells. Myeloid progenitor cells comprise one or more of: common myeloid progenitor cells (CMP); and the committed myeloid progenitors: erythroid / megakaryocytic progenitor (MEP), granulocyte / monocyte progenitors (GMP); and megakaryocyte progenitor (MKP).

[0055] Common Myeloid Progenitor cells (CMP) are a hematopoietic progenitor subset that can give rise to all lineages of myeloerythroid cells, but lack the potential to differentiate into lymphoid lineages. The CMP cells of both humans and mice stain negatively for the markers Thy-1 (CD90), IL-7Ra (CD127); and with a panel of lineage markers, which lineage markers may include CD2; CD3; CD4; CD7; CD8; CD10; CD1 1 b; CD14; CD19; CD20; CD56; and glycophorin A (GPA) in humans and CD2; CD3; CD4; CD8; CD19; IgM; Ter1 10; Gr-1 in mice. The cells are CD34 positive, and CD38 positive. In humans, the CMP is also characterized as IL-3Rocl0CD45RA . In the mouse the CMP are Sca-1 negative, (Ly-6E and Ly-6A), c-kithi, and FcyRl0.

[0056] Common lymphoid progenitors, CLP, express low levels of c-kit (CD117) on their cell surface. CLP cells express high levels of the IL-7 receptor alpha chain (CDw127). Murine CLPs express low levels of Sca-1 (Ly-6E and Ly-6A, see van de Rijn (1989) Proc Natl Acad Sci 86:4634-4638). Human CLPs express low levels of CD34. Human CLP cells may be characterized as CD38 positive and CD10 positive. The CLP subset also has the phenotype of lacking expression of lineage specific markers, exemplified by B220, CD4, CD8, CD3, Gr-1 and Mac-1. Human CLP cells are characterized as lacking expression of Thy-1 , a marker that is characteristic of hematopoietic stem cells. The phenotype of the CLP may be further characterized as Mel-14 , CD43l0, HSAl0, CD45+and common cytokine receptor y chain positive.

[0057] Aged. As used herein, the term aged refers to the effects, or the characteristics, of increasing age, particularly with respect to the bias of hematopoietic stem cells towards cells of the myeloid lineage. The rate of aging is species specific, where a human may be aged at about 50 years; and a rodent at about 2 years. In general terms, a natural progressive decline in body systems starts in early adulthood, but it becomes most evident several decades later. Onearbitrary way to define old age more precisely in humans is to say that it begins at conventional retirement age, around about 60, around about 65 years of age. Another definition sets parameters for aging coincident with the loss of reproductive ability, which is around about age 45, more usually around about 50 in humans, but may, however, vary with the individual. In addition to chronologic aging, individuals may suffer from a similar phenotype due to inflammation, genetic causes, and the like.

[0058] “CCR5-CCL5 Axis” refers to the interaction between the chemokine receptor CCR5, and its cognate ligand, CCL5. CCL5, also called RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted), belongs to CC subfamily of chemokines. Many inflammatory cells can express CCL5. While CCL5 can bind to CCR1 , CCR3, CCR4 and CCR5, it has the highest affinity for CCR5 (also known as CD195). A number of functions have been ascribed to the CCL5 / CCR5 axis, including cell proliferation, migration, angiogenesis, metastasis and survival. Activation of CCR5 has been reported to include PI3K / AKT, NF-KB, HIF-a, RAS- ERK-MEK, JAK-STAT and TGF-p-smad pathways.

[0059] CCL5 belongs to the CC subfamily of chemokines, due to its adjacent cysteines near N terminus. It is an 8kDa protein acting as a classical chemotactic cytokine or chemokine, comprising 68 amino acids. CCL5 is proinflammatory chemokine, recruiting leukocytes to the site of inflammation. The refseq for the human protein may be found in Genbank, accession NP_001265665, and NP_002976. After the binding of CCL5 to CCR5, phosphoinositide 3-kinase (PI3K) is phosphorylated and subsequently, the phosphorylated PI3K phosphorylates protein kinase B (PKB; also known as Akt) on the serine 473. Then, the Akt / PKB complex phosphorylates and inactivates a serine / threonine protein kinase GSK-3.

[0060] CCR5 (C-C chemokine receptor type 5) is a receptor for the chemokine CCL5. It is expressed on immune cells. It is also known for its role in HIV infection, as the virus uses CCR5 to enter and infect host cells. Antagonists of CCR5 are substances that block this receptor, preventing its normal function. The refseq for human CCR5 may be accessed at Genbank, NP_000570, NP_001093638.

[0061] The CCR5 protein belongs to the beta chemokine receptors family of integral membrane proteins. It is a G protein-coupled receptor, which functions as a chemokine receptor in the CC chemokine group. CCR5's cognate ligands include CCL3, CCL4 (also known as MIP 1 a and 1 p, respectively), and CCL3L1 . CCR5 interacts with CCL5 (a chemotactic cytokine protein also known as RANTES). CCR5 is predominantly expressed on T cells, macrophages, dendritic cells, eosinophils, microglia and a subpopulation of either breast or prostate cancer cells. CCR5 may play a role in inflammatory responses to infection, though its exact role in normal immune functionis unclear. Regions of this protein are also crucial for chemokine ligand binding, the functional response of the receptor, and HIV co-receptor activity.

[0062] Antagonist agents (inhibitors) of the CCR5 / CCL5 axis decrease activity of CCR5. In some embodiments an agent binds to CCR5 and inhibits activation of CCR5 by its ligand. In some embodiments an agent binds to CCR5 and blocks interaction thereof with CCL5. In some embodiments an agent binds to CCL5 and inhibits activation of CCR5 by its ligand. In some embodiments an agent binds to CCL5 and blocks interaction thereof with CCR5. In some embodiments an agent is an antibody. It may be noted that an anti-CCR5 antibody useful in depletion will specifically bind to CCR5, but may not inhibit its activity.

[0063] In some embodiments an antagonist agent is a small molecule. Maraviroc, which binds to the CCR5 receptor, blocks the interaction between the receptor and HIV, thus preventing the virus from entering and infecting immune cells. Other small molecule antagonist agents useful in the methods of the disclosure include VCH-286, AZD-5672, AK-220, Aplaviroc, Vicriviroc, TAK- 779 (Takeda), SCH-C (SCH351 125), B07 (Dong et al., 2012. Bioorganic & Med Chem Let 22: 3284), etc. Cenicriviroc and BMS-813160 are antagonists of both CCR2 and CCR5.

[0064] Peptide antagonist agents of interest include, for example, soluble forms of CCR5 that act as competitive inhibitors for binding between CCR5 and CCL5; non-activating CCL5 proteins and fragments, etc. Exemplary peptide antagonists include, for example: DAPTA which comprises amino acids 185-192 of the HIV-1 SF-2 env protein; amino terminus-modified forms of RANTES, including AOP-RANTES, PCS-RANTES, Met-RANTES, and NNY-RANTES; and vMIP-ll.

[0065] RNA interference also finds use, e.g. in short hairpin RNA (shRNAs), short interfering RNA (siRNA), and microRNA (miRNA) specific for CCR5 sequences. AgoshRNA is a relatively new type of shRNA that is smaller than typical shRNAs and is able to be expressed in monocytes unlike their predecessors. In embodiments, the CCR5 antagonist is: miR-103; miR-107; shRNA 1005 as described in Shimizu et al., 2010, Blood 1 15:1534-1544; or an RNA-based inhibitor of CCR5 as described in United States Patent Application Publication Number US2016289681 .

[0066] In one embodiment, the CCR5 antagonist is an anti-CCR5 aptamer, for example as described in Zhou et al., 2015. Chem Bio. 22:379-390.

[0067] Alternative strategies include genetic manipulation of cells, e.g. using CRISPR-Cas9: to disrupt the CCR5 gene, rendering the receptor non-functional.

[0068] Monoclonal antibodies are useful as antagonists of the CCR5 / CCL5 axis. Monoclonal antibodies specific for CCR5 are also useful as agents to deplete CCR5+cells, e.g. CCR5+HSC. Antibodies that bind to CCR5 include, for example, PRO 140 (Leronlimab), which binds to CCR5 and prevents HIV from entering cells. In some embodiments, the antagonist agent is Leronlimabor a variant thereof. In some embodiments, the antagonist agent is HGS004 or a variant thereof. In some embodiments, the antagonist is HGS101 or a variant thereof.

[0069] Other antibodies that specifically bind to human CCR5 are known in the art and commercially available, for example MAB182 (R&D systems); HEK / 1 / 85a (Miltenyi Biotech), anti CD195 (Invitrogen); eBioT21 / 8 (T21 / 8) (eBioscience™); clone 3A9 (BD Pharmingen™) and the like.

[0070] Monoclonal antibodies specific for human CCL5 are known in the art and commercially available, for example clone 8.PP.11 (LS Bio); clones 947A1 H11 , 21445, VL-1 (Thermo Fisher Scientific); clone 21445 (R&D Systems); clone JM03-45 (HUABIO); Clone REA346 (Miltenyi Biotech); MAB278 (R&D Systems); and the like.

[0071] In some embodiments, methods of selective immunodepletion comprise administering an effective dose of agents in addition to those targeting the CCR5 / CCL5 axis, including antibodies specific for CD117; antibodies and agents that block CD47 interaction with SIRPa; antibodies specific for an additional my-HSC selective marker, e.g. CD150, CD62p, NEO, etc. In some embodiments, the additional my-HSC selective marker is selected from the group consisting of: CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1. In some embodiments, the additional my-HSC selective marker is selected from the group consisting of: CD150 (Slamfl ), CD62p, and NEO1 .

[0072] Agents of interest that bind to CD150 include antibodies specific for human CD150. Such antibodies are known in the art and are commercially available, for example and without limitation SLAMF1 / SLAM / CD150 Antibody LS-C204428; A12 monoclonal antibody; SLAM.4; and Clone REA151 .

[0073] Agents of interest that bind to CD62p (P-selectin) include antibodies specific for human CD62p. Such antibodies are known in the art and commercially available, for example and without limitation inclacumab; Crizanlizumab; HuEP5C7; Clone REA389; clone AK-6; clone Psel.KO.2.12; etc.

[0002] Agents of interest that bind to NEO1 include antibodies specific for human NEO1. Such antibodies are known in the art and commercially available, for example and without limitation Neogenin Antibody (RM0124-3G55), Neogenin Antibody (221519), Neogenin Antibody (AF1079), etc.

[0074] Agents of interest that specifically bind to CD1 17 include antibodies that specifically bind to human CD117, and c-kit ligand. CD1 17 is a receptor tyrosine kinase type III, which binds to stem cell factor (a substance that causes certain types of cells to grow), also known as "steel factor" or "c-kit ligand". When this receptor binds to stem cell factor (SCF) it forms a dimer that activates its intrinsic tyrosine kinase activity, that in turn phosphorylates and activates signal transduction molecules that propagate the signal in the cell. See, for example, the human refseqentries Genbank NM_000222; NP_000213. CD117 is an important cell surface marker used to identify certain types of hematopoietic (blood) progenitors in the bone marrow. Hematopoietic stem cells (HSC), multipotent progenitors (MPP), and common myeloid progenitors (CMP) express high levels of CD1 17. A number of antibodies that specifically bind human CD117 are known in the art and commercially available, including without limitation SR1 , 2B8, ACK2, YB5- B8, 57A5, 104D2, etc. Of interest is the humanized form of SR1 , AMG 191 , described in US Patent no. 8,436,150, and 7,915,391 which is an aglycosylated lgG1 humanized antibody.

[0075] As used herein, the term “anti-CD47 agent” or “agent that interferes with the binding between CD47 and SIRPa” refers to any agent that reduces the binding of CD47 (e.g., on a target cell) to SIRPa (e.g., on a phagocytic cell). Non-limiting examples of suitable anti-CD47 reagents include high affinity SIRPa polypeptides, anti-SIRPa antibodies, and anti-CD47 antibodies or antibody fragments. In some embodiments, a suitable anti-CD47 agent (e.g. an anti-CD47 antibody, a SIRPa reagent, etc.) specifically binds CD47 to reduce the binding of CD47 to SIRPa.

[0076] Anti-human CD47 antibodies suitable for clinical use include, without limitation, magrolimab (hu5F9-G4, see U.S. Patent no. 9,017,675), AK1 17; AO-176; CC-90002; IBI188; IMC-002; SHR-1603; SRF231 ; STI-6643; TJ011133; and ZL-1201. Soluble SIRPa agents include, for example, Evorpacept (ALX148), and CV1 -Fc (see, for example, Weiskopf et al. (2013) Science 341 (6141 ): 88-91 ). Such antibodies may comprise an Fc region sequence.

[0077] In some embodiments, an anti-CD47 agent is a “high affinity SIRPa reagent”, which includes SIRPa -derived polypeptides and analogs thereof (e.g., CV1 -hlgG4, and CV1 monomer, ALX148). High affinity SIRPa reagents are described in international application PCT / US13 / 21937, which is hereby specifically incorporated by reference. High affinity SIRPa reagents are variants of the native SIRPa protein. The amino acid changes that provide for increased affinity are localized in the d1 domain, and thus high affinity SIRPa reagents comprise a d1 domain of human SIRPa, with at least one amino acid change relative to the wild-type sequence within the d1 domain. Such a high affinity SIRPa reagent optionally comprises additional amino acid sequences, for example antibody Fc sequences; portions of the wild-type human SIRPa protein other than the d1 domain, including without limitation residues 150 to 374 of the native protein or fragments thereof, usually fragments contiguous with the d1 domain; and the like. High affinity SIRPa reagents may be monomeric or multimeric, i.e. dimer, trimer, tetramer, etc. In some embodiments, a high affinity SIRPa reagent is soluble, where the polypeptide lacks the SIRPa transmembrane domain and comprises at least one amino acid change relative to the wild-type SIRPa sequence, and wherein the amino acid change increases the affinity of the SIRPa polypeptide binding to CD47, for example by decreasing the off-rate by at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or more.

[0078] Optionally a SIRPa reagent is a fusion protein, e.g., fused in frame with a second polypeptide. In some embodiments, the second polypeptide is capable of increasing the size of the fusion protein, e.g., so that the fusion protein will not be cleared from the circulation rapidly. In some embodiments, the second polypeptide is part or whole of an immunoglobulin Fc region. The Fc region aids in phagocytosis by providing an “eat me” signal, which enhances the block of the “don’t eat me” signal provided by the high affinity SIRPa reagent. In other embodiments, the second polypeptide is any suitable polypeptide that is substantially similar to Fc, e.g., providing increased size, multimerization domains, and / or additional binding or interaction with Ig molecules.

[0079] In some embodiments, a subject anti-CD47 agent is an antibody that specifically binds SIRPa (i.e. , an anti-SIRPa antibody) and reduces the interaction between CD47 on one cell (e.g., an infected cell) and SIRPa on another cell (e.g., a phagocytic cell). Suitable anti-SIRPa antibodies can bind SIRPa without activating or stimulating signaling through SIRPa because activation of SIRPa would inhibit phagocytosis. Instead, suitable anti-SIRPa antibodies facilitate the preferential phagocytosis of inflicted cells over normal cells. Those cells that express higher levels of CD47 (e.g., infected cells) relative to other cells (non-infected cells) will be preferentially phagocytosed. Thus, a suitable anti-SIRPa antibody specifically binds SIRPa (without activating / stimulating enough of a signaling response to inhibit phagocytosis) and blocks an interaction between SIRPa and CD47. Suitable anti-SIRPa antibodies include fully human, humanized or chimeric versions of such antibodies. Humanized antibodies are especially useful for in vivo applications in humans due to their low antigenicity. Similarly caninized, felinized, etc. antibodies are especially useful for applications in dogs, cats, and other species respectively. Antibodies of interest include humanized antibodies, or caninized, felinized, equinized, bovinized, porcinized, etc., antibodies, and variants thereof.

[0080] Anti-SIRPa antibodies in clinical and preclinical trials for human use include, for example, CC-95251 ; BYON4228; SIRPa-targeting antibody BR105; Bl 770371 and BI-765063 / OSE172 (Boehringer Ingelheim); and GS-189 (FSI-189) (Gilead Sciences).

[0081] As used herein, "antibody" includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies. The term "antibody" also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')z, Fab, Fv and rlgG. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term “entire” antibody is used to refer to an antibody comprising both variable regions and constant regions, i.e. an Fc region.

[0082] Selection of antibodies for stem cell depletion may be based on a variety of criteria, including selectivity, affinity, cytotoxicity, etc. The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein sequences at least two times the background and more typically more than 10 to 100 times background. In general, antibodies of the present invention bind antigens on the surface of target cells in the presence of effector cells (such as natural killer cells or macrophages). Fc receptors on effector cells recognize bound antibodies. The cross-linking of Fc receptors signals the effector cells to kill the target cells by cytolysis or apoptosis. In one embodiment, the induction is achieved via antibody-dependent cellular cytotoxicity (ADCC).

[0083] "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g. as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g. at 25°C.

[0084] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or with DNA encoding the antigen. Methods of preparing polyclonal antibodies are known to the skilled artisan. The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, an appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.

[0085] Human antibodies can be produced using various techniques known in the art, including phage display libraries. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.

[0086] Antibodies also exist as a number of well-characterized fragments produced by digestion with various peptidases. Thus, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-Cm by a disulfide bond. The F(ab)'2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2dimer into an Fab' monomer. The Fab'monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries.

[0087] A "humanized antibody" is an immunoglobulin molecule that contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0088] Antibodies of interest may be tested for their ability to induce ADCC (antibody-dependent cellular cytotoxicity). Antibody-associated ADCC activity can be monitored and quantified through detection of either the release of label or lactate dehydrogenase from the lysed cells, or detection of reduced target cell viability (e.g. annexin assay). Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-1 1 -dUTP nick end labeling (TUNEL) assay (Lazebnik et al., Nature: 371 , 346 (1994). Cytotoxicity may also be detected directly by detection kits known in the art, such as Cytotoxicity Detection Kit from Roche Applied Science (Indianapolis, Ind.). Preferably, the antibodies of the present invention induce at least 10%, 20%, 30%, 40%, 50%, 60%, or 80% cytotoxicity of the target cells.

[0089] In some embodiments, the antibody is conjugated to an effector moiety. The effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a cytotoxic moiety. Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, saporin, auristatin-E and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes toantibodies. Targeting the cytotoxic moiety to transmembrane proteins serves to increase the local concentration of the cytotoxic moiety in the targeted area.

[0090] A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, including pet and laboratory animals, e.g. mice, rats, rabbits, etc. Thus, the methods are applicable to both human therapy and veterinary applications. In one embodiment the patient is a mammal, preferably a primate. In other embodiments the patient is human.

[0091] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of a first and a second agent. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0092] "Concomitant administration" of a known therapeutic agent with a pharmaceutical composition of the present invention means administration of a first and a second agent at such time that both will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the agents with respect to each other. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular compositions of the present invention.

[0093] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0094] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0095] "Pharmaceutically acceptable excipient "means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0096] The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0097] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0098] The phrase “determining the treatment efficacy” and variants thereof can include any methods for determining that a treatment is providing a benefit to a subject. The term “treatment efficacy” and variants thereof are generally indicated by alleviation of one or more signs or symptoms associated with the disease and can be readily determined by one skilled in the art. “Treatment efficacy” may also refer to the prevention or amelioration of signs and symptoms of toxicities typically associated with standard or non-standard treatments of a disease. Determination of treatment efficacy is usually indication and disease specific and can include any methods known or available in the art for determining that a treatment is providing a beneficial effect to a patient. For example, evidence of treatment efficacy can include but is not limited to remission of the disease or indication. Further, treatment efficacy can also include general improvements in the overall health of the subject, such as but not limited to enhancement of patient life quality, increase in predicted subject survival rate, decrease in depression or decrease in rate of recurrence of the indication (increase in remission time). (See, e.g., Physicians' Desk Reference (2010).)Disruption of the CCL5-CCR5 Axis

[0099] Compositions and methods are provided herein for rebalancing the immune system of a mammalian subject, by disruption of the CCL5-CCR5 axis. In some embodiments, an antagonist agent of CCR5 or CCL5 is administered to the individual. In some embodiments, the agent inhibits the interaction between CCL5 and CCR5, for example by binding to CCL5 and / or CCR5. In some embodiments, CCR5 expressing HSC are depleted in vivo or in vitro. The result of this rebalancing can be a relative enhancement of circulating lymphocyte populations, including lymphocyte progenitor populations, and decreased myeloid cell populations. The rebalanced immune system can have an improved capacity to respond to novel infections, including vaccinations, and has reduced inflammaging properties.

[0100] In an embodiment, an individual is treated with one or a cocktail of antagonist agents specific for one or both of CCR5 and CCL5. In some embodiments an agent specifically binds to and inhibits activity of CCR5. In some embodiments an agent specifically binds to and inhibits activity of CCL5. Agents of interest include drugs approved for human clinical use. In some embodiments the antagonist agent is a monoclonal antibody, e.g. Leronlimab, HGS004, orHGS101 , which specifically binds to CCR5, etc. In some embodiments the antagonist is a small molecule, e.g. Maraviroc, Aplaviroc, Vicriviroc, etc. In some embodiments, cells of interest, e.g. HSC, are modified to decrease expression of CCR5, e.g. by genome editing, RNAi, etc.

[0101] In some embodiments, the CCL5-CCR5 axis is disrupted by in vivo or in vitro selective immunodepletion of CCR5+HSC. The methods comprise contacting a population of cells, e.g. cells in bone marrow, comprising HSC with an effective dose of one or more agents that specifically bind to CCR5. In some embodiments the agent is an antibody. In some embodiments the anti-CCR5 antibody is conjugated to a toxin.

[0102] In an embodiment, an individual is treated with an agent, e.g. an antibody, specific for CCR5 that is effective to provide for selective depletion, which enriches the population of bal- HSC to my-HSC in the subject by at least 1 .5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more. For example, the number of CCR5+ HSC, or pHSC, may be reduced by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more relative to CCR5 HSC.

[0103] After a period of time sufficient for rebalancing, e.g. after about 1 week, after about 2 weeks, after about 3 weeks, the ratio of the number of lymphoid progenitors in bone marrow, e.g. common lymphoid progenitors, to the number of myeloid progenitors, e.g. common myeloid progenitors, may be increased at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more. The number of circulating naive T cells relative to the total circulating lymphocyte population may be increased at least 1 .5-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, or more.

[0104] In some embodiments, methods of selective immunodepletion comprise administering an effective dose of an agent specific for CD117 in combination with a CCR5 selective agent(s). In some embodiments, methods of selective immunodepletion comprise administering an effective dose of an agent that blocks CD47 interaction with SIRPa, in combination with the CCR5 selective agent(s). In some embodiment, methods of selective immunodepletion comprise administering an effective dose of an agent specific for CD117, and an agent that blocks CD47 interaction with SIRPa, in combination with the CCR5 selective agent(s). In some embodiments one or all of the agents (CCR5 selective agent, CD1 17 specific agent, agent that blocks CD47 interaction with SIRPa) is an antibody. In other embodiments, an antibody specific for CCR5 is combined with one or more antibodies specific for an additional my-HSC selective marker, e.g. CD150, etc.

[0105] In some embodiments the antibody specific for any of CCR5, CCL5, CD1 17, CD47, etc. is a humanized monoclonal antibody. An antibody may comprise an Fc region sequence. In some embodiments, a single dose, or multiple doses, of the antibody is administered in vivo. In some embodiments the dose of antibody is delivered by intravenous infusion. The effective dose of the antibody may be up to about 50 mg / kg, up to about 25 mg / kg, up to about 10 mg / kg; up to about5 mg / kg; up to about 1 mg / kg; up to about 0.1 mg / kg. In some embodiments an antibody dose is from about 0.1 mg / kg to about 25 mg / kg, from about 0.5 mg / kg to about 15 mg / kg, from about 1 to about 5 mg / kg. The antibody is optionally conjugated to a cytotoxic agent.

[0106] The effective dose of an agent that blocks CD47 interaction with SIRPa, for example an anti-CD47 antibody, anti-SIRPa antibody, or soluble SIRPa polypeptide, will depend on the individual and the specific agent, but will generally be at least about 50 ig / kg body weight, at least about 250 .g / kg, at least about 500 pig / kg, at least about 750 p.g / kg, at least about 1 mg / kg, and up to about 2.5 mg / kg, up to about 5 mg / kg, up to about 7.5 mg / kg, up to about 10 mg / kg, up to about 15 mg / kg, up to about 25 mg / kg, up to about 50 mg / kg, up to about 100 mg / kg. In some embodiments the agent is a CV1 (high affinity SIRPa) monomer or CV1 microbody dimer. In other embodiments the agent is an anti-CD47 antibody. In other embodiments the agent is an anti-SIRPa antibody.

[0107] The depleting agents can be administered daily, twice daily, every other day, every third day, etc. for a period of time sufficient to affect the desired selective depletion, which may be at least about 1 day, up to about 2 days, up to about 3, 4, 5, 6, 7, 8 or more days. In some embodiments from 4-7 days is sufficient. In some embodiments a single dose is administered. In other embodiments a plurality of doses is administered, e.g. 2, 3, 4, 5 or more. The agents may be formulated together or separately, but are usually administered concomitantly.

[0108] The administration of the agents can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. Administration is preferably parenteral, e.g. intravenous.

[0109] The compositions containing CCR5 antagonists or CCR5 selective agents for depletion, e.g. antibodies, soluble SIRPa, etc. can be administered for therapeutic treatment. Compositions are administered to a patient in an amount sufficient to selectively deplete my-HSC, as described above. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. The particular dose required for a treatment will depend upon the medical condition and history of the mammal, as well as other factors such as age, weight, gender, administration route, efficiency, etc.Formulations

[0110] For disruption of the CCL5-CCR5 axis, one or more agents are formulated in a pharmaceutical composition. The agents can be formulated separately or together, usually separately. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (e.g., Ansel et al., Pharmaceutical Dosage Formsand Drug Delivery; Lieberman, Pharmaceutical Dosage Forms (vols. 1 -3, 1992), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981 ; Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)). As is known in the art, adjustments for patient condition, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0111] In one embodiment, the pharmaceutical compositions are in a water-soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly useful are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0112] The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.

[0113] The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges. It is recognized that compositions of the invention when administered orally, should be protected from digestion. This is typically accomplished either by complexing the molecules with a composition to render them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are well known in the art.

