Cell-mediated Control Research Articles

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils

ABSTRACT High-grade serious ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here, we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ~ 50% of the treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

Syndecan-1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD1 checkpoint immunotherapy.

Tumor cell-originated events prevent efficient antitumor immune response and limit the application of anti-PD1 checkpoint immunotherapy. We show that syndecan-1 (SDC1) has a critical role in the regulation of T cell-mediated control of tumor growth. SDC1 inhibition increases the permeation of CD8+ T cells into tumors and triggers CD8+ T cell-mediated control of tumor growth, accompanied by increased proportions of progenitor-exhausted and effector-like CD8+ T cells. SDC1 deficiency alters multiple signaling events in tumor cells, including enhanced IFN-γ-STAT1 signaling, and augments antigen presentation and sensitivity to T cell-mediated cytotoxicity. Combinatory inhibition of SDC1 markedly potentiates the therapeutic effects of anti-PD1 in inhibiting tumor growth. Consistently, the findings are supported by the data from human tumors showing that SDC1 expression negatively correlates with T cell presence in tumor tissues and the response to immune checkpoint blockade therapy. Our findings suggest that SDC1 inhibits antitumor immunity, and that targeting SDC1 may promote anti-PD1 response for cancer treatment.

Multi-omics analysis of SIV-specific CD8+ T cells in multiple anatomical sites.

CD8+ T cells exert immunological pressure against immunodeficiency lentiviruses. In previous studies, we examined the TCR repertoire of CD8+ T cells specific for a single SIV immunodominant epitope, Gag-CM9, throughout SIV infection or after vaccination, and across multiple anatomic sites. We identified both tissue specific TCR sequences and TCRs shared by multiple anatomical sites. Here we use single cell RNA sequencing to evaluate if the tissue localization or TCR sequence of a CM9-specific CD8+ T cell corresponds with unique transcriptomics. CM9-specific CD8+ T cells were sorted from blood, lymph nodes, spleen, and liver from SIV infected rhesus macaques with progressive SIV infection and in animals who spontaneously control SIV replication after cessation of antiretroviral therapy. The cells were processed through a single cell sequencing protocol, creating a TCR amplified library and an RNA gene expression library corresponding to individual cells. Gene set enrichment analysis revealed no distinct transcriptional profiles for CM9 specific CD8+ T cells between different anatomical sites and between cells with shared or tissue specific TCRs. Similarly, no clear transcriptional profiles were associated with clonotypes which were shared across individual animals. However, CM9 specific CD8+ T cells from posttreatment controllers did exhibit enrichment of pathways associated with cellular activation compared to progressively infected animals, suggesting that altered transcription in distinct cellular pathways in antigen specific CD8+ T cells may associate with viral control. Together, these studies represent a thorough analysis of the relationship between anatomical and clonal origin, and the transcriptional profile of antigen specific CD8+ T cells and unravel pathways that may be important for CD8+ T cell mediated control of SIV replication.

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ∼50% of treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

Type I IFN signaling in the absence of IRGM1 promotes M. tuberculosis replication in immune cells by suppressing T cell responses

Polymorphisms in the IRGM gene are associated with susceptibility to tuberculosis in humans. A murine ortholog of Irgm, Irgm1, is also essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. Multiple processes have been associated with IRGM1 activity that could impact the host response to Mtb infection, including roles in autophagy-mediated pathogen clearance and expansion of activated T cells. However, what IRGM1-mediated pathway is necessary to control Mtb infection in vivo and the mechanistic basis for this control remains unknown. We dissected the contribution of IRGM1 to immune control of Mtb pathogenesis in vivo and found that Irgm1 deletion leads to higher levels of IRGM3-dependent type I interferon signaling. The increased type I interferon signaling precludes T cell expansion during Mtb infection. The absence of Mtb-specific T cell expansion in Irgm1−/− mice results in uncontrolled Mtb infection in neutrophils and alveolar macrophages, which directly contributes to susceptibility to infection. Together, our studies reveal that IRGM1 is required to promote T cell-mediated control of Mtb infection in neutrophils, which is essential for the survival of Mtb-infected mice. These studies also uncover new ways type I interferon signaling can impact TH1 immune responses.

Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr), but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a, and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner and is associated with effective parasite control during chronic stage. Conditional invalidation of Transporter associated with Antigen Processing (TAP)-mediated major histocompatibility complex (MHC) class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulates the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections.

Abstract 2188: Tirzepatide treatment restores antitumor immunity in a model of obesity-driven cancer

Abstract Obesity is a widely and increasingly prevalent disease and a risk factor for several cancers. Recent work has shown that weight loss following bariatric surgery is potently protective against obesity-associated cancers. However, little is known about the potential anticancer effects of weight loss achieved through incretin-based therapies. We therefore interrogated the effects of the dual glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptor agonist tirzepatide (TZP) on body weight and tumor progression in a mouse model of obesity and transplanted colon cancer cells. Moreover, we tested whether TZP-mediated weight loss can reverse obesity-associated suppression of CD8+ T cell immunity. Male C57BL/6J mice were fed either 60 kcal% fat diet (D12492) or a sucrose-matched 10 kcal% fat diet (D12450J) for ≥15 weeks to generate diet induced obese (DIO) or control mice, respectively. Once DIO mice reached a body weight of 47 g they were randomized to begin TZP treatment or receive a vehicle control every other day for 3 weeks (when stable body weights were reached for TZP mice). All mice then received subcutaneous injections with 2.5 × 105 MC38 cells and continued to receive TZP or vehicle. To determine the role of CD8+ T cell mediated tumor control in driving the effects of TZP treatment, mice received either CD8-depleting antibodies or an isotype control at days -2, 0, 2 relative to tumor cell injection and subsequently every 4 days. Tumor mass was determined by digital scale. Fasting blood glucose, body weight loss, and body composition were monitored to determine the metabolic effects of TZP. Immune cell populations were measured by full spectrum flow cytometry. We found TZP reduced body weight, fasting blood glucose, and adiposity relative to DIO mice, as was expected. These metabolic improvements were accompanied by reduction of tumor mass in TZP mice relative to DIO mice. DIO mice had increased levels of exhausted T cells which were normalized by TZP treatment. Depletion of CD8+ cells did not alter the tumor mass of DIO mice, indicating negligible CD8+ T cell-mediated tumor control. In contrast, CD8+ cell depletion in control and TZP mice increased in tumor masses to the levels comparable to those of DIO mice. These results demonstrate that TZP can mitigate obesity-associated acceleration of tumor growth and restore markers of effective antitumor T cell immunity. Critically, we find that restoration of CD8+ T cell function is essential for TZP’s ability to limit tumor growth in DIO mice. Taken together, these results demonstrate that the metabolic improvements achieved by TZP treatment are accompanied by mitigation of obesity-driven immunosuppression. These findings shed important light on the potential for TZP and other incretin-based therapies to limit obesity-associated cancer progression, and are especially timely given the growing population of patients utilizing incretin-based therapies. Citation Format: Michael F. Coleman, Elaine M. Glenny, Tori L. McFarlane, Hannah M. Malian, Claire E. Gates, Fangxin Chen, Alyssa N. Ho, Violet A. Kiesel, Stephen D. Hursting. Tirzepatide treatment restores antitumor immunity in a model of obesity-driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2188.

Abstract 584: Synergistic action of ATF6 and EZH2 in sensing metabolic stress to boost anti-tumor immunity

Abstract Although immune checkpoint blockade (ICB) marked a breakthrough in melanoma treatment, half of patients fail to respond, underscoring the crucial need for complementary adjuvant and neo-adjuvant therapies. Due to the low overall response rate and the stark contrast in outcomes between patients that respond and those that fail to respond, identifying the defining characteristics of immune checkpoint blockade (ICB) response is essential for enhancing the efficacy of melanoma treatments. To recapitulate and investigate the ICB-responsive phenotype in vitro, melanoma cells were metabolically adapted to an environment lacking glucose which successfully drove the dependence on oxidative metabolism and restored MHC-I expression, two established characteristics of ICB responsive tumors. Further, metabolic adaptations significantly enhance the tumor sensitivity to T-cell killing. Surprisingly even in JAK1 and IFNAR1 deficient cells, metabolic adaptation restored MHC I antigen presentation, indicating an interferon-independent mechanism of MHC-I regulation. Proteomic analysis of three metabolically conditioned melanoma cells lines (human and mouse) identified downregulation of the histone methyltransferase EZH2 and the activating transcription factor ATF6 as key corollaries of MHC-I expression. Interestingly, EZH2 and ATF6 activity were found to be inversely related in metabolically adapted cells and patient tumor samples. These studies mechanistically explore the epigenomic control of antigen presentation machinery and the transcriptional targets of ATF6 controlling sensitivity T cell killing. Here, through in vitro and in vivo studies, we demonstrate that constitutive activation of ATF6, and thereby activation of the adaptive unfolded protein response (UPR), sensitizes melanoma to immune control. These data suggest that EZH2 and ATF6 act synergistically to balance immune mediated killing during metabolic stress whereby loss of EZH2 increases MHC-I antigen presentation and activation of ATF6 sensitizes tumor cells to T cell-mediated control. Ongoing therapeutic studies combining EZH2 inhibition, ATF6 activation, and ICB in pre-clinical models further suggest targeting the synergy between EZH2 and ATF6 is a putative adjuvant or neo-adjuvant target to improve the clinical response of ICB. Citation Format: Jacob L. Edmondson, Megan R. Reed, Daniel Fil, Billie Heflin, Brian Koss, Alan J. Tackett. Synergistic action of ATF6 and EZH2 in sensing metabolic stress to boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 584.

Abstract 1358: Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity

Abstract Immune checkpoint receptors (ICRs) negatively regulate antitumor immune responses. While significant advances have been made in understanding the mechanisms of well-established ICRs such as PD-1 and CTLA-4, other checkpoint proteins remain incompletely characterized and may be targeted to synergistically boost antitumor immunity. One such protein is V-domain immunoglobulin suppressor of T-cell activation (VISTA), an ICR whose expression curtails the function of several lymphoid and myeloid lineages, including tumor-reactive cytotoxic T cells. Globally blocking VISTA elicits potent T cell-mediated antitumor immunity and systemic myeloid cell-mediated inflammatory responses. Mechanistically, VISTA has been shown to inhibit T cell activation by acting as an inhibitory ligand on antigen-presenting cells. However, the impact of T cell-intrinsic VISTA on modulating tumor-reactive T cell responses has not been thoroughly investigated, and it remains unclear whether blocking T cell-specific VISTA is adequate for revitalizing T cell-mediated tumor control. In this study, we utilized VISTA conditional knockout mice models to examine the relative contribution of T cell-intrinsic VISTA versus myeloid-derived VISTA in the regulation of antitumor immunity. We present evidence that blocking VISTA on T cells alone is insufficient to induce strong antitumor immune responses, whereas blocking VISTA on myeloid cells counteracts its dominant role in orchestrating T cell suppression. Citation Format: Sachin Patnaik, Elizabeth Delaney, Cassandra Gilmour, Keman Zhang, Amin Zakeri, Hieu M. Ta, Prerana B. Parthasarathy, Tyler Alban, Timothy Chan, Li Lily Wang. Elucidating the contribution of T cell-intrinsic VISTA to the suppression of antitumor T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1358.

Abstract 6860: Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment

Abstract Radiation therapy (RT) has long been a cornerstone in cancer treatment, yet the mechanisms underlying its impact on the immune system remain incompletely understood. RT is recognized for its ability to induce DNA damage within cancer cells, leading to cancer cell death. For those cancer cells that do not die as a result of radiation, tumor infiltration by immune cells is closely associated with treatment outcomes. T cell infiltration is a dynamic process, with subsets taking up residence in the tumor while the majority recirculate through lymph nodes into the peripheral blood. T regulatory cells (Tregs) have been recognized as potent suppressors of anti-tumor immune responses, dampening immune-mediated clearance of cancer cells. While RT can initially contribute to an improved anti-tumor immune response, this beneficial effect is often counterbalanced by the presence of suppressive Tregs. Tregs can infiltrate the tumor microenvironment, ultimately impeding and overwhelming the immune response generated by RT. Our objective was to gain insights into the migratory patterns of Tregs to and from tumor sites and to elucidate how RT influences the dynamics of Treg recirculation. Utilizing the established photoconvertible Kaede mouse model we can track photoconverted cells following RT. Following photoconversion, photoconverted T cells were observable within the tumor and subsequently moved to the TdLN and on to other NdLN at later time points. In the TdLN of mice bearing MC38, Moc1, and Moc2 tumors, Tregs represented a significantly higher proportion of photoconverted cells compared to non-Treg CD4 or CD8 T cells. To validate their active departure from the tumor site, mice underwent FTY720 treatment, resulting in a reduction in both photoconverted Treg and non-Treg migration to the TdLN. When RT was administered to the tumor, there was a decrease in photoconverted Treg and non-Treg CD4 T cells within the TdLN, consistent with the expected cytotoxic effects on the irradiated tumor upstream. To focus on recruitment back from the TdLN, mice were analyzed after photoconversion of the TdLN. This revealed that a majority of TdLN Tregs exhibited a preference for relocating to the tumor, with only a minority migrating to the NdLN. The newly entered Tregs were analyzed in the tumor, at D1 and D3 post RT, and we found that these Tregs exhibited increased expression of CCR8, CD69, KLRG1 and Ly6C as compared to those in non-irradiated tumors, suggesting that these are recent entrants with limited suppressive capacity. These findings illuminate the intricate dynamics between RT, Tregs, and the use of site-specific photoconversion to track immune cell movement and changes over time to understand treatment responses. Our ongoing studies focus on the interplay between the Treg and the tumor environment that dictates the success of CD8 T cell mediated control of residual cancer cells following RT. Citation Format: David J. Friedman, Tiffany Blair, Gwen Kramer, Aanchal Preet Kaur, Jason Baird, Alejandro Alice, Megan Potuznik, Marka R. Crittenden, Michael J. Gough. Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6860.

Active Tuberculosis Is Associated with Depletion of HIV-Specific CD4 and CD8 T Cells in People with HIV.

Infection with Mycobacterium tuberculosis (Mtb) in people with HIV (PWH) is associated with depletion of Mtb-specific CD4 T cell responses, increased risk of progression to active tuberculosis (TB) disease, and increased immune activation. Although higher HIV viral loads have been reported in Mtb/HIV co-infection, the extent to which Mtb infection and TB disease impact the frequency and phenotype of HIV-specific T cell responses has not been well described. We enrolled a cohort of PWH in Kenya across a spectrum of Mtb infection states, including those with no evidence of Mtb infection, latent Mtb infection (LTBI), and active pulmonary TB disease, and evaluated the frequency, immune activation, and cytotoxicity phenotype of HIV-specific CD4 and CD8 T cell responses in peripheral blood by flow cytometry. We found evidence of depletion of HIV-specific CD4 and CD8 T cells in people with TB, but not with LTBI. Expression of the immune activation markers human leukocyte antigen-DR isotype (HLA-DR) and Ki67 and of the cytotoxic molecules granzyme B and perforin were increased in total CD4 and CD8 T cell populations in individuals with TB, although expression of these markers by HIV-specific CD4 and CD8 T cells did not differ by Mtb infection status. These data suggest that TB is associated with overall increased T cell activation and cytotoxicity and with depletion of HIV-specific CD4 and CD8 T cells, which may contribute to further impairment of T cell-mediated immune control of HIV replication in the setting of TB.

Elevated glutamate impedes anti-HIV-1 CD8 + T cell responses in HIV-1-infected individuals on antiretroviral therapy

CD8 + T cells are essential for long-lasting HIV-1 control and have been harnessed to develop therapeutic and preventive approaches for people living with HIV-1 (PLWH). HIV-1 infection induces marked metabolic alterations. However, it is unclear whether these changes affect the anti-HIV function of CD8 + T cells. Here, we show that PLWH exhibit higher levels of plasma glutamate than healthy controls. In PLWH, glutamate levels positively correlate with HIV-1 reservoir and negatively correlate with the anti-HIV function of CD8 + T cells. Single-cell metabolic modeling reveals glutamate metabolism is surprisingly robust in virtual memory CD8 + T cells (TVM). We further confirmed that glutamate inhibits TVM cells function via the mTORC1 pathway in vitro. Our findings reveal an association between metabolic plasticity and CD8 + T cell-mediated HIV control, suggesting that glutamate metabolism can be exploited as a therapeutic target for the reversion of anti-HIV CD8 + T cell function in PLWH.

Sex-Biased T-cell Exhaustion Drives Differential Immune Responses in Glioblastoma.

Immunotherapies in patients with GBM have been unsuccessful due to a variety of factors, including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-biased T-cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve the therapeutic efficacy of immunotherapy in GBM. See related commentary by Alspach, p. 1966. This article is featured in Selected Articles from This Issue, p. 1949.

Impact of Inter- and Intra-Donor Variability by Age on the Gel-to-Tissue Transition in MMP-Sensitive PEG Hydrogels for Cartilage Regeneration.

Matrix metalloproteinase (MMP)-sensitive hydrogels are promising for cartilage tissue engineering due to cell-mediated control over hydrogel degradation. However, any variability in MMP, tissue inhibitors of matrix metalloproteinase (TIMP), and/or extracellular matrix (ECM) production among donors will impact neotissue formation in the hydrogels. The goal for this study was to investigate the impact of inter- and intra-donor variability on the hydrogel-to-tissue transition. Transforming growth factor β3 was tethered into the hydrogel to maintain the chondrogenic phenotype and support neocartilage production, allowing the use of chemically defined medium. Bovine chondrocytes were isolated from two donor groups, skeletally immature juvenile and skeletally mature adult donors (inter-donor variability) and three donors within each group (intra-donor group variability). While the hydrogel supported neocartilaginous growth by all donors, donor age impacted MMP, TIMP, and ECM synthesis rates. Of the MMPs and TIMPs studied, MMP-1 and TIMP-1 were the most abundantly produced by all donors. Adult chondrocytes secreted higher levels of MMPs, which was accompanied by higher production of TIMPs. Juvenile chondrocytes exhibited more rapid ECM growth. By day 29, juvenile chondrocytes had surpassed the gel-to-tissue transition. On the contrary, the adult donors had a percolated polymer network indicating that despite higher levels of MMPs the gel-to-transition had not yet been achieved. The intra-donor group variability of MMP, TIMP, and ECM production was higher in adult chondrocytes but did not impact the extent of the gel-to-tissue transition. In summary, age-dependent inter-donor variations in MMPs and TIMPs significantly impact the timing of the gel-to-tissue transition in MMP-sensitive hydrogels.

KIR-HLA interactions extend human CD8+ T cell lifespan in vivo.

BACKGROUNDThere is increasing evidence, in transgenic mice and in vitro, that inhibitory killer cell immunoglobulin-like receptors (iKIRs) can modulate T cell responses. Furthermore, we have previously shown that iKIRs are an important determinant of T cell-mediated control of chronic viral infection and that these results are consistent with an increase in the CD8+ T cell lifespan due to iKIR-ligand interactions. Here, we tested this prediction and investigated whether iKIRs affect T cell lifespan in humans in vivo.METHODSWe used stable isotