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The inhibitory receptor PD-1 suppresses T cell activation by recruiting the phosphatase SHP-2. However, mice with a T-cell-specific deletion of SHP-2 do not have improved antitumor immunity. Here we showed that mice with conditional targeting of SHP-2 in myeloid cells, but not in T cells, had diminished tumor growth. RNA sequencing (RNA-seq) followed by gene set enrichment analysis indicated the presence of polymorphonuclear myeloid-derived suppressor cells and tumor-associated macrophages (TAMs) with enriched gene expression profiles of enhanced differentiation, activation and expression of immunostimulatory molecules. In mice with conditional targeting of PD-1 in myeloid cells, which also displayed diminished tumor growth, TAMs had gene expression profiles enriched for myeloid differentiation, activation and leukocyte-mediated immunity displaying >50% overlap with enriched profiles of SHP-2-deficient TAMs. In bone marrow, GM-CSF induced the phosphorylation of PD-1 and recruitment of PD-1-SHP-2 to the GM-CSF receptor. Deletion of SHP-2 or PD-1 enhanced GM-CSF-mediated phosphorylation of the transcription factors HOXA10 and IRF8, which regulate myeloid differentiation and monocytic-moDC lineage commitment, respectively. Thus, SHP-2 and PD-1-SHP-2 signaling restrained myelocyte differentiation resulting in a myeloid landscape that suppressed antitumor immunity.

BK virus (BKV), a human polyomavirus that remains latent in renal epithelial cells, can be reactivated after hematopoietic stem cell transplantation (HSCT) leading to hemorrhagic cystitis. The incidence of BK viremia is higher after Umbilical cord blood transplantation (UCBT) than HSCT from adult donors. Data regarding the role of immune recovery after UCBT in BKV reactivation is lacking. We examined the correlation between the development of BK viremia and immune reconstitution in 27 adult recipients of UCBT. The incidence of BK viremia was 52% and developed most frequently within the first 8 weeks after the transplantation, but persisted in seven patients at 6 months, and three patients at 1-year post UCBT. Detection of BK viremia 1 year after transplant was negatively associated with the number of CD8+ cells (p = 0.03) and CD8+CD45RO+ cells (p = 0.05) at 6 months, and the number of CD4+ (p = 0.03) and CD4+CD45RO+ cells (p = 0.03) at 12 months after UCBT. Conversely, BK viremia at 6 and 12 months was positively correlated with the number of T regulatory (Treg) cells at 1 month (p = 0.005 and p = 0.016, respectively). Because UCB Treg have highly potent immunosuppressive function, our findings indicate that sustained BK viremia in UCBT recipients might be associated with the increase of Treg cells early after transplantation, which mediate impaired and delayed reconstitution of CD4+ and CD8+ T effector cells.

Immune checkpoint therapies aiming to enhance T cell responses have revolutionized cancer immunotherapy. However, although a small fraction of patients develops durable anti-tumor responses, the majority of patients display only transient responses, underlying the need for finding auxiliary approaches. Tumor microenvironment poses a major metabolic barrier to efficient anti-tumor T cell activity. As it is now well accepted that metabolism regulates T cell fate and function, harnessing metabolism may be a new strategy to potentiate T cell-based immunotherapies.

Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to nuclear hormone superfamily, with three distinct types identified: PPARapha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus play critical roles in the differentiation and fate of immune cells. PPARα is involved in lipid and carbohydrate metabolism and PPARα agonists, such as fibrates, have been used for the treatment of hypertriglyceridemia and cardiovascular diseases. PPARα has an anti-inflammatory role during infection, and similar to PPARγ, affects the polarization of macrophages. In acute myelogenous leukemia (AML), PPARα mutations correlate with chemoresistance, poor treatment outcomes and unfavorable prognosis. In experimental tumor models, it has been proposed that PPARα agonists might enhance anti-tumor T cell responses during PD-1 blocking immunotherapy. To dissect the mechanistic role of PPARα in tumor immunity, we used mice with global deletion of PPARα and examined tumor growth and profile of the immunological landscape, using various syngeneic tumor models. Significantly larger B16-F10 melanoma and MC-17 fibrosarcoma tumors were observed in PPARα KO mice compared with wild-type control, suggesting that PPARα deletion attenuated the immunological response against cancer. To dissect the role of PPARα in key populations of the innate and adaptive immune system involved in anti-tumor responses, we analyzed the immunological landscape of tumor, tumor draining lymph nodes (TDLN) and spleen, 14-16 days after tumor implantation. Assessment of CD4 + and CD8 + T cells, CD11b +F4/80 + tumor-associated macrophages (TAMs), CD11b +Ly6C hiLy6G - monocytic myeloid derived suppressor cells (M-MDSC), and CD11b +Ly6C loLy6G + polymorphonuclear myeloid derived suppressor cells (PMN-MDSC), by using flow cytometry, showed no quantitative differences between the two experimental groups. Functionally, MDSC from PPARα KO and WT mice showed comparable immunosuppressive properties as determined by suppression assay using splenocytes from OTI transgenic mice. However, PPARα KO TAMs demonstrated a less activated state, as determined by the lower expression levels of MHC-II that is critical for antigen presentation, and CD86 that is critical for T cell costimulation and prevention of T cell anergy and exhaustion. In agreement with these properties of TAMs, CD4 + T cells from TDLN of PPARα KO mice had diminished expression of activation markers, including PD-1, PD-L1 and ICOS, and numerically decreased central memory-like CD4 + T cells (T CM), compared to control tumor bearing mice. Furthermore, CD69, an emerging marker of T cell exhaustion, was significantly upregulated in CD4 + and CD8 + T cells from the TDLN of PPARα KO mice. To determine whether PPARα ablation altered the cell intrinsic properties of myeloid cells and/or T cells resulting in impaired anti-tumor function, we examined in vitro responses of isolated populations. In response to activation via TCR/CD3 and CD28, PPARα deficient T cells had no significant differences in expansion and cytokine production compared to control. In contrast, PPARα deficient Ly6C + monocytes isolated from the bone marrow displayed diminished responses to TLR-mediated signaling as determined by production of IL-6 and TNFα. Our in vitro and in vivo findings reveal a dominant role of PPARα in regulating the fate of innate immune cells thereby altering T cell responses and anti-tumor function. Our findings have implications for the development of new therapeutic approaches to enhance innate immune cell function for the improvement of cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

PD-1 is a T cell inhibitor for which blocking agents have achieved success as anti-cancer therapeutics. The current view is that cancer limits host immune responses by upregulating PD-L1 in the tumor microenvironment thereby causing PD-1 ligation and inactivation of CD8+ Teff cells. Recently, we determined that PD-1 alters the differentiation of myeloid progenitors during cancer-mediated emergency myelopoiesis. We found that PD-1 is expressed in granulocyte/macrophage progenitors (GMP), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSC) that promote tumor growth. In tumor-bearing mice with myeloid-specific PD-1 ablation, accumulation of GMP and MDSC was prevented, while output of effector myeloid cells was increased. PD-1-mediated T cell inactivation is attributed to the function of SHP-2 phosphatase, which is activated by recruitment to PD-1 cytoplasmic tail. Temporal activation of SHP-2 is critical for myeloid cell fate. Activating SHP-2 mutations prevent myeloid cell differentiation and lead to the accumulation of immature myelocytes and development of leukemia. To determine whether PD-1-mediated inhibition of anti-tumor immunity relies on SHP-2-mediated effects in T cells or myeloid cells, we generated mice with conditional targeting of the Ptpn11 gene (encoding for Shp-2) and selectively eliminated Shp-2 in T cells (Shp-2fl/flLckCre) or myeloid cells (Shp-2fl/flLysMCre). No significant difference in tumor growth was observed between control Shp2fl/fl and Shp-2fl/flLckCre mice bearing B16-F10 melanoma. Strikingly, Shp-2fl/flLysMCre mice had significantly diminished tumor growth that was not further decreased by anti-PD-1 antibody, in contrast to control Shp-2fl/fl mice in which anti-PD-1 treatment significantly reduced tumor size. To determine how Shp-2 ablation affected the properties of myeloid cells, we examined CD11b+Ly6ChiLy6G- monocytic (M-MDSC), CD11b+Ly6CloLy6G+ polymorphonuclear (PMN-MDSC), CD11b+F4/80+ tumor-associated macrophages (TAM) and CD11c+MHCII+ dendritic cells (DC). No quantitative differences were observed in these myeloid subsets in tumor bearing mice among the different groups. However, M-MDSC from Shp-2fl/flLysMCre mice had elevated expression of CD86 and IFNγ, consistent with effector differentiation. Suppression assays, by measuring antigen-specific responses of OTI transgenic T cells, showed significantly attenuated suppressor function of MDSC isolated from tumor-bearing Shp-2f/fLysMCre mice compared to control or Shp-2f/fLckCre mice. CD38 is a key mediator of MDSC-mediated immunosuppression. It is an ADP-ribosyl cyclase that has ectoenzyme and receptor functions, is induced early during differentiation of myeloid progenitors by retinoic acid receptor alpha (RARα) signaling, and mediates T cell immunosuppression. Because Shp-2 is involved in the differentiation of myeloid progenitors, we examined CD38 expression. We found that expression of CD38 was significantly reduced in MDSC from Shp-2fl/flLysMCre mice compared to control and Shp-2fl/flLckCre-tumor bearing mice. Since the suppressive potency of MDSC is decreased by autophagy, and SHP-2 has been implicated in regulating autophagy in cancer cells, we examined autophagy of MDSC in our system. Assessment of autophagy in ex vivo isolated MDSC, using Cyto-ID that stains the autophagosome membrane and indicates autophagic activity, showed enhanced autophagy in MDSC isolated from tumor bearing Shp-2fl/flLysMCre mice compared to control or Shp-2fl/flLckCre mice. Enhanced autophagy was also detected in bone marrow-derived MDSC from Shp-2fl/flLysMCre mice as determined by accumulation of LC3B-II and p62 during culture under conditions of starvation-induced stress. Consistent with the diminished MDSC suppressor function, myeloid cell-specific Shp-2 ablation in tumor-bearing mice induced an increase of CD8+ T cells showing an effector phenotype with improved functionality, despite preserved expression of PD-1 and Shp-2. Together these results indicate that inhibition of PD-1-mediated SHP-2 activation in myeloid progenitors, thereby preventing the accumulation of immature immunosuppressive MDSC and promoting the differentiation of effector myeloid cells, might be a previously unidentified mechanism by which PD-1 blockade mediates anti-tumor function. Disclosures No relevant conflicts of interest to declare.

PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell-specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1-blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell-specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell-specific PD-1 ablation, myeloid cell-specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell-specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1-deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.

Umbilical cord blood transplantation (UCBT) is an alternative for patients who need hematopoietic stem cell transplant (HSCT), but lack HLA-matched adult donors. Rabbit anti-thymoglobulin (ATG) has been used in UCBT conditioning to achieve T cell depletion, but ATG-induced immunosuppression is associated with delayed immune reconstitution, increased infectious complications and higher non-relapse mortality. In a clinical trial of reduced intensity double-unit UCBT (dUCBT), we substituted low dose total body irradiation (TBI) for ATG to determine whether dUCBT without ATG would alter kinetics and quality of immune reconstitution. Thirty-one patients with hematopoietic malignancies and a median age of 58 yr were treated with Flu/Mel/TBI, followed by dUCBT and GVHD prophylaxis with tacrolimus and sirolimus. We examined reconstitution of immune cell populations, thymic regeneration by quantifying T cell receptor excision circles (TREC) and serum cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-γ, TNF-α, IL-12p70, GM-CSF) using the LUNARISTM Human 11-Plex Cytokine Biochip384 from AYOXXA Biosystems. Assessments were done prior to and at 1, 2, 3, 6, 12 and 24 months after dUCBT. Results are based on 28 evaluable patients. The 2-yr overall survival and progression-free survival were 53% and 48%. Median time to neutrophil and platelet engraftment was 25 and 52 days, respectively. Before UCBT, the median values for most leukocyte subsets were below normal limits, except for monocytes. CD3+ cells remained depressed until 2 months post-transplant when gradually began to re-emerge. However, CD4+ and CD8+ subsets had distinct reconstitution kinetics (Figure 1). CD4+ T cells declined at 1 month but gradually increased and exceeded pre-transplant levels by 9 months after UCBT. In contrast, at 9 months, CD8+ lymphocytes remained depressed to 50% of pre-transplant levels, but increased thereafter and reached normal values by 2-yr. NK cells and monocytes reached normal values at 3 months post-UCBT. B cells were mostly undetectable for the first 6 months, followed by a 10-fold increase at 9 months and exceeded the upper normal limit by 2-yr. TREC, which were within normal range before transplant, decreased after UCBT but remained detectable between 1-6 months and recovered to normal levels by 9 months. Among cytokines, only IL-8, IL-6 and TNF-α displayed significant changes. IL-8 and IL-6 peaked at day 100 and 9 months, and subsequently declined. In contrast, TNF-α increased by day 100 and remained elevated thereafter. To evaluate functional immunity, we assessed correlations between viral reactivation and reconstitution of immune cell populations and thymic function. Nineteen patients experienced 24 clinically significant viral reactivations or infections, with 1-year cumulative incidence of 62%, which was comparable to 53% observed in dUCBT cohorts receiving ATG-containing conditioning. Although there was no difference in CMV, EBV, adenovirus and VZV reactivation, there was a significant increase in the incidence of HHV-6 reactivation. HHV-6 viremia was observed in 24 of 28 (86%) patients during the first month after dUCBT. Six of these 24 (25%) patients developed HHV-6-related encephalitis. There was a correlation between development of encephalitis and HHV-6 viral load ≥50.000 copies/ml (p=0.007). Pre-transplant TREC levels ≥1.200 copies/ml negatively correlated with subsequent HHV-6 reactivation (p=0.04), indicating that baseline reserve of thymic function has a significant role in post-transplant immune reconstitution. On days 30, 60 and 100 post-transplant, higher TREC levels correlated with lack of HHV-6 viremia (p Figure 1 Disclosures Defilipp: Incyte: Research Funding. Politikos:Angiocrine Bioscience Inc: Research Funding. Armand:Otsuka: Research Funding; Roche: Research Funding; Affimed: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Infinity: Consultancy; Sigma Tau: Research Funding; ADC Therapeutics: Consultancy; Tensha: Research Funding; Genentech: Research Funding; Pfizer: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Koreth:Amgen: Consultancy; Equillium: Consultancy; Cugene: Consultancy. Avigan:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexel: Consultancy; Takeda: Consultancy. Rosenblatt:Celgene: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Parexel: Consultancy; Imaging Endpoint: Consultancy; Partner Tx: Other: Advisory Board; Dava Oncology: Other: Education; BMS: Research Funding. Chen:Abbvie: Consultancy; Incyte: Consultancy; Magenta: Consultancy; Takeda: Consultancy; Kiadis: Consultancy. Soiffer:Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Jazz: Consultancy; Cugene: Consultancy. Cutler:Kadmon: Consultancy; Incyte: Consultancy; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Aleta Biotherapeutics: Consultancy; Celgene: Consultancy; Avrobio: Consultancy; LifeVault Bio: Consultancy; Draper Labs: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.