[0114] The compositions for administration will commonly comprise an antibody or other agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0115] Compositions are administered to a patient in an amount sufficient to alter the balance of my-HSC to bal-HSC, as described above. The number of CCR5+HSC, relative to CCR5 HSC, may be reduced. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. The particular dose required for a treatment will depend upon the medical condition and history of the mammal, as well as other factors such as age, weight, gender, administration route, efficiency, etc.USES

[0116] The methods disclosed herein provide for a rebalancing of immune systems to reduce the number of myeloid-biased hematopoietic stem cells, e.g. CCR5+HSC, and generally to increase the production of lymphoid cells relative to myeloid cells. An imbalance that skews to myeloid bias is associated with aging and the elderly. By rebalancing, the ability of the individual to respond to novel pathogens can be improved, for example to SARS-CoV2, monkeypox, etc., and other pathogens not previously encountered by the subject. The ability of an individual to respond to vaccination is also improved.

[0117] In addition to improving the individual’s adaptive immune responses, the expansion of myeloid-biased HSCs with age can contribute to aberrant immune inflammatory responses. Myeloid-biased HSCs are pro-inflammatory, producing or eliciting inflammatory cytokines (TNF- a, IL-1 , IL-6, etc.), in response to microbes or endogenous antigens. The morbidity and mortality of elderly patients infected with new respiratory pathogens such as new strains of influenza and SARS-CoV-2 is not only because of poor and delayed adaptive immune response, but also the inflammatory consequences. These heightened inflammatory responses driven by expansion of my-HSCs in the elderly also contribute to chronic inflammatory disease, which can occur in the absence of a pathogenic source. Rebalancing of the immune system through depletion of myeloid-biased HSCs allows for a more functional immune response, by increasing the generation of new T and B cells and reducing the production of inflammatory myeloid cells.

[0118] Rebalancing of myeloid-biased hematopoietic stem cells has relevance to blood and solid cancers. The diminution of adaptive immunity and the confounding presence of a more inflammatory milieu in the aged contributes to an inability to recognize and eliminate newly arising cancers. Rebalancing restores surveillance systems required for transformed and partially transformed cells that drive cancer, and reduces the generation of myeloid cells that suppress tumor immunity.

[0119] In some embodiments, the disclosure provides compositions and methods for use in a therapeutic method of rebalancing the immune system in a human subject, e.g. an aged human subject, in need thereof. These methods bring the body from a pathological state back into its normal, healthy state; or prevent development of a pathological state. In some embodiments, the disclosure provides compositions and methods for use in a therapeutic method of improved response to infection and / or vaccination. In some embodiments, the disclosure provides compositions and methods for use in a therapeutic method in reducing inflammation, e.g. inflammation associate with infection, inflammaging, etc. In some embodiments, the disclosure provides compositions and methods for use in in a therapeutic method for improving surveillance of cancer cells. In some embodiments, the disclosure provides compositions and methods for use in in a therapeutic method that reduces the population of myeloid cells that suppress tumor immunity.

[0120] In some embodiments, the disclosure provides compositions and methods for rebalancing the immune system of individuals suffering or at risk of a hematologic malignancy. Examples of hematologic malignancies and pre-malignancies that may be treated using the subject methods include leukemias, lymphomas, and myelomas, including but not limited to acute biphenotypic leukemia, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia (APL), biphenotypic acute leukemia (BAL) blastic plasmacytoid dendritic cell neoplasm, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL) (called small lymphocytic lymphoma (SLL) when leukemic cells are absent), acute monocytic leukemia (AMOL), Hodgkin's lymphomas, NonHodgkin's lymphomas (e.g. chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma (FL), Mantle cell lymphoma (MCL), Marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), Hairy cell leukemia, Post-transplant lymphoproliferative disorder (PTLD), Waldenstrom's macroglobulinemia / lymphoplasmacytic lymphoma, hepatosplenic-T cell lymphoma, and cutaneous T cell lymphoma (including Sezary's syndrome)), multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms. In particular embodiments, the subject methods find utility in treatment of leukemias, e.g. acute biphenotypic leukemia, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, chronic lymphocytic leukemia (CLL), acute monocytic leukemia (AMOL).

[0121] Individuals selected for treatment may include, for example, individuals diagnosed with clonal hematopoiesis of indeterminate potential (CHIP), pre-malignant AML patients or MDS patients, naive AML patients who are ineligible for standard induction chemotherapy or allogeneic hematopoietic cell transplant due to age and / or co-morbidities; previously untreated intermediate and high risk myelodysplastic syndrome (MDS) patients; and MDS patients who are relapsed and / or refractory to frontline hypomethylating agents.

[0122] A pre-malignancy or pre-leukemia condition of interest includes myelodysplastic syndrome (MDS), which is group of clonal hematopoietic stem cell disorders typified by peripheral cytopenia, dysplastic hematopoietic progenitors, a hypercellular or hypocellular bone marrow, and a high risk of conversion to acute myeloid leukemia. Symptoms are referable to the specific cell line most affected and may include fatigue, weakness, pallor (secondary to anemia), increased infections and fever (secondary to neutropenia), and increased bleeding and bruising (secondary to thrombocytopenia). Diagnosis is by blood count, peripheral smear, and bone marrow aspiration and biopsy. Treatment with venetoclax, azacitidine or decitabine may help; if acute myeloid leukemia supervenes, it is treated per the usual protocols.

[0123] The syndrome is unified by the presence of distinct mutations of hematopoietic stem cells, most frequently in genes involved in RNA splicing. Myelodysplastic syndromes are characterized by ineffective and dysplastic hematopoiesis and include the following: Refractory anemia: Anemia with reticulocytopenia; normal or hypercellular marrow with erythroid hyperplasia, and dyserythropoiesis; blasts < 5% of nucleated marrow cells; Refractory anemia with ringed sideroblasts: Same as refractory anemia with reticulocytopenia, except that ringed sideroblasts are > 15% of nucleated marrow cells; Refractory cytopenia with multilineage dysplasia: Cytopenia not restricted to red cells; prominent dysplasia of white cell precursors and megakaryocytes; Refractory cytopenia with multilineage dysplasia and ringed sideroblasts: With ringed sideroblasts that are > 15% of nucleated marrow cells; Refractory anemia with excess blasts (RAEB): Cytopenia of > 2 cell lines with morphologic abnormalities of hematopoietic cells; hypercellular marrow with dyserythropoiesis and dysgranulopoiesis; blasts 5 to 9% (RAEB-I) or 10 to 19% (RAEB-II) of nucleated marrow cells; Myelodysplastic syndrome, unclassified: MDS that does not fall into any defined category; MDS with isolated del(5q): Typically severe anemia and thrombocytosis, with deletion of the long arm of chromosome 5; Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML): Mixed myelodysplastic / myeloproliferative neoplasms; absolute monocytosis (> 1000 / mcL [> 1 / L]) in blood; significant increase in marrow monocyte precursors; Chronic neutrophilic leukemia: Characterized by neutrophilia and absence of the Philadelphia chromosome and the BCR-ABL1 fusion gene.

[0124] Risk of MDS increases with age due to the acquisition of somatic mutations that can promote clonal expansion and dominance of a particular hematopoietic stem cell, and possiblydue to exposure to environmental toxins such as benzene, radiation, and chemotherapeutic agents (particularly long or intense regimens and those involving alkylating agents, hydroxyurea, and / or topoisomerase inhibitors). Chromosomal abnormalities (eg, deletions, duplications, structural abnormalities) are often present.

[0125] A condition of particular interest for treatment is CHIP, which is a pre-malignant expansion of mutated blood stem cells. Hematopoietic stem cells (HSCs) randomly accumulate somatic mutations during aging. While most of these mutations have no consequence, rare fitnessincreasing mutations may allow an HSC to clonally expand. This age-associated expansion is termed clonal hematopoiesis of indeterminate potential (CHIP). It is found in 10-30% of those older than 70, most commonly occurs due to mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1, and can be detected by sequencing of peripheral blood or bone marrow cells. These mutations are thought to provide a selective advantage to the hematopoietic stem cells in which they occur, and are detectable as clones in peripheral blood samples because the mutated stem cells maintain the ability to differentiate into circulating granulocytes, monocytes, and lymphocytes.

[0126] While CHIP is a pre-malignant expansion of mutated blood stem cells that also associates with non-hematological disorders, these mutations are also founding mutations for hematological neoplasms such as acute myeloid leukemia, it is unsurprising that CHIP associates with increased risk of developing these cancers. CHIP also associates with increased risk of atherosclerotic cardiovascular disease and death due to non-malignant causes.

[0127] The presence of CHIP can be determined by methods known in the art, for example by analyzing a patient sample(s) comprising hematopoietic cells. The cells can be isolated from a bone marrow, blood or blood-derived sample. A plurality of cells in the sample(s) are analyzed for the presence of clonality, usually by high throughput sequencing of polynucleotides isolated from the cell, for example whole exome sequencing, targeted sequencing of frequently mutated genes, etc. The number of cells analyzed may be at least 102, at least 103, at least 104, at least 105or more. The sequencing can be performed on bulk blood cells, e.g. PBLs, or on selected cell populations, e.g. myeloid cells, stem and progenitor cells, etc. The presence of CHIP can be defined by the presence of somatic mutations, where the most frequently mutated genes include, for example, DNMT3A, TET2, ASXL1, SF3B1, and GNB1. The variant allele fraction (VAF) can be determined, i.e. as the fraction of alleles present in the plurality of cells that comprise a specific somatic mutation. An individual is determined to be a CHIP carrier if the VAF is_>0.08, >0.09, >0.1 , >0.125, >0.15, >0.175, >0.2 or more. In some embodiments a cut-off of a VAF >0.2 is used to define an individual as having CHIP. In some embodiments, a subject treated according to a method described herein experiences a reduction in VAF as a result of the treatment. In some embodiments, the subject has a VAF >0.2 prior to the treatment. In some embodiments, the subject has a VAF >0.1 prior to the treatment. In some embodiments, the VAF is reduced to lessthan 0.2 as a result of the treatment. The data can be compared to measurements from a control normal cell population. The data can be normalized for comparison.Kits

[0128] In some embodiments a kit is provided, comprising an effective dose of the one or more agents disclosed herein for disruption of the CCL5-CCR5 axis. A kit may further comprise additional agents, e.g. antibodies specific for CD1 17, anti-CD47 agents, anti-SIRPa agents, antibodies specific for my-HSC markers, etc. The kit may comprise for example, a container, a dose of one or more of the antibodies or agents, a syringe and / or a vial, and instructions for use thereof. In some embodiments the kit comprises instructions for administration to treat a disease.Experimental Example 1

[0129] Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all types of blood cells. Past research has identified that long-term hematopoietic stem cells (LT- HSCs) experience a loss in potency with old age and become more myeloid biased (My-bi). Several chemokines’ role in hematopoiesis has been examined to yield general conclusions about their effects on proliferation, retention and mobilization of hematopoietic progenitors; however, the chemokine CCL5 and its receptor CCR5’s effects on HSC aging remains largely unknown. Here, we show that expression of CCR5 increases with age in murine and human HSCs. We further show CCR5’s influence in inducing myeloid bias in HSCs through transplantation assays in which HSCs negative for CCR5 expression generate lower frequency of myeloid cells. Additionally, blocking CCL5’s functional activity and binding to CCR5 results in higher frequency of naive T cells and mature B cells in peripheral blood.

[0130] Here, we identify a new marker for my-HSCs, CCR5. CCR5 is a CC chemokine receptor that mainly binds the chemokine CCR5, and others such as CCR1 and CCR3. It is also the main coreceptor for the entry of human immunodeficiency virus strains HIV-1 and HIV-2.

[0131] CCR5 marks an increasing population of mouse and human HSCs with age. To determine whether CCR5 marks a population of HSCs that increase with age, as seen with other my-HSC markers, we used flow cytometry to measure relative CCR5 protein levels on cell surfaces of young (<4 months-old) and old (>18 months-old) Lin_cKIT+SCA1+(KLS) cells, multipotent progenitor populations, multipotent progenitor subset a (MPPa) and multipotent progenitor subset b (MPPb), and phenotypic HSCs (hereafter referred to as pHSCs) defined by marker expression Lin_c-KIT+SCA1 +CD48-FLK2-CD150+CD34_. (Fig 1A) We observed that the frequency of CCR5+KLS cells and pHSCs increased significantly with age in mice (Fig 1 B).

[0132] To determine whether the ligand protein levels show the same trend of increase with age as the receptor, we performed ELISA on whole bone marrow samples and observed an increasein concentration of CCL5 with age, from 245.863pg / ml to 702.274 pg / ml. (Fig 1C) Overall, CCR5 is expressed in a progressively increasing trend in both pHSCs and progenitors. Similarly, we analyzed human bone marrow samples with flow cytometry and observed that the subpopulation of CCR5+ HSCs, identified by marker expression CD34+CD90+CD45RA CD38' , also seem to expand with age, however, in a gradual trend (Fig 1 C). However, since the human samples of different ages have different processing methods, we cannot comment on the significance of this trend and whether it correletes with that in mouse HSCs, as the processing methods may affect expression of cell surface markers.

[0133] CCR5+ HSCs co-express known my-HSC markers and respond to myeloablative stress. To characterize the CCR5+ HSC population we examined other cell-surface markers that are expressed by this subpopulation of HSCs on flow cytometry. We observed that the frequency of CCR5+ cells is greater in my-HSCs (CD150highpHSCs) compared to bal-HSCs (CD150lowpHSCs) and that CCR5+ pHSCs express other known my-bi markers such as Neogeninl , CD62p, and CD41 (Fig 2B).

[0134] Next, we examined whether CCR5+and CCR5" hematopoietic stem and progenitor cells (HSPCs) have a difference in response to myeloablative stress, in young and old mice. We treated young and old mice with 150mg / kg 5-FU and analyzed KLS cells in the bone marrow on flow cytometry 4 days post- end of treatment, a timepoint with high HSC proliferation. We observed that post- 5FU challenge, the frequency of CD150highKLS increase as expected in both young and old mice. (Fig 2C) Further, we showed that post-5-FU challenge CCR5+KLS frequency increases significantly in young mice but not in old mice. (Fig 2C) We could not analyze CCR5+ pHSCs due to low cell count, as most pHSCs were depleted post-challenge.

[0135] Mouse CCR5+ HSPCs show myeloid bias upon transplantation. \Ne next examined whether the rescue of the blood system by CCR5+ and CCR5- HSCs show any differences in the frequency of myeloid and lymphoid populations through transplantation assays. First, we sorted CCR5+ and CCR5- KLS from old CD45.2+ mice and transplanted into young CD45.1 + recipients following lethal irradiation. We analyzed peripheral blood of the recipients every 4 weeks starting 4 weeks post-transplant and observed that throughout the first 4 months, the PB of the mice that received CCR5+ KLS cells had a higher frequency of donor-derived CD1 1 b+ myeloid cells.

[0136] Next, to determine whether the difference in frequency was caused by long-term CCR5+ HSCs, we performed a secondary transplant, transplanting cells from the bone marrow of the recipients to other young recipient mice. Throughout the secondary transplant, despite the recipients that received cells from the CCR5+ group having higher frequency of donor-derived myeloid cells in PB in average, this difference was not significant. 4 months post-transplantation, the CCR5+ groups had higher frequency of myeloid cells and lower frequency of lymphoid cellsin average (calculated as the total of T cells, B cells, and NK cells), although the difference in frequency was not statistically significant. To determine whether the difference in frequency was caused by the difference in my-bi pHSCs in the BM of the recipient mice, we analyzed the BMs on flow cytometry 4 months post-secondary transplantation and observed that the CCR5+ recipients had a significantly higher frequency of my-bi, CD15Ohl0h, pHSCs in their bone marrow. This indicated that the transplantation of CCR5+ / - KLS not only changes the make-up of peripheral blood throughout a primary transplant but alters the frequency of biased pHSCs in the bone marrow. Moreover, post-secondary transplant, the CCR5- group had a higher frequency of donor derived common lymphoid progenitors (CLPs), possible indicative of the expansion of bal- HSCs following the depletion of my-HSCs. The increase in lymphoid restricted progenitors further explains the higher frequency of lymphoid cells post-transplant. (Fig 3A)

[0137] Next, to determine whether there is a subset of biased HSCs in young mice that can rejuvenate the blood of aged mice, or whether bias is acquired with age, we transplanted CCR5+ and CCR5- KLS cells from young CD45.2+ donors to old CD45.1 + recipients. We similarly analyzed peripheral blood of recipients on flow cytometry, starting 4 weeks post-transplantation, and observed that old recipients of young CCR5+ KLS cells consistently had higher average frequency of donor-derived CD1 1 b+ myeloid cells in peripheral blood, although the difference was only statistically significant at time point 12 weeks post-transplant. (Fig 3B)However this transplant was not followed through to secondary.

[0138] Human CCR5+ HSCs show myeloid bias upon transplantation. Furthermore, to determine whether CCR5 marks a my-bi population of human HSCs, we also transplanted human HSCs from the bone marrow of an 85 year-old donor to young NSG mice following sub-lethal irradiation. We observed that mice that received CCR5+ human HSCs had higher frequency of donor derived myeloid cells throughout the primary transplant. To see if long- term CCR5+ human HSCs were conferring myeloid bias we performed serial transplantation by transplanting CD34 enriched cells from the recipients of the primary transplant to a different set of young NSG mice. We observed that 16 weeks post-secondary transplant, the CCR5+ group had significantly higher frequency of donor derived myeloid cells compared to the CCR5- group. Although the group also had a higher mean frequency lymphoid cells, the difference was not significant. We further analyzed the bone marrows of recipient mice 16 weeks post-secondary transplant and observed that the CCR5+ group had significantly higher frequency of donor derived CD41 + HSCs, a marker of myeloid bias. (Fig 4A)

[0139] Disruption of the CCL5-CCR5 axis enhances immune response. Finally, we examined the effects of disrupting the CCL5-CCR5 axis through antibody injections to both young and old mice. First, we utilized a CCL5 neutralizing monoclonal antibody (CCL5 mAb) and treated mice every 5 days for 4 weeks with a dosage of 2.5mg / kg. Then we utilized maraviroc, a CCR5 antagonist which binds the receptor and blocks binding of ligands, commercially available as an HIV drug,and treated mice for 5 days with a dosage of 10Omg / kg. At the end of the CCL5 mAb treatment, we analyzed peripheral blood and bone marrow on flow cytometry. Old treated mice had significantly higher frequency of mature B cells and CLPs, compared to old untreated, there were no significant differences in young treated and untreated animals. (Fig 5C) Similarly, at the end of the 5-day maraviroc treatment, old treated mice had significantly higher frequency of mature circulating B cells (CD19+B220+lgD+lgM+), and additionally naive CD4 and CD8 T cells (CD44- CD62L+); however, the difference in their CLP frequencies were not significant. (Fig 5B) We further tested whether the disruption of the CCL5- CCR5 axis causes changes in immune response by treating mice with LPS post CCL5 mAb treatment and observed that treated old mice had higher frequencies of lymphoid cells circulating during acute inflammatory challenge. (FIG. 6).

[0140] Previous studies have shown that subsets of LT-pHSCs have differences in their reconstitution potential, with several marker expressions contributing to more myeloid hematopoietic lineages. The ligand CCL5 was previously shown to be overexpressed in whole bone marrows of age mice and contribute to my-HSCs through KO experiments. Additionally HSCs expressing chemokine receptors, such as CCR2, have been shown to contribute to emergency myelopoiesis upon ischemic injury. Here, we additionally show that, both CCR5+ and CCR5- HSCs can be serially transplanted; however, CCR5+ HSCs, which expand with age in mice and human, reconstitute a higher frequency of myeloid progeny. Furthermore, the disruption of the CCL5-CCR5 axis increases the frequency of lymphoid progenitors and lymphoid progeny in peripheral blood, also in aged mice that have been challenged with inflammatory stimuli. (FIG. 6). Furthermore, CCR5+ KLS cells from young mice overexpressed genes involved in myeloid differentiation, Tyrobp, Dapk2 and Hmgn2, compared to CCR5- KLS cells. (FIG. 7) These cells also overexpressed Lef 1 and Srgn, associated with acute myeloid leukemia.

[0141] Similarly, CCR5+ KLS cells from old mice overexpressed Aif1 , involved in myeloid differentiation, and CCR2, previously shown to induce myeloid bias. (FIG. 7) The expression of CCR5 in my-HSCs could help elucidate the distinct HSC types within the CD150h'9hHSC compartment that are biased. Additionally, the identification of cell-surface markers in biased HSCs is useful in the isolation of clonal HSCs that have been shown to contribute to myeloid bias and myeloid malignancies with age.Materials and methods:

[0142] Mouse Hematopoietic Stem Cell Isolation by Flow Cytometry: Tibia and femur were removed from the mice and crushed with mortar and pestle in FACS buffer (2% fetal bovine serum (FBS) in phosphate-buffered saline (PBS). Selection for cKIT expressing cells in the pellet was performed through incubation with c-Kit antibody conjugated to APC, followed by incubation with APC magnetic beads. Cells were washed and MACS-isolated with LS magnetic columns.Remaining cells were stained with antibodies against lineage markers: CD3e, CD4, CD8a, B220, CD19, Gr-1 , TER119, CD1 1 b (Biotin or BV786), additional markers necessary to isolate HSCs: IL7ra (AF700), c-Kit (APC-eFluor), Sca-1 (BUV395), FLT3 (PerCP-eFluor), CD150 (BV650), CD34 (BV71 1 ), progenitor markers: CD16 / 32 (PE), CD105 (PE-Cy5), Neogeninl (AF488), CD62p (PE-Cy7), CD41 (BV510), and CCR5 (PE-Cy7 or BV421 ). After wash in FACS buffer, cells were stained for streptavidin (BUV737) for secondary stain and sytox red (APC) was used as viability stain. Flow cytometry analysis was used to quantify CCR5 expressions in HSCs and other cells in the BM in mice of different ages.

[0143] Human Hematopoietic Stem Cell Isolation by Flow Cytometry:

[0144] Young Donors (aged 21 ): Cryopreserved CD34+ hematopoietic stem and progenitor cells (HSPCs) were obtained from AllCells. These cells were isolated from human bone marrow aspirated by magnetic bead enrichment using the manufacturer’s protocol. After enrichment, cells were cryopreserved in freezing medium and stored in liquid nitrogen until use. Upon receival, the cells were thawed according to the manufacturer’s recommendations and used directly for staining and flow cytometry analysis.

[0145] Old Donors (aged 46-85): Fresh femoral head samples were obtained. Bone marrow was scraped from the femoral head and cut into smaller pieces with a blade. Dissociated samples were transferred into 50ml_ tubes filled with FACS buffer and left on the shaker for 10 minutes. Samples were spun and fat cells in the supernatant were discarded, and rest of the supernatant was saved. Pellet containing hard bone marrow tissue was processed again following the same steps, and remaining fat cells were discarded. The remaining samples from both steps were filtered through 70um strainers, and centrifuged . Supernatant was discarded and pellets were further processed. Selection for CD34 expressing cells was performed with CD34 magnetic beads and MACS-isolated with LS columns.

[0146] For both young and old donors cells were stained with antibodies against lineage markers: CD3, CD4, CD8, CD14, CD19, CD20, CD56, CD235a (PE-Cy5), additional markers: CD34 (APC- Cy7), CD38 (APC), CD45RA (BV785), CD90 (FITC), CD123 (PE), CD105 (PE-Cy7), CD41 (BV510), CD71 (BV650), CD11 b (BV711 ), IL7RA (AF700), FLT3 (PercP- Cy5.5), live / dead stain (PI) and CCR3 or CCR5 (BV421 ). Flow cytometry analysis was used to quantify CCR3 and CCR5 expressions in HSCs and other cells in the samples.

[0147] HSPC Transplantation from mice to mice'. 3-mo-old female B6.SJL- Ptprc Pepc / BoyJ (CD45.1 +) recipient mice were lethally irradiated at a split dose of 9 Gy in 4-h intervals. CCR5+ and CCR5- KLS were isolated from BM of 20-to 24-mo-old female (CD45.2+) mice on flow cytometry. Per mouse, 200 cells were transplanted retro-orbitally (RO) to recipient mice, in addition to 250,000 Sca-1 depleted support cells were transplanted to recipients from donor whole bone marrow. 16 weeks post-transplant, the mice were sacrificed by lethal anesthesia andcells in the BM were analyzed using the mouse hematopoietic stem cell isolation by flow cytometry protocol. For secondary transplants, 500,000 whole bone marrow cells from BM of primary recipients were transplanted into lethally irradiated CD45.1 + secondary recipients. The same protocol was used to transplant from young to young mice and young to old mice, except old recipients were irradiated with a lower split dose of 8 Gy in 4-h intervals.

[0148] HSC transplantation from human to mice: 3-mo-old female NSG recipient mice were irradiated at a dose of 2 Gy. CCR5+ and CCR5- HSCs were isolated from BM of 85-year-old male femoral head sample. Per mouse, 100 cells were transplanted via RO to recipient mice.

[0149] Peripheral Blood Analysis: Starting post 4-weeks of transplantation, PB of recipient mice was collected through heparinized capillaries and suspended into EDTA. Samples were treated with ACK Lysis buffer twice for 5 minutes at room temperature to lyse red blood cells. To analyze chimerism and cell populations, the remaining pellet of cells from recipient mice were stained. The recipient mice that received human cells were stained with CD45 (BV785), HLA-ABC (PE), GPA / CD235a (BV421 ), CD3 (PE-Cy5), CD4 (BV711), CD8 (AF700), CD11 b (FITC), CD14 (BUV395), CD19 (APC-Cy7), CD56 (BV510), Viability (APC).

[0150] Antibody treatments: CCL5 mAb : 3-to 4-mo-old and 20-to 24-mo-old mice were given IP injections of CCL5 mAb, 50mg per mouse, once a week for a month. BM and PB of mice were analyzed via flow cytometry 1 day post-end of treatment. Maraviroc: 3-to 4-mo-old and 20-to 24- mo-old mice were given IP injections of Maraviroc dissolved in 10% DMSO + 40%PEG300 + 5% Tween80 + 45% PBS, 10Omg / kg per mice, for 5 consecutive days, PB of mice were analyzed via flow cytometry 1 day post-end of treatment and BM of mice were analyzed 4 days post-end of treatment. These were compared to untreated old mice. To determine naive T cell and mature B cell populations, samples were analyzed on flow cytometry after staining with T cell panel: CD3 (BV510), CD4 (PE-Cy7), C8 (PE), CD62L (BV71 1 ), CD44 (AF488), PD1 (BV421 ), Viability (APC) and separately with B cell panel: B220 (APC Cy-7), CD19 (BUV786), IgD (AF700), IgM (FITC), CD21 / 35 (BV421 ), CD43 (PE Cy7), CD93 (PE), CD23 (PerCP-eFluor), CD62L (BV71 1 ), CD27 (BV510), Viability (APC).

[0151] Acute Inflammatory response with LPS Treatment: 3-to 4-mo-old and 20- to 24-mo-old untreated mice or mice treated with CCL5 mAb were given one IP injection of 0.5ug / g LPS once. Peripheral blood was analyzed before treatment, 72-h, 1 -week and 2-weeks post-treatment. Samples were analyzed using the peripheral blood analysis protocol detailed above.

[0152] Myeloablative Stress with 5-FU Treatment: 3-to 4-mo-old and 20- to 24-mo-old mice were given one IP injection of 50 mg / kg of 5-FU. BM of mice were analyzed 4-days post-end of treatment with mouse hematopoietic stem cell isolation by flow cytometry protocol detailed above.

[0153] PNA Sequencing: Heatmaps were generated on R.

[0154] Statistical Analysis and graphing software: Data was statistically analyzed with GraphPadPrism software and experimental design graphics were created using Biorender. Parametricunpaired t-test was used for each plot, with two-tailed p values showing * for p < 0.05, ** for p < 0.01 , for p < 0.001 and **** for p < 0.0001 . Error bars indicate + / - SD.Example 2CCR5 marks a subset of hematopoietic stem cells that are myeloid-primed and expand with age

[0155] Hematopoietic stem cells (HSCs) are multipotent self-renewing cells that give rise to all types of blood cells. Past research has identified that long-term hematopoietic stem cells (LT- HSCs) in young mice and humans produce a balanced output of lymphoid and myeloid cells, while in old age they are largely replaced by myeloid biased HSCs (my-HSC). It has not yet been determined whether this transition results from epigenetic changes in a single population of HSC or if two or more subsets of HSCs exist that gain or lose dominance with age via processes of selection. Whether epigenetic change or competition, several characteristics of each may exist to ensure that the appropriate subset is placed in niches that support them. HSC can be mobilized into the blood and home selectively to target tissues via expression of ‘homing receptors’, but these molecules do not determine their intra-organ migration to appropriate niches. Chemokines are the class of molecules that determine intra-organ migration of cells. Here, we show that the chemokine receptor CCR5 is mainly expressed on my-HSCs, and therefore the frequency of CCR5+HSCs increases with age. Aged HSCs negative for CCR5 expression generate lower frequency of myeloid cells than lymphoid cells upon transplantation into recipients. Additionally, disruption of the CCL5-CCR5 signaling axis with blocking antibodies changes frequency of lymphoid populations in peripheral blood of aged mice, supporting research that shows the depletion of my-HSCs can result in the rejuvenation of adaptive immunity.

[0156] Analysis of phenotypic markers have yielded knowledge regarding fate and function of HSCs under different conditions, including in aging systems. Aging of long-term HSCs is marked by the expansion of myeloid-biased HSCs (my-HSCs), resulting in reduced lymphoid output, which contributes to impaired adaptive immune function in older individuals. Selective depletion of my-HSCs in aged mice, using antibodies against markers enriched in my-HSC populations, can result in the expansion of the residual balanced HSC pool, and rejuvenate the adaptive immune system by restoring the production of naive lymphocytes.

[0157] Here, we identify a new marker that distinguishes a subset of HSCs that are predisposed to myeloid differentiation, CCR5. CCR5 is a Cysteine-Cysteine chemokine receptor that mainly binds the chemokine CCL5, and others such as CCL1 and CCL3. It is also the main cell surface coreceptor for the entry of human immunodeficiency virus strains HIV-1 and HIV-2. While its role in HSCs has been elucidated as a homing receptor, influencing migratory response, for instance mediating homing in the thymus, its role in influencing fate bias has been limitedly explored. Previous studies have suggested that the CCL5-CCR5 signaling axis mediates myelopoiesis andthat exposure to CCR5 ligands can cause myeloid skewing of stem and progenitor cells, whether by transdifferentiation or selective expansion of already biased my-HSC. Our study demonstrates that CCR5 marks a subset of HSCs that expand with age; and shows transcriptional and functional characteristics associated with my-HSCs. Further, we demonstrate that antibodybased disruptions of the CCL5-CCR5 signaling axis in aged mice with single antibodies can change peripheral blood populations to resemble characteristics of young animals.Results

[0158] CCR5 marks a population of mouse HSCs that co-express my-HSC markers and increase with age. To determine whether CCR5 marks a population of HSCs that increase with age, we used flow cytometry to measure relative CCR5 levels on phenotypic HSCs (referred to as pHSCs from here on), which include both long term HSCs and short term HSCs and multipotent progenitors (MPP) cells from young (<4 months-old) and aged (>18 months-old) mice. We observed that CCR5 was expressed at low levels on a subset of pHSCs (FIG. 8A). The frequency of CCR5+cells increased significantly with age in the KLS population (a subset of cells enriched for murine HSCs), pHSCs and MPP subsets A and B but not C (FIG. 8B). To assess whether the ligand levels show the same trend of increase with age as the receptor, we performed ELISA on whole bone marrow samples from young and aged mice and observed an increase in concentration of CCL5 levels with age (FIG. 8C).

[0159] It was previously shown that my-HSCs that co-express certain cell surface markers, specifically CD62p, CD41 , Neogenin-1 , and CD150, expand with age. We assessed whether CCR5+pHSCs co-express these markers. Flow cytometry analysis showed that CCR5+pHSCs from aged mice co-express Neogeninl , CD41 and CD62p. While Neogeninl was expressed highly in both CCR5 and CCR5+pHSCs, CD41 and CD62p were expressed significantly higher in CCR5+pHSCs than CCR5 pHSCs (FIG. 8D, FIG. 12A). These results show that one or more subsets of my-HSCs may be enriched in CCR5+pHSCs.

[0160] To transcriptionally characterize these cells, we performed bulk RNA sequencing on KLS cells from aged mice which had higher expression of CCR5 as previously stated. Gene Set Enrichment Analysis (GSEA) revealed that CCR5+KLS cells from the aged mice exhibit a significantly higher enrichment score for pathways associated with myeloid cell differentiation in comparison to CCR5 KLS cells. Additionally, we observed that CCR5 KLS cells from aged mice exhibit higher enrichment score for pathways related to B cell differentiation and the regulation of T cell differentiation in comparison to CCR5+KLS cells (FIG. 8E-G). These results supported that transcriptionally, CCR5+stem and progenitor cells are primed towards myeloid differentiation.

[0161] A higher fraction of CCR5+HSCs are in the cell division cycle than CCR5 HSCs. To further characterize CCR5+and CCR5 subsets, we focused on their cell cycle dynamics duringnative hematopoiesis. Because pHSCs are not representative of functionally pure long-term HSCs (LT-HSC, which are enriched in GO of cell cycle), analyzing cell cycle dynamics of those populations could reflect frequency of LT-HSCs within CCR5+and CCR5 populations instead of true cell cycle dynamics. To address this, we utilized an additional marker, Hoxb5, a transcriptional factor expressed on LT-HSCs, and analyzed cell cycle dynamics of CCR5+Hoxb5+or CCR5 Hoxb5+cell. We used we Hoxb5-tri-mCherry mice, in which LT-HSCs are marked with mCherry under Hoxb5 regulation. Initially, we showed that both CCR5+and CCR5 pHSC populations express Hoxb5 (FIG. 9A and FIG. 9B). Then, we analyzed the cell cycle status of these cells in young and old mice using Ki67 and DAPI staining (FIG. 9C). A higher frequency of CCR5 LT-HSCs compared to CCR5+LT-HSCs from both young and aged mice were in GO. Consistently, a higher frequency of CCR5+pHSCs were in G2 / S and G1 phase in young and aged mice, although the difference in G1 phase frequency did not reach statistical significance in young mice (FIG. 9D). Overall, these findings show that CCR5+pHSCs cycle more actively.

[0162] Following this characterization of CCR5+and CCR5 HSPCs in undisturbed settings, we examined whether CCR5+and CCR5 pHSCs differ in their response to myeloablative stress. We treated young and aged mice with 150mg / kg 5-FU and analyzed changes in CCR5+and CCR5 populations using flow cytometry 5 days post treatment, a timepoint where due to cell cycle induction of surviving primitive HSCs, there is high HSC proliferation (FIG. 9F). The frequency of CCR5+pHSCs increased post 5-FU challenge in young mice, although the increase did not reach statistical significance in old mice. Additionally, because 5-FU can change marker expression, we analyzed how CCR5+and CCR5 populations change within LT-HSCs, given Hoxb5’s specificity as a marker. The frequency of CCR5 cells within LT-HSCs also increased post 5-FU challenge in young mice. Overall, these findings support that CCR5+cells are affected most by 5-FU as they are cycling more and respond to myeloablative stress by expansion.

[0163] Mouse CCR5+HSPCs show myeloid bias in serial transplantation assay. Next, we examined whether the transplantation of CCR5+and CCR5 pHSCs result in differences in the frequency of myeloid and lymphoid populations in peripheral blood (PB). We sorted and transplanted 200 CCR5+or 200 CCR5 KLS cells from aged CD45.2+mice into young CD45.1+recipients following lethal irradiation and analyzed hematopoietic output through primary and secondary transplantations (FIG. 10A). The recipient mice were also given 250,000 support cells that are CD45.2 / 45.1+. PB of the recipients was analyzed every 4 weeks starting 4 weeks posttransplantation. Notably, because CCR5+and CCR5 cells have differential expression of other my-HSC markers, including CD150h'9h(FIG. 8D), the different transplanted populations may be further enriched for other biased populations. We observed that throughout the first 4 months, PB of the mice that received CCR5+KLS cells had a significantly higher output of donor-derived myeloid cells, determined by CD1 1 b+granulocytes and monocytes (FIG. 10B). Consequently,the frequency of lymphoid cells was lower in CCR5+recipients throughout the weeks, determined by total T, B, and NK cell frequency, although this difference was not statistically significant (FIG. 10C). The donor chimerism was low for both groups, potentially due to low-engraftment potential of aged HSCs in young mice, as shown in previous studies (FIG. 10D). However, difference in myeloid output between recipient groups was significant, despite very low chimerism.

[0164] To determine whether the difference in frequency was caused by long-term CCR5+HSCs, we performed a secondary transplant, transplanting whole bone marrow cells from the primary recipients to lethal ly irradiated young secondary recipients. Throughout the secondary transplant, we observed that the PB of the mice that received CCR5+KLS cells had a persistently higher frequency of donor-derived myeloid cells (FIG. 10E), similar to a report of retained balanced vs myeloid bias at the HSC cellular level. Although the difference in the frequency of lymphoid cells was not significant at all times assayed throughout the secondary transplant, it stayed consistently higher in recipients of CCR5 cells (FIG. 10F). In addition, at week 8 of the secondary transplant, CCR5+donor HSCs showed a significant increase in total donor chimerism, coinciding with the highest myeloid output and diminished lymphoid production in these recipients (FIG. 10G).

[0165] To address whether the differences in lineage output in PB were caused by the differences in donor derived stem and progenitor populations in the BM of the recipient mice, we analyzed the BM cells by flow cytometry 4 months post-secondary transplantation. Recipients of CCR5+cells had a significantly higher frequency of CCR5+stem and progenitors than recipients of CCR5 cells (FIG. 10H). BM analysis further showed that the CCR5+recipients had higher frequency of common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs). In contrast, for some of the CCR5 recipients, megakaryocyte-erythroid progenitors and megakaryocyte progenitors were higher than in CCR5+recipients, although the difference was not statistically significant. This suggests that the CCR5 population may be more heterogenous than the CCR5+population. Additionally, we observed a slight reduction in common lymphoid progenitors (CLPs) in CCR5+transplanted recipients; however this difference was not significant (FIG. 10H). This suggests that CCR5 might be affecting lymphoid production more downstream from common lymphoid progenitors (CLPs), causing differences in lymphoid frequencies in PB, or that statistical significance may not reached due to the heterogeneity of the CCR5 cells. Overall, the increased frequency of CMPs and GMPS, along with the slight reduction in CLPs in CCR5+recipients, could explain the myeloid-skewed output in their PB. Notably, these results showcase functional differences in the broader CCR5+and CCR5 stem and progenitor cell populations, although it should be noted that transplanted cells were KLS rather than pHSCs, and consequently chimerism throughout both the primary and secondary cells are low (FIG. 13C) .

[0166] Disruption of the CCL5-CCR5 signaling axis alters immune cell frequencies. Given that CCR5 marks cells with a predominant myeloid output, and previous work has shown that antibody-based depletion of my-HSCs can increase the frequency of lymphoid cells, we tested whether antibody or antagonist mediated disruption of the CCL5-CCR5 signaling axis in aged mice could rejuvenate their immune response potential. We utilized a CCL5-neutralizing monoclonal antibody to treat aged mice with intra-peritoneal (IP) injections every 5 days for 4 weeks with a dosage of 2.5mg / kg, along with lgG2A treated controls. Additionally, we utilized the CCR5 antagonist, Maraviroc, and treated aged mice daily for 5 days with a dosage of 200mg / kg, along with controls that received Maraviroc vehicle without the drug. At the end of treatments, we analyzed peripheral blood and bone marrow by flow cytometry to analyze populations of cells that are known to change with age (FIG. 1 1 A).

[0167] Untreated old mice had lower frequencies of naive T cells (CD3+CD4 / 8+CD44 / lowCD62L+) and mature B cells (CD19+B220+lgD+lgM+) in their PB, compared to young animals (FIG. 11 B and FIG. 11 C). Treatment with anti-CCL5 significantly increased the levels of observed naive T cells and mature B cells compared to IgG treated controls. The same treatment did not significantly affect levels of memory T cells, although they were generally decreased in the treated group (CD3+CD4 / 8+CD44+CD62L+). Frequency of aged B cells (B220+CD19+lgM+CD43 CD93 CD23 CD21 / 35 ) (FIG. 11 B and FIG. 11 C) were higher in old untreated animals compared to young. Treatment with Maraviroc similarly increased mature B cell frequencies, however it did not significantly change naive T cell frequencies compared to the administration of maraviroc vehicle (FIG. 11 B and FIG. 1 1 C) . Maraviroc did not change the frequency of the cell populations that increased with age, including memory T and aged B cells.

[0168] To evaluate how disruption of the CCL5-CCR5 signaling axis affects stem and progenitor populations, we analyzed bone marrow from treated animals. Untreated aged mice had higher frequency of CD150h'9hmy-HSCs than young mice, as expected. Anti-CCL5 and Maraviroc treated old mice had a lower frequency of CD150h'9hmy-HSCs compared to their respective IgG treated and vehicle controls, and consequently had higher frequency of CD150lowpHSCs (FIG. 11 D and FIG. 14C). Neither anti-CCL5 treatment nor Maraviroc increased frequency of CLPs, which are found in higher frequency in young mice bone marrow (FIG. 1 1 D). This result aligns with the transplantation results where CCR5+ and CCR5- cell recipients did not have differences in the frequency of donor derived CLPs, which may indicate that the effects of the CCL5-CCR5 signaling axis on lineage bias is downstream of CLPs. Overall, these results demonstrated that disruption of the CCR5-CCL5 signaling axis impacts progenitor populations and lineage output, as reflected by increased frequencies of naive T and mature B cells, and a decrease in CD150h'9hmy-HSCs, particularly for aged mice treated with anti-CCL5.

[0169] We further tested whether the disruption of the CCL5-CCR5 signaling axis causes changes in long-term innate immune responses. Because anti-CCL5 treatment led to significantincreases in naive T and mature B cell populations, we treated aged mice with anti-CCL5 for a month, and untreated aged mice with PBS. Post-treatment, we gave IP injections of 0.5 mg / kg LPS to anti-CCL5 treated and untreated control mice to trigger an acute inflammatory response. We observed that untreated aged mice had higher frequencies of circulating myeloid cells during the acute inflammatory response compared to their treated counterparts (Fig 4E). Myeloid frequencies increased during the first week following LPS challenge in aged untreated mice; however myeloid frequencies of treated aged mice stayed consistent. We also analyzed frequencies of total T and B cells throughout inflammatory challenge to see overall changes in lymphocyte populations. Compared to changes in myeloid frequencies, (Cd11 b CD8+) T and (Cd11 b B220+) B cells were higher in frequency in the treated aged mice, however this difference did not reach statistical significance throughout all weeks. Previous studies have shown that CCL5 levels increase post-LPS treatment, amongst other inflammatory chemokines; hence, blocking interactions of this ligand might provide a route to suppress pro-inflammatory responses in aged systems.

[0170] Finally, through flow cytometry staining we have shown that CCR5 is on the surface of human HSCs (FIG. 15). While it seems that there might be an increase of CCR5+human HSCs with age, going from young to middle aged, we did not compare data for statistical significance as young and aged donors were collected / processed differently, further detailed in the methods, which can affect expression of cell surface markers.

[0171] Previous studies have shown that subsets of LT-HSCs have intrinsic differences in their reconstitution potential. Cells with my-HSC associated marker expression contribute to more myeloid hematopoietic lineages. Subsets of myeloid-biased LT-HSCs have been shown to clonally expand with age in both human and mice, and to impair immune function in old individuals. The chemokine CCL5 is upregulated in whole bone marrow and plasma of aged mice. CCL5 is shown herein to be associated with an age-related expansion of subpopulations of myeloid primed HSCs, which skews blood cell towards myeloid lineages.

[0172] The disclosure herein demonstrates that the frequency of CCR5+stem and progenitor cells increases with age; and may be also increased in young animals following myeloablative stress. It is also shown that aged CCR5+pHSCs contribute predominantly to myeloid lineages during blood reconstitution of irradiated recipients. Bulk-RNA sequencing data shows that CCR5+KLS cells from young and aged mice overexpress genes involved in myeloid differentiation, including Dapk2 and Aif 1 , as well as CCR2, which has been previously shown to mark my-HSCs and contribute to emergency myelopoiesis (FIG. 15).

[0173] It is shown herein that disrupting the CCL5-CCR5 signaling axis results in short term changes of leukocyte populations, marked by increase of naive T and mature B cells in some cases, and reduction in the expansion of myeloid cells during acute inflammatory response. Previous literature showed that aged mice have even more increased levels of these cells afterantibody based depletion of HSCs expressing several my-HSC markers (Neogeninl , CD41 , and CD62p), supplemented by anti-cKIT and anti-CD47 treatment which have been shown to optimize my-HSC depletion. Overall, these results show that the CCL5-CCR5 axis can be a pathway for therapeutic targeting to overcome expansion of my-HSCs.Materials and methods:

[0174] Mice. Hoxb5-tri-mCherry with CD45.2 C57BU6J background mice were used for cell cycle analysis and 5-FU experiments. CD45.2 C57BL / 6J mice were used as donors for transplants as well as for analysis and other experiments. 18+ months old mice are referred to as aged and 2-4 months old mice are referred to as young. Mice were bred at our animal facility according to NIH guidelines. 8-to-12-week old female B6.SJL- Ptprca Pepcb / BoyJ mice (Jackson Laboratory) were used as recipients for transplantations. Supporting cells were collected from B6.SJL-Ptprca Pepcb / BoyJ x C57BL / 6J (F1 mice CD45.1 + / CD45.2+). All animal protocols were approved by the Stanford University Administrative Panel on Laboratory

[0175] Animal Care. Mouse Bone Marrow Processing and Hematopoietic Stem Cell Sorting and Analysis on Flow Cytometry. Tibia, femur, and pelvis were removed from the mice and crushed with mortar and pestle in FACS buffer (2% fetal bovine serum (FBS) in phosphate-buffered saline (PBS)). Selection for c-KIT expressing cells in the pellet was performed through incubation with anti-c-KIT APC-eFluor780 (Thermo) for 15 minutes at 4C, followed by incubation with anti-APC magnetic beads for 15 mins at 4C. Cells were washed and MACS-isolated using LS magnetic columns (Miltenyi Biotec). Remaining cells were stained with antibodies against lineage markers: CD3e, CD4, CD8a, B220, CD19, Gr-1 , TER119, CD1 1 b (BV786), additional markers necessary to isolate HSCs and progenitors: IL7ra (AF700), c-Kit (APC-eFluor), Sca-1 (BUV395), FLT3 (PerCP-eFluor), CD150 (BV650), CD34 (BV71 1 ), progenitor markers: CD16 / 32 (PE), CD105 (PE-Cy5), CD62p (PE-Cy7), CD41 (BV510), and CCR5 (PE-Cy7 or BV421 ). Neogeninl was detected using a polyclonal primary antibody (R&D Systems, Catalog #: AF1079). Samples were washed with FACS buffer and cells were stained with a donkey anti-goat Alexa Fluor 488 conjugated antibody for secondary staining. SYTOX Red Dead Cell Stain (Life Technologies) was used as viability stain.

[0176] ELISA. Mice were euthanized and femurs were collected. Syringe with PBS was used to flush out the bone marrow from the femurs. Sample was centrifuged and pellet of cells were used for ELISA following protocol from Mouse / Rat CCL5 / RANTES Quantikine ELISA Kit (MMR00, R&D Systems).

[0177] RNA-sequencing. Mice were euthanized and bone marrow was processed as described above. CCR5+and CCR5 KLS cells were sorted into 1 .5 ml Eppendorf tubes preloaded with 1 ,000 pl of TRIzol LS Reagent (ThermoFisher, Cat. #10296028). RNA was extracted using the TRIzol method, including chloroform phase separation and isopropanol precipitation, followed bypurification on QIAGEN miRNeasy Mini columns (QIAGEN, Cat. #217004). RNA purity and integrity were assessed using a NANODROP1000 spectrophotometer and an Agilent Bioanalyzer 2100 with a PICO chip.

[0178] Library preparation was performed using the KAPA mRNA HyperPrep Kit (KAPA, Cat. #KK8580) in conjunction with the IDT for Illumina Dual Index Adapter Kit (IDT, Cat. #20021454) according to the manufacturer’s protocol. Briefly, mRNA was isolated using magnetic oligo-dT beads and fragmented by heat and magnesium. First-strand cDNA synthesis was conducted with random priming, followed by combined second-strand synthesis and A-tailing. Adapter ligation, library amplification, and clean-ups were performed with KAPA Pure Beads. Strand specificity was achieved by incorporating dUTP in the second strand, which prevents its amplification.

[0179] Final libraries were assessed for quality and size distribution using an Agilent Bioanalyzer 2100 High Sensitivity DNA Chip. Equimolar amounts of cDNA libraries from each sample were pooled and sequenced on the Illumina NovaSeq 6000 platform with an S4 flowcell. FASTQ files were generated using the bcl2fastq2 Conversion Software v2.19. Sequence data were aligned and normalized using the STAR aligner or DRAGEN RNA pipeline to map reads to the Mus musculus transcriptome (GRCm38, RefSeq). Differential expression analysis was conducted using DESeq2.

[0180] Cell Cycle Analysis. Hematopoietic stem cells from young and old / - / oxb5-tri-mCherry mice were isolated and enriched for c-KIT as described above. Pellet of cells were stained with cell surface antigens: Lineage (BV786), c-KIT (APC-Cy7), Sca-1 (BV711 ), Flt3 (PerCP-eFluor), CD150 (BV650), CD34 (FITC), CCR5(PE-Cy7) for 30 minutes. Cells were washed 2 times with 1 mL Staining Buffer (from BD Fixation / Permeabilization kit) and pelleted by centrifugation. Cells were resuspended in Fixation / Permeabilization solution (from BD Fixation / Permeabilization kit) for 20 minutes at 4 °C, and washed 2 times in 1 mL of 1 X Perm / Wash buffer (from BD Fixation / Permeabilization kit). Finally, cells were stained for intracellular Ki67 (APC) and 5 pl of 20 pg / ml DAPI for 30 minutes at 4 °C in the dark. Stained cells were washed 2 times with 1 mL of 1 X Perm / Wash buffer and analyzed on flow cytometry.

[0181] Transplantations, peripheral blood, and bone marrow analysis. 3-mo-old female B6.SJL- Ptprc^Pepd0 / Boy J (CD45.1 +) recipient mice were lethally irradiated at a split dose of 9 Gy in 4- h intervals. CCR5+ and CCR5- KLS were isolated from BM of aged C57BL / 6J (CD45.2+) mice on flow cytometry. Per mouse, 200 cells were transplanted retro-orbitally (RO) to recipient mice, in addition to 250,000 Sca-1 depleted support cells were transplanted to recipients from donor whole bone marrow.

[0182] To isolate support cells from B6.SJL-Ptprca Pepcb / BoyJ x C57BU6J (F1 mice CD45.1 + / CD45.2+), bone marrow was processed as described. Cells were incubated with SCA depletion beads (Miltenyi Biotec) for 20 mins at 4°C. Cell suspension was washed with FACSbuffer and LS magnetic columns (Miltenyi Biotec) were used to separate SCA-depleted cells. The flow-through fraction, containing the SCA-1 -depleted support cells, was used for transplantation.

[0183] Starting post 4-weeks of transplantation, PB of recipient mice was collected through heparinized capillaries and suspended into 500ul_ PBS with 2mM EDTA. Samples were treated with ACK Lysis buffer (Thermo) twice for 5 minutes at room temperature to lyse red blood cells. To analyze chimerism and cell populations, the remaining pellet of cells from recipient mice were stained with leukocytes were stained with antibodies against CD45.2 (BV421 ), CD45.1 (BV785), Teri 19 (BV737), CD11 b (FITC), Gr-1 (BV395), CD8 (PE), CD4 (PE-Cy7), NK1 .1 (PE-Cy5), and B220 (APC-Cy7). The percentage of donor chimerism in the peripheral blood was defined as the percentage of CD45.2+cells among total live cells.

[0184] 16 weeks post-transplant, mice were sacrificed by lethal anesthesia. For secondary transplant, 500,000 whole bone marrow cells from the primary recipients were transplanted into lethally irradiated young B6.SJL- Ptprc Pepd / BoyJ (CD45.1 ) recipients. At the end of secondary transplant, mice were euthanized and bone marrow from femurs, tibias, and pelvises were processed as previously described. For HSC and progenitor analysis, cells were stained with antibodies against lineage markers: CD3e, CD4, CD8a, B220, CD19, Gr-1 , TER1 19, CD11 b (BV737-conjugated Streptavidin or BV510), and additional markers necessary to analyze HSCs and progenitors populations: CD45.2 (BV421), CD45.1 (BV785), c-KIT (APC-eFluor870), Sca-1 (BV395), CD150 (BV650), FLT3 (PerCP-eFluor710), CD41 (BV510 or PE), CD34 (BV71 1 or AF488), CD16 / 32 (PE), IL7Ra (AF700), and CCR5 (PE-Cy7).

[0185] Mice injections. Anti-CCL5: Young and aged mice were given IP injections of 2.5mg / kg monoclonal CCL5 antibody (R&D Systems MAB478-500), once a week for 4 weeks. Control mice were given IP injections of rat lgG2A isotype control of the same concentration. BM and PB of mice were analyzed via flow cytometry 1 day post-end of treatment. Maraviroc. Young and aged mice were given IP injections of 200mg / kg Maraviroc (MedChem Express) dissolved in 10% DMSO + 40%PEG300 + 5% Tween80 + 45% PBS, for 5 consecutive days. Control mice were given IP injections of vehicle solvent containing 10% DMSO + 40%PEG300 + 5% Tween80 + 45% PBS. PB of mice was analyzed via flow cytometry 1 day post-end of treatment and BM of mice were analyzed 5 days post-end of treatment.

[0186] For anti-CCL5 and Maraviroc treatments samples were analyzed on flow cytometry after staining with T cell panel: CD3 (BV510), CD4 (PE-Cy7), C8 (PE), CD62L (BV71 1 ), CD44 (AF488), PD1 (BV421 ), Viability (APC) and separately with B cell panel: B220 (APC Cy-7), CD19 (BUV786), IgD (AF700), IgM (FITC), CD21 / 35 (BV421 ), CD43 (PE Cy7), CD93 (PE), CD23 (PerCP-eFluor), CD62L (BV71 1 ), CD27 (BV510), Viability (APC).

[0187] LPS: young and aged untreated mice or mice treated with monoclonal CCL5 antibody, as detailed, were given IP injection of 0.5ug / g LPS once. Peripheral blood was analyzed before treatment, 72-h, 1 -week and 3-weeks post-treatment, with staining for antibodies against: Teri 19(BV737), CD11 b (FITC), Gr-1 (BV395), CD8 (PE), CD4 (PE-Cy7), NK1.1 (PE-Cy5), and B220 (APC-Cy7). 5-FU: Young and old / - / o b5-tri-mCherry mice were given IP injection of 50 mg / kg of 5-FU once. BM of mice were analyzed 4-days post-end of treatment on flow cytometry as detailed above.

[0188] Human Hematopoietic Stem Cell Sorting and Analysis on Flow Cytometry. Young Donors (aged 21 -30): Cryopreserved CD34+ hematopoietic stem and progenitor cells (HSPCs) were obtained from AllCells. These cells were isolated from human bone marrow aspirated by magnetic bead enrichment using the manufacturer’s protocol. After enrichment, cells were cryopreserved in freezing medium and stored in liquid nitrogen until use. Upon receival, the cells were thawed according to the manufacturer’s recommendations and used directly for staining and flow cytometry analysis.

[0189] Old Donors (aged 43-91 ): Fresh femoral head samples were obtained. Bone marrow was scraped from the femoral head and cut into smaller pieces with a blade. Dissociated samples were transferred into 50mL tubes filled with FACS buffer and left on the shaker for 10 minutes. Samples were spun and fat cells in the supernatant were discarded, and rest of the supernatant was saved. Pellet containing hard bone marrow tissue was processed again following the same steps, and remaining fat cells were discarded. The remaining samples from both steps were filtered through 70um strainers, and centrifuged . Supernatant was discarded and pellets were further processed. Selection for CD34 expressing cells was performed with CD34 magnetic beads and MACS-isolated with LS columns.

[0190] For both young and old donors cells were stained with antibodies against lineage markers: CD3, CD4, CD8, CD14, CD19, CD20, CD56, CD235a (PE-Cy5), additional markers: CD34 (APC- Cy7), CD38 (APC), CD45RA (BV785), CD90 (FITC), CD123 (PE), CD105 (PE-Cy7), CD41 (BV510), CD71 (BV650), CD11 b (BV711 ), IL7RA (AF700), FLT3 (PercP- Cy5.5), live / dead stain (PI) and CCR3 or CCR5 (BV421 ). Flow cytometry analysis was used to quantify CCR3 and CCR5 expressions in HSCs and other cells in the samples.

[0191] Statistical Analysis and graphing software'. Data was statistically analyzed with GraphPadPrism software. Parametric unpaired t-test was used for each plot, with two-tailed p values showing * for p < 0.05, ** for p < 0.01 , *** for p < 0.001 and **** for p < 0.0001 . The ROUT method with Q = 1 was used to remove outliers. Error bars indicate mean + / - SEM.References

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[0241] Embodiment 1. A method of rebalancing myeloid-biased hematopoietic stem cells (my- HSC) relative to balanced hematopoietic stem cells (bal-HSC) in the immune system of a mammalian subject the method comprising: administering to the individual a therapy disrupt the CCL5-CCR5 axis.

[0242] Embodiment 2. The method of embodiment 1 , wherein the therapy comprises administering an effective dose of a CCR5 antagonist.

[0243] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the CCR5 antagonist is an antibody specific for CCR5.

[0244] Embodiment 4. The method of embodiment 3, wherein the antibody is Leronlimab or human monoclonal antibody CCR5 mAb.

[0245] Embodiment 5. The method of embodiment 1 or embodiment 2, wherein the CCR5 antagonist is a small molecule that inhibits CCR5.

[0246] Embodiment 6. The method of embodiment 5, wherein the small molecule is selected maraviroc, VCH-286, AZD-5672, AK-220, aplaviroc, vicriviroc and cenicriviroc.

[0247] Embodiment 7. The method of embodiment 1 or embodiment 2, wherein the CCR5 antagonist is a soluble peptide.

[0248] Embodiment 8. The method of embodiment 1 or embodiment 2, wherein the therapy comprises genome editing of HSC.

[0249] Embodiment 9. The method of embodiment 1 or embodiment 2, wherein the CCR5 antagonist is an RNAi.

[0250] Embodiment 10. The method of embodiment 1 or embodiment 2, wherein the therapy comprises administering an agent specific for CCR5 in a dose effective to deplete CCR5+HSC.

[0251] Embodiment 11 . The method of embodiment 10, further comprising administering to the subject an agent that blocks CD47 interaction with SIRPa.

[0252] Embodiment 12. The method of embodiment 10 or embodiment 11 , further comprising administering to the subject an agent that specifically binds to CD117.

[0253] Embodiment 13. The method of any of embodiments 1-12, wherein the subject is an elderly human.

[0254] Embodiment 14. The method of any of the previous embodiments, wherein following the treatment, there is an enrichment of bal-HSC to my-HSC of from at least 1 .5-fold to at least 15- fold.

[0255] Embodiment 15. The method of any of the previous embodiments, wherein following the treatment, the ratio of the number of lymphoid progenitors in bone marrow of the subject to the number of myeloid progenitors in bone marrow of the subject is increased at least 1 .5-fold fold to at least 15-fold.

[0256] Embodiment 16. The method of any of the previous embodiments, wherein following the treatment the number of circulating naive T cells relative to the total circulating lymphocyte population is increased at least 1.5-fold to at least 15-fold.

[0257] Embodiment 17. The method of any of the previous embodiments, wherein the balance of immune cells including one or more of: the relative number of one or more of naive T cells, exhausted T cells, ABC, myeloid progenitors and lymphoid progenitors, is determined before treatment.

[0258] Embodiment 18. The method of any of the previous embodiments, wherein following the treatment, the basal circulating level of an inflammaging marker selected from: one or more of IL-1 a, CXCL5, IL1 RL1 , IL-23, IL-1b, CXCL2, IL-31 , IL-5, GM-CSF, is decreased at least 2-fold.

[0259] Embodiment 19. The method of any of the previous embodiments, wherein following the treatment, an antigen-specific CD8+ T cell response is increased at least 1.5-fold.

[0260] Embodiment 20. The method of any of the previous embodiments, wherein following the treatment, an antigen-specific antibody response is increased at least 1 .5-fold.

[0261] Embodiment 11-1 . A method of rebalancing myeloid-biased hematopoietic stem cells (my- HSC) relative to balanced hematopoietic stem cells (bal-HSC) in the immune system of a mammalian subject, the method comprising: administering to the subject an agent that disrupts the CCL5-CCR5 axis.

[0262] Embodiment li-2. The method of embodiment 1 , wherein the agent reduces CCR5 activity, and / or blocks a binding interaction between CCR5 and CCL5.

[0263] Embodiment II-3. The method of embodiment 1 or embodiment 2, wherein the agent depletes CCR5+hematopoietic stem cells.

[0264] Embodiment II-4. The method of any of embodiments 1 -3, wherein the agent is a CCR5 antagonist.

[0265] Embodiment II-5. The method of any of embodiment 1 -3, wherein the agent is a CCL5 antagonist.

[0266] Embodiment 11-6. The method of any one of embodiments 1-4, wherein the agent is an antibody specific for CCR5.

[0267] Embodiment II-7. The method of embodiment 6, wherein the antibody is Leronlimab or a variant thereof, HGS004 or a variant thereof, or HGS101 or a variant thereof.

[0268] Embodiment II-8. The method of embodiment 4, wherein the CCR5 antagonist is a small molecule that inhibits CCR5.

[0269] Embodiment II-9. The method of embodiment 8, wherein the small molecule is selected from maraviroc, VCH-286, AZD-5672, AK-220, aplaviroc, vicriviroc and cenicriviroc.

[0270] Embodiment 11-10. The method of embodiment 4, wherein the CCR5 antagonist is a soluble peptide.

[0271] Embodiment 11-11. The method of embodiment 10, wherein the peptide is selected from DAPTA, AOP-RANTES, PCS-RANTES, Met-RANTES, NNY-RANTES, vMIP-ll.

[0272] Embodiment 11-12. The method of any one of embodiments 1-3 and 5, wherein the agent is an antibody specific for CCL5.

[0273] Embodiment 11-13. The method of any one of embodiments 1-3, wherein the agent is a CCR5 antagonist, and wherein the CCR5 antagonist is an RNAi; or wherein the agent is a CCL5 antagonist, and wherein the CCL5 antagonist is an RNAi.

[0274] Embodiment 11-14. The method of any one of embodiments 1 -3, wherein the agent comprises genome editing of HSC, and wherein the genome editing results in decreased expression of CCR5 and / or CCL5.

[0275] Embodiment 11-15. The method of any one of embodiments 1-14, wherein the agent is administered in a dose effective to deplete CCR5+HSC.

[0276] Embodiment 11-16. The method of embodiment 15, further comprising administering to the subject an agent that blocks CD47 interaction with SIRPoc.

[0277] Embodiment 11-17. The method of embodiment 15 or embodiment 16, further comprising administering to the subject an agent that specifically binds to CD117.

[0278] Embodiment 11-18. The method of any of embodiments 15-17, further comprising administering an antibody specific for a second my-HSC specific marker.

[0279] Embodiment 11-19. The method of embodiment 18, wherein the marker is selected from the group consisting of CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.

[0280] Embodiment II-20. The method of any of embodiments 1-19, wherein the subject is a human.

[0281] Embodiment 11-21 . The method of any of embodiments 1 -20, wherein the subject is aged.

[0282] Embodiment II-22. The method of any of the previous embodiments, wherein following the administering step, there is an enrichment of bal-HSC to my-HSC of at least 1 .5-fold to at least 15-fold.

[0283] Embodiment II-23. The method of any of the previous embodiments, wherein following the administering step, there is an enrichment of CCR5 HSC to CCR5+HSC of at least 1.5-fold to at least 15-fold.

[0284] Embodiment II-24. The method of any of the previous embodiments, wherein following the administering step, the ratio of the number of lymphoid progenitors in bone marrow of the subject to the number of myeloid progenitors in bone marrow of the subject is increased.

[0285] Embodiment II-25. The method of any of the previous embodiments, wherein following the administering step the number of circulating naive T cells relative to the total circulating lymphocyte population is increased.

[0286] Embodiment II-26. The method of any one of the previous embodiments, wherein following the administering step a basal circulating level of an inflammaging marker is decreased, optionally wherein the inflammaging marker is selected from the group consisting of IL-1 a, CXCL5, IL1 RL1 , IL-23, IL-1 b, CXCL2, IL-31 , IL-5, GM-CSF, and a combination thereof.

[0287] Embodiment II-27. A method of rebalancing myeloid-biased hematopoietic stem cells (my-HSC) relative to balanced hematopoietic stem cells (bal-HSC) in a hematopoietic stem cell composition, the method comprising:

[0288] depleting CCR5+HSC from the population.

[0289] Embodiment II-28. The method of embodiment 27, wherein the depleting step is performed in vitro.

[0290] Embodiment II-29. The method of embodiment 28, wherein the HSC population is administered to an individual in need thereof.

[0291] Embodiment II-30. A composition for use in the methods of any of embodiments 1 -24.

[0292] Embodiment 11-31 . A kit for use in the methods of any of embodiments 1 -24.

[0293] Embodiment II-32. A method for preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective dose of an agent that disrupts the CCR5- CCL5 axis, wherein the administering results in a reduction of myeloid-biased hematopoietic stem cells (my-HSC) relative to balanced hematopoietic stem cells (bal-HSC), thereby preventing or treating the disease or disorder.

[0294] Embodiment II-33. The method of embodiment 32, wherein the subject is at risk of infection.

[0295] Embodiment II-34. The method of embodiment 33, wherein the subject has received or will receive a vaccine for preventing the infection.

[0296] Embodiment II-35. The method of embodiment 34, wherein the administering results in an improved immune response to the vaccine.

[0297] Embodiment II-36. The method of embodiment 32, wherein the subject has or is at risk of developing cancer or a hematological malignancy.

[0298] Embodiment 11-37. The method of embodiment 36, wherein the administering results in an improved anti-cancer immune response.

[0299] Embodiment 11-38. The method of embodiment 32, wherein the subject has or is at risk of developing clonal hematopoiesis of indeterminate potential (CHIP).

[0300] Embodiment 11-39. The method of embodiment 38, wherein the administering results in reduced clonal expansion of HSCs.

[0301] Embodiment II-40. The method of embodiment 32, wherein the subject has or is at risk of developing a chronic inflammatory disorder.

[0302] Embodiment 11-41 . The method of embodiment 40, wherein the administering results in a reduction in at least one inflammaging marker, optionally wherein the inflammaging marker is basal circulating level of a cytokine selected from the group consisting of IL-1 a, CXCL5, IL1 RL1 , IL-23, IL-1b, CXCL2, IL-31 , IL-5, GM-CSF, and a combination thereof.

[0303] Embodiment II-42. The method of any one of embodiments 32-41 , wherein the subject is human.

[0304] Embodiment II-43. The method of any one of embodiments 32-42, wherein the subject is aged.

[0305] Embodiment II-44. The method of any of embodiments 32-43, wherein the agent reduces CCR5 activity, and / or blocks a binding interaction between CCR5 and CCL5.

[0306] Embodiment II-45. The method of any of embodiments 32-44, wherein the agent depletes CCR5+hematopoietic stem cells.

[0307] Embodiment II-46. The method of any of embodiments 32-45, wherein the agent is a CCR5 antagonist.

[0308] Embodiment II-47. The method of any of embodiments 32-45, wherein the agent is a CCL5 antagonist.

[0309] Embodiment II-48. The method of any of embodiments 32-45, wherein the agent is an antibody specific for CCR5.

[0310] Embodiment II-49. The method of embodiment 48, wherein the antibody is Leronlimab or a variant thereof, HGS004 or a variant thereof, or HGS101 or a variant thereof.

[0311] Embodiment II-50. The method any of embodiments 32-45, wherein the agent is a small molecule that inhibits CCR5.

[0312] Embodiment 11-51 . The method of embodiment 50, wherein the small molecule is selected from maraviroc, VCH-286, AZD-5672, AK-220, aplaviroc, vicriviroc and cenicriviroc.

[0313] Embodiment II-52. The method of any of embodiments 32-45, wherein the agent is a soluble peptide.

[0314] Embodiment II-53. The method of embodiment 52, wherein the peptide is selected from DAPTA, AOP-RANTES, PCS-RANTES, Met-RANTES, NNY-RANTES. vMIP-IL

[0315] Embodiment II-54. The method of any of embodiments 32-45, wherein the agent is an antibody specific for CCL5.

[0316] Embodiment II-55. The method of any of embodiments 32-45, wherein the agent is a CCR5 antagonist, and wherein the CCR5 antagonist is an RNAi; or wherein the agent is a CCL5 antagonist, and wherein the CCL5 antagonist is an RNAi.

[0317] Embodiment II-56. The method of any of embodiments 32-45, wherein the agent comprises genome editing of HSC, and wherein the genome editing results in decreased expression of CCR5 and / or CCL5.

[0318] Embodiment li-57. The method of any of embodiments 32-56, wherein the agent is administered in a dose effective to deplete CCR5+HSC.

[0319] Embodiment II-58. The method of any of embodiments 32-57, further comprising administering to the subject an agent that blocks CD47 interaction with SIRPa.

[0320] Embodiment II-59. The method of any of embodiments 32-58, further comprising administering to the subject an agent that specifically binds to CD1 17.

[0321] Embodiment II-60. The method of any of embodiments 32-59, further comprising administering an antibody specific for a second my-HSC specific marker.

[0322] Embodiment 11-61 . The method of embodiment 60, wherein the marker is selected from the group consisting of CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.

[0323] Embodiment li-62. The method of any of embodiments 32-61 , wherein following the administering step, there is an enrichment of bal-HSC to my-HSC of at least 1 .5-fold to at least 15-fold.

[0324] Embodiment li-63. The method of any of embodiments 32-62, wherein following the administering step, there is an enrichment of CCR5 HSC to CCR5+HSC of at least 1 .5-fold to at least 15-fold.

[0325] Embodiment li-64. The method of any of embodiments 32-63, wherein following the administering step, the ratio of the number of lymphoid progenitors in bone marrow of the subject to the number of myeloid progenitors in bone marrow of the subject is increased.

[0326] Embodiment li-65. The method of any of embodiments 32-64, wherein following the administering step the number of circulating naive T cells relative to the total circulating lymphocyte population is increased.

[0327] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.

[0328] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims

[0329] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

Claims

WHAT IS CLAIMED IS:1 . A method of rebalancing myeloid-biased hematopoietic stem cells (my-HSC) relative to balanced hematopoietic stem cells (bal-HSC) in the immune system of a mammalian subject, the method comprising: administering to the subject an agent that disrupts the CCL5-CCR5 axis.

2. The method of claim 1 , wherein the agent reduces CCR5 activity, and / or blocks a binding interaction between CCR5 and CCL5.

3. The method of claim 1 or claim 2, wherein the agent depletes CCR5+hematopoietic stem cells.

4. The method of any of claims 1 -3, wherein the agent is a CCR5 antagonist.

5. The method of any of claim 1 -3, wherein the agent is a CCL5 antagonist.

6. The method of any one of claims 1 -4, wherein the agent is an antibody specific for CCR5.

7. The method of claim 6, wherein the antibody is Leronlimab or a variant thereof, HGS004 or a variant thereof, or HGS101 or a variant thereof.

8. The method of claim 4, wherein the CCR5 antagonist is a small molecule that inhibits CCR5.

9. The method of claim 8, wherein the small molecule is selected from maraviroc, VCH- 286, AZD-5672, AK-220, aplaviroc, vicriviroc and cenicriviroc.

10. The method of claim 4, wherein the CCR5 antagonist is a soluble peptide.11 . The method of claim 10, wherein the peptide is selected from DAPTA, AOP-RANTES, PCS RANTES, Met-RANTES, NNY-RANTES, vMIP-ll.

12. The method of any one of claims 1 -3 and 5, wherein the agent is an antibody specific for CCL5.

13. The method of any one of claims 1 -3, wherein the agent is a CCR5 antagonist, and wherein the CCR5 antagonist is an RNAi; or wherein the agent is a CCL5 antagonist, and wherein the CCL5 antagonist is an RNAi.

14. The method of any one of claims 1 -3, wherein the agent comprises genome editing of HSC, and wherein the genome editing results in decreased expression of CCR5 and / or CCL5.

15. The method of any one of claims 1 -14, wherein the agent is administered in a dose effective to deplete CCR5+HSC.

16. The method of claim 15, further comprising administering to the subject an agent that blocks CD47 interaction with SIRPa.

17. The method of claim 15 or claim 16, further comprising administering to the subject an agent that specifically binds to CD1 17.

18. The method of any of claims 15-17, further comprising administering an antibody specific for a second my-HSC specific marker.

19. The method of claim 18, wherein the marker is selected from the group consisting of CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.

20. The method of any of claims 1 -19, wherein the subject is a human.21 . The method of any of claims 1 -20, wherein the subject is aged.

22. The method of any of the previous claims, wherein following the administering step, there is an enrichment of bal-HSC to my-HSC of at least 1 .5-fold to at least 15-fold.

23. The method of any of the previous claims, wherein following the administering step, there is an enrichment of CCR5 HSC to CCR5+HSC of at least 1 .5-fold to at least 15-fold.

24. The method of any of the previous claims, wherein following the administering step, the ratio of the number of lymphoid progenitors in bone marrow of the subject to the number of myeloid progenitors in bone marrow of the subject is increased.

25. The method of any of the previous claims, wherein following the administering step the number of circulating naive T cells relative to the total circulating lymphocyte population is increased.

26. The method of any one of the previous claims, wherein following the administering step a basal circulating level of an inflammaging marker is decreased, optionally wherein the inflammaging marker is selected from the group consisting of IL-1 a, CXCL5, IL1 RL1 , IL-23, IL- 1 b, CXCL2, IL-31 , IL-5, GM-CSF, and a combination thereof.

27. A method of rebalancing myeloid-biased hematopoietic stem cells (my-HSC) relative to balanced hematopoietic stem cells (bal-HSC) in a hematopoietic stem cell composition, the method comprising: depleting CCR5+HSC from the population.

28. The method of claim 27, wherein the depleting step is performed in vitro.

29. The method of claim 28, wherein the HSC population is administered to an individual in need thereof.

30. A composition for use in the methods of any of claims 1 -24.31 . A kit for use in the methods of any of claims 1-24.

32. A method for preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective dose of an agent that disrupts the CCR5-CCL5 axis, wherein the administering results in a reduction of myeloid-biased hematopoietic stem cells (my- HSC) relative to balanced hematopoietic stem cells (bal-HSC), thereby preventing or treating the disease or disorder.

33. The method of claim 32, wherein the subject is at risk of infection.

34. The method of claim 33, wherein the subject has received or will receive a vaccine for preventing the infection.

35. The method of claim 34, wherein the administering results in an improved immune response to the vaccine.

36. The method of claim 32, wherein the subject has or is at risk of developing cancer or a hematological malignancy.

37. The method of claim 36, wherein the administering results in an improved anti-cancer immune response.

38. The method of claim 32, wherein the subject has or is at risk of developing clonal hematopoiesis of indeterminate potential (CHIP).

39. The method of claim 38, wherein the administering results in reduced clonal expansion of HSCs.

40. The method of claim 32, wherein the subject has or is at risk of developing a chronic inflammatory disorder.41 . The method of claim 40, wherein the administering results in a reduction in at least one inflammaging marker, optionally wherein the inflammaging marker is basal circulating level of a cytokine selected from the group consisting of IL-1 a, CXCL5, IL1 RL1 , IL-23, IL-1 b, CXCL2, IL-31 , IL-5, GM-CSF, and a combination thereof.

42. The method of any one of claims 32-41 , wherein the subject is human.

43. The method of any one of claims 32-42, wherein the subject is aged.

44. The method of any of claims 32-43, wherein the agent reduces CCR5 activity, and / or blocks a binding interaction between CCR5 and CCL5.

45. The method of any of claims 32-44, wherein the agent depletes CCR5+hematopoietic stem cells.

46. The method of any of claims 32-45, wherein the agent is a CCR5 antagonist.

47. The method of any of claims 32-45, wherein the agent is a CCL5 antagonist.

48. The method of any of claims 32-45, wherein the agent is an antibody specific forCCR5.

49. The method of claim 48, wherein the antibody is Leronlimab or a variant thereof, HGS004 or a variant thereof, or HGS101 or a variant thereof.

50. The method any of claims 32-45, wherein the agent is a small molecule that inhibits CCR5.51 . The method of claim 50, wherein the small molecule is selected from maraviroc, VCH- 286, AZD-5672, AK-220, aplaviroc, vicriviroc and cenicriviroc.

52. The method of any of claims 32-45, wherein the agent is a soluble peptide.

53. The method of claim 52, wherein the peptide is selected from DAPTA, AOP-RANTES, PCS-RANTES, Met-RANTES, NNY-RANTES, vMIP-ll.

54. The method of any of claims 32-45, wherein the agent is an antibody specific for CCL5.

55. The method of any of claims 32-45, wherein the agent is a CCR5 antagonist, and wherein the CCR5 antagonist is an RNAi; orwherein the agent is a CCL5 antagonist, and wherein the CCL5 antagonist is an RNAi.

56. The method of any of claims 32-45, wherein the agent comprises genome editing of HSC, and wherein the genome editing results in decreased expression of CCR5 and / or CCL5.

57. The method of any of claims 32-56, wherein the agent is administered in a dose effective to deplete CCR5+HSC.

58. The method of any of claims 32-57, further comprising administering to the subject an agent that blocks CD47 interaction with SIRPa.

59. The method of any of claims 32-58, further comprising administering to the subject an agent that specifically binds to CD117.

60. The method of any of claims 32-59, further comprising administering an antibody specific for a second my-HSC specific marker.61 . The method of claim 60, wherein the marker is selected from the group consisting of CD150 (Slamfl ), CD61 (Itgb3), CD41 (Itga2b), CD62p, and NEO1.

62. The method of any of claims 32-61 , wherein following the administering step, there is an enrichment of bal-HSC to my-HSC of at least 1 .5-fold to at least 15-fold.

63. The method of any of claims 32-62, wherein following the administering step, there is an enrichment of CCR5 HSC to CCR5+HSC of at least 1 .5-fold to at least 15-fold.

64. The method of any of claims 32-63, wherein following the administering step, the ratio of the number of lymphoid progenitors in bone marrow of the subject to the number of myeloid progenitors in bone marrow of the subject is increased.

65. The method of any of claims 32-64, wherein following the administering step the number of circulating naive T cells relative to the total circulating lymphocyte population is increased.

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