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The majority of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC) (R/M) do not benefit from anti-PD-1 therapy. Hypoxia induced immunosuppression may be a barrier to immunotherapy. Therefore, we examined the metabolic effect of anti-PD-1 therapy in a murine MEER HNSCC model as well as intratumoral hypoxia in R/M patients. In order to characterize the tumor microenvironment in PD-1 resistance, a MEER cell line was created from the parental line that are completely resistant to anti-PD-1. These cell lines were then metabolically profiled using seahorse technology and injected into C57/BL6 mice. After tumor growth, mice were pulsed with pimonidazole and immunofluorescent imaging was performed to analyze hypoxia and T cell infiltration. To validate the preclinical results, we analyzed tissues from R/M patients (n=36) treated with anti-PD-1 mAb, via immunofluorescent imaging for number of CD8+ T cells (CD8), Tregs and the percent area (CAIX) and mean intensity (I) of carbonic anhydrase IX in tumor. We analyzed disease control rate (DCR), progression free survival (PFS), and overall survival (OS) using proportional odds and proportional hazards (Cox) regression. We found that anti-PD-1 resistant MEER has significantly higher oxidative metabolism, while there was no difference in glycolytic metabolism. Intratumoral hypoxia was significantly increased and CD8+ T cells decreased in anti-PD-1 resistant tumors compared with parental tumors in the same mouse. In R/M patients, lower tumor hypoxia by CAIX/I was significantly associated with DCR (p=0.007), PFS, and OS, and independently associated with response (p=0.028) and PFS (p=0.04) in a multivariate model including other significant immune factors. During PD-1 resistance, tumor cells developed increased oxidative metabolism leading to increased intratumoral hypoxia and a decrease in CD8+ T cells. Lower tumor hypoxia was independently associated with increased efficacy of anti-PD-1 therapy in patients with R/M HNSCC. To our knowledge this is the first analysis of the effect of hypoxia in this patient population and highlights its importance not only as a predictive biomarker but also as a potential target for therapeutic intervention.

Several non-redundant features of the tumour microenvironment facilitate immunosuppression and limit anticancer immune responses. These include physical barriers to immune infiltration, the recruitment of suppressive immune cells and the upregulation of ligands on tumour cells that bind to inhibitory receptors on immune cells. Recent insights into the importance of the metabolic restrictions imposed by the tumour microenvironment on antitumour T cells have begun to inform immunotherapeutic anticancer strategies. Therapeutics that target metabolic restrictions, such as low glucose levels, a low pH, hypoxia and the generation of suppressive metabolites, have shown promise as combination therapies for different types of cancer. In this Review, we discuss the metabolic aspects of the antitumour T cell response in the context of immune checkpoint blockade, adoptive cell therapy and treatment with oncolytic viruses, and discuss emerging combination strategies.Immunotherapeutic approaches to cancer can be affected by metabolic restrictions that limit the potency of anticancer T cell responses. In this Review, DePeaux and Delgoffe discuss the metabolic features of the tumour microenvironment that limit anticancer immune responses, as well as emerging therapeutic approaches to target these.

BackgroundOncolytic viruses (OVs) are an attractive way to increase immune infiltration into an otherwise cold tumor. While OVs are engineered to selectively infect tumor cells, there is evidence that they can infect other non-malignant cells in the tumor. We sought to determine if oncolytic vaccinia virus (VV) can infect lymphocytes in the tumor and, if so, how this was linked to therapeutic efficacy.MethodsTo investigate infection of lymphocytes by VV, we used a GFP reporting VV in a murine head and neck squamous cell carcinoma tumor model. We also performed in vitro infection studies to determine the mechanism and consequences of VV lymphocyte infection by VV.ResultsOur findings show that VV carries the capacity to infect proportions of immune cells, most notably T cells, after intratumoral treatment. Notably, this infection is preferential to terminally differentiated T cells that tend to reside in hypoxia. Infection of T cells leads to both virus production by the T cells as well as the eventual death of these cells. Using a mouse model which overexpressed the antiapoptotic protein Bcl2 in all T cells, we found that reducing T cell death following VV infection in MEER tumors reduced the number of complete regressions and reduced survival time compared with littermate control mice.ConclusionsThese findings suggest that OVs are capable of infecting more than just malignant cells after treatment, and that this infection may be an important part of the OV mechanism. We found that exhausted CD8+ T cells and regulatory CD4+ T cells were preferentially infected at early timepoints after treatment and subsequently died. When cell death in T cells was mitigated, mice responded poorly to VV treatment, suggesting that the deletion of these populations is critical to the therapeutic response to VV.

BackgroundCheckpoint blockade immunotherapy has dramatically changed cancer treatment; however, these therapies depend on the presence of a pre-existing immune infiltrate. Unfortunately, some patients have few to no infiltrating immune cells, highlighting the need for therapies that can generate antigenic stimuli. Oncolytic viruses, which infect and lyse tumor cells while leaving healthy tissue unharmed, are an attractive means to provide these signals, although the mechanisms of action of these engineered viral therapies remain incompletely understood. Virally induced immunogenic death causes an influx of tumor- and virus- specific effector CD8+ T cells. Many oncolytic viruses also decrease tumor-infiltrating suppressive immune populations, such as regulatory T cells (Treg), however the mechanism for this is unknown. Here we show that an oncolytic strain of vaccinia virus (VV) infects tumor infiltrating Tregs, in contrast to the prevailing idea that oncolytic viruses only infect tumor cells. Infection leads to viral-mediated Treg depletion that is required for tumor regression.MethodsUsing a mouse model of head and neck squamous cell carcinoma (MEER), a VV-resistant line was generated through serial treatment of a VV-sensitive MEER line. At varied time points post-intratumoral treatment with VV, tumor infiltrating lymphocytes (TIL) were isolated from both the VV-resistant and VV-sensitive lines and analyzed by flow cytometry.ResultsOne day post-treatment of VV-sensitive MEER tumors, tumor isolated Tregs were infected by VV as determined by viral GFP expression. Infection was confirmed in vitro with purified Tregs. Four days post-treatment, tumor infiltrating Treg counts were reduced, and active caspase 3 staining was increased, suggesting that infection lead to Treg death. At 7 days post-treatment, the remaining Tregs in the VV-sensitive tumors acquired a fragile phenotype (IFN?+ Nrp1-). This was not observed in the VV-resistant MEER line. Fragile Tregs are less suppressive and indeed we observed an increase in pro-inflammatory cytokine production from CD8+ and Tconv (CD4+ Foxp3-) T cells in the VV-sensitive tumors compared to VV-resistant. We then engineered oncolytic VV to be susceptible to Cre mediated inactivation. Infection of various murine transgenic Cre lines confirmed the importance of non-tumoral immune infection for therapeutic efficacy, with a particular emphasis on Treg infection.ConclusionsThese data reveal a previously unappreciated mechanism of action of oncolytic virus immunotherapy, in which new tumor immunity accompanies the viral mediated loss and phenotypic change of regulatory populations. Importantly, as this treatment is delivered intratumorally the loss of Tregs is tumor specific, resulting in targeted Treg deletion without systemic autoimmunity.

BackgroundWhile CD8+ cytotoxic T cells are clearly critical for identification and elimination of cancer cells, factors concentrated within the tumor microenvironment drive altered differentiation of these cells to a hypofunctional, short-lived state termed T cell exhaustion1 (figure 1a). Exhaustion is a progressive lineage, and it is now clear that terminally exhausted T (tTexh) cells are not the targets of checkpoint blockade immunotherapy but may serve as factors that limit immunotherapeutic efficacy.2–6 Compared directly, tumor-infiltrating CD8+ tTexh cells bear notable phenotypic similarity to CD4+Foxp3+ regulatory T (Treg) cells in expression of immunosuppressive molecules suggesting beyond loss of effector function, tTexh cells may be directly anti-functional and constrain tumor-specific immunity. Thus, we hypothesize that tTexh cells potentiate the suppressive microenvironment of solid tumor and that strategies to limit their generation or reprogram their immunosuppressive nature will improve control of tumor progression.MethodsT cell populations were isolated from murine tumor lines, B16-F10 melanoma, Ptenflox/floxBrafLSL.V600ETyr2Cre.ERT2–derived Clone 24 melanoma, MEER head and neck carcinoma, and MC38 adenocarcinoma. T cell-specific CD39 (Entpd1) deletion was accomplished by crossing Entpd1flox/flox mice to Cd4Cre or E8iGFP-Cre-ERT2. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery. Tumor hypoxia was alleviated by CRISPR-Cas9-directed deletion of mitochondrial genes in B16-F10 or by treatment with axitinib or metformin.ResultsWhen sorted directly from tumor, CD8+PD-1hiTim-3+ tTexh cells, but not progenitor PD-1intTim-3– pTexh cells, induce marked suppression of T cell effector responses, comparable to CD4+Foxp3+ Treg cells from the same environment (figure 1b-c). The ectonucleotidase, CD39, increases as cells progressively differentiate and is associated with terminal exhaustion.7 8 CD8+ T cell-restricted deletion of CD39 restricts regulatory functions of tTexh cells (figure 1b), improving tumor control and augmenting response to checkpoint blockade (figure 1d). CD39 expression correlates with hypoxia exposure and tTexh cells sorted from tumors engineered to be less hypoxic or treated with hypoxia-mitigating agents displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, supports adenosine generation, and limits therapeutic efficacy.ConclusionsOur data support a model that as CD8+ T cells progress to terminal exhaustion, hypoxia exposure enforces the upregulation of CD39, providing tTexh cells a mechanism to suppress proinflammatory processes and promote tumor progression. These findings suggest tTexh cells are not solely dysfunctional but rather are deleterious to antitumor immunity and may need to be drastically reprogrammed or depleted to improve patient outcomes.ReferencesBlank CU, et al. Defining “T cell exhaustion”. Nat Rev Immunol 2019;19:665–674.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20:326–336.Blackburn SD, et al. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad Sci USA 2008;105:15016–15021.Sade-Feldman M, et al. Defining T Cell states associated with response to checkpoint immunotherapy in Melanoma. Cell 2018;175:998–1013.e20.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Siddiqui I, et al. Intratumoral Tcf1+PD-1+CD8+ T Cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 2019;50:195–211.e10.Canale FP, et al. CD39 expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11:e1005177.Abstract 679 Figure 1(a) Schematic depicting differentiation of CD8+ T cells to terminal exhaustion in cancer and subsequent suppression of local immune responses by expression of ectonucleosidase, CD39; (b) When assayed directly ex vivo, CD8+ terminally exhausted T (tTexh) cells, but not progenitor exhausted T (pTexh) cells, suppress effector functions as effectively as CD4+Foxp3+ Treg isolated from the same environment. Deletion of CD39 alleviates tTexh-mediated suppression; (c) CD8+ T cell suppression correlates with expression of CD39 on tTexh from various tumor models. (d) CD8+ T cell-specific deletion of CD39 slows tumor growth and improves immune response to checkpoint blockade-resistant tumors. Data are pooled from ≥3 experiments. Statistics are two-way ANOVA with multiple comparisons or Pearson correlation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

BackgroundOncolytic viruses are an underappreciated immunotherapy capable of inflaming the tumor microenvironment (TME), vaccinating a patient against their own tumor, and delivering gene therapy to the TME. However, apart from the oncolytic HSV T-vec, these therapies have not seen widespread use, due in part to incomplete understanding of their immunologic mechanisms of action. We sought to determine features of oncolytic vaccinia virus (VV) response and resistance using subclones of the HPV+ head and neck cancer model MEER rendered sensitive or resistant to VV.MethodsA VV sensitive MEER tumor resisting treatment was serially passaged in mice and treated with VV until a stably resistant line was generated (Fig1). Sensitive or resistant MEER tumors were implanted, treated with a single intratumoral dose of VV, and harvested 4–7 days later for cytometric analysis. A genetically encoded TGFβ inhibitor was recombined into oncolytic VV (VV-TGFβi).ResultsWe used serial in vivo passaging to generate a VV-resistant MEER line (MEERvvR) from one sensitive to VV (MEERvvS, figure 1) and compared their immune infiltrate. While VV promoted acute cytokine production and cytotoxicity in conventional T cells, the major determining factor between sensitivity and resistance was the phenotype of Treg cells. At baseline, Treg cells in MEERvvS had lower Nrp1 expression and higher IFNγ-STAT1 signaling compared to MEERvvR, indicative of Treg 'fragility'. VV treatment induced MEERvvS Treg cells to become immunostimulatory and produce IFNγ (figure 2). RNAseq revealed MEERvvR produced more TGFβ than MEERvvS cells, suggesting these tumors directly stabilize Treg cells. To determine if MEERvvR could be sensitized to VV, we engineered oncolytic vaccinia to produce a genetically-encoded TGFβ inhibitor which binds TGFβRII, preventing TGFβ1-3 binding (VV-TGFβi). When MEERvvR were treated with VV-TGFβi, elite responses were restored, with commensurate increase in survival (figure 3) associated with increased STAT1 signaling in Treg cells.ConclusionsResistance to oncolytic vaccinia is controlled by Treg cell phenotype; tumors harboring more fragile Treg cells respond exquisitely to VV. An oncolytic vaccinia engineered to produce a novel TGFβi could remodel the TME to be less supportive of Tregs, rendering resistant tumors sensitive to VV. Our data highlight the importance of Treg cell status in resistance to oncolytic virus therapy and suggest TGFβ can be effectively targeted through an inhibitor encoded within the virus. Importantly, this TME directed production of the TGFβi carries no toxicity previously associated with systemic TGFβ inhibition, suggesting a viral approach to TGFβ inhibition can be an effective strategy support broader immunotherapy response.Abstract 743 Figure 1Strategy used to generate a vaccinia resistant MEER (MEERvvR) from vaccinia sensitive MEER (MEERvvS)Abstract 743 Figure 2IFNγ production in Treg cells in MEERvvS and MEERvvR after treatment with PBS or control vaccinia (VV-Ctrl)Abstract 743 Figure 3Survival of VV-resistant MEER treated with PBS, control vaccinia (VV-Ctrl), or vaccinia engineered to deliver a potent inhibitor of TGFβ (VV-TGFβi)

BackgroundBlockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors; yet only in a subset of patients.1–3 Limited success with checkpoint blockade therapy suggests other cell extrinsic or intrinsic mechanisms may be dampening an effective immune response. Cytotoxic CD8+ T cells (CTL) encountering chronic antigen and metabolic restriction can differentiate to a terminally exhausted state (Texh), marked by hyporesponsiveness and metabolic, epigenetic, and transcriptional dysfunction.4–8 While enrichment of this population in tumor is a negative prognostic factor,9–10 it remains unclear whether Texh are simply non-functional or instead possess tolerogenic or suppressive properties. Transcriptional profiling of tumor-infiltrating PD-1int (progenitor exhausted) CTL versus PD-1hiTIM-3+ (terminally exhausted; Texh), reveals that exhausted cells express a pattern of genes associated with immune suppression. We hypothesize that Texh potentiate the suppressive microenvironment of solid tumor by autoregulation and inhibition of local immune responses.MethodsT cell populations were isolated from murine melanoma–B16-F10 or a lab-generated melanoma clone of the spontaneous BREF/PTEN model–by expression of inhibitory receptors and assayed in tandem in microsuppression assays. Murine melanoma clones with inhibited oxidative metabolism were generated by CRISPR-Cas9 deletion and validated for ablated mitochondrial respiration by extracellular flux analysis. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery.ResultsWhen sorted directly from tumor, PD-1hiTim3+ Texh, but not progenitor exhausted PD-1int CTL, induce marked suppression of T cell effector responses, comparable to Foxp3+ Treg from the same environment. Expression of the ectonucleotidase, CD39, is uniquely expressed in Texh and increases as T cells differentiate towards exhaustion. Genetic deletion of CD39 in Texh eliminates the regulatory phenotype of tumor-infiltrating Texh and enforced CD39 expression on effector T cells can inhibit T cell receptor signaling and downstream function. CD39 expression correlates with exposure to hypoxia and Texh sorted from tumors engineered to be less hypoxic displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, contributes to generation of immunosuppressive adenosine, and has been previously associated with terminal exhaustion.11–13 ConclusionsOur data support a model that as CTL progress to terminal exhaustion, hypoxic exposure enforces the upregulation of CD39, providing Texh a mechanism to suppress proinflammatory processes. These findings suggest Texh are not solely dysfunctional but rather are deleterious to anti-tumor immunity and may need to be drastically reprogrammed or deleted in order to alleviate immunosuppressive functions.ReferencesWolchok JD. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med 2017; 377, 1345–1356.Hellmann MD, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an open-label, phase 1, multicohort study. Lancet Oncol 2017; 18, 31–41.Robert C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015; 372, 2521–2532.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Blackburn SD, Shin H, Freeman GJ & Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc. Natl. Acad. Sci 2008;105:15016–15021.Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165.Najjar YG, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019; 4.Loo K, et al. Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy. JCI Insight 2017; 2.Daud AI, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J. Clin. Invest 2016;126:3447–3452.. Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun 2018;9:2724.Canale FP, et al. CD39 Expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11, e1005177.

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8 + T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell–intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates. Delgoffe and colleagues show that continuous TCR signaling and hypoxia increase Blimp-1, which suppresses PGC-1α-dependent mitochondrial reprogramming and increases reactive oxygen species generation. Such conditions promote T cell exhaustion.

Regulatory T (T reg ) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells 1 , 2 . Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME 3 , which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function 4 – 6 . At the same time, T reg cells maintain a strong suppression of effector T cells within the TME 7 , 8 . As previous studies suggested that T reg cells possess a distinct metabolic profile from effector T cells 9 – 11 , we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral T reg cells are linked. Here we show that T reg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of T reg cells in vitro. T reg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. T reg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1—a lactate transporter—in T reg cells reveals that lactate uptake is dispensable for the function of peripheral T reg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, T reg cells are metabolically flexible: they can use ‘alternative’ metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations. The tumour microenvironment is low in glucose and high in the alternative metabolite lactate, which regulatory T cells are shown here to use, maintaining their ability to suppress effector immune cells.

CD8 + T cells are critical for elimination of cancer cells. Factors within the tumor microenvironment (TME) can drive these cells to a hypofunctional state known as exhaustion. The most terminally exhausted T (tT ex ) cells are resistant to checkpoint blockade immunotherapy and might instead limit immunotherapeutic efficacy. Here we show that intratumoral CD8 + tT ex cells possess transcriptional features of CD4 + Foxp3 + regulatory T cells and are similarly capable of directly suppressing T cell proliferation ex vivo. tT ex cell suppression requires CD39, which generates immunosuppressive adenosine. Restricted deletion of CD39 in endogenous CD8 + T cells resulted in slowed tumor progression, improved immunotherapy responsiveness and enhanced infiltration of transferred tumor-specific T cells. CD39 is induced on tT ex cells by tumor hypoxia, thus mitigation of hypoxia limits tT ex suppression. Together, these data suggest tT ex cells are an important regulatory population in cancer and strategies to limit their generation, reprogram their immunosuppressive state or remove them from the TME might potentiate immunotherapy. Exhausted CD8 + T cells with diminished effector functions accumulate in tumors. Here, the authors show that hypoxia induces a suppressive phenotype in exhausted T cells and that interfering with hypoxia-mediated CD39 expression limits immunosuppression in the tumor and augments immunotherapy, resulting in arrest of tumor growth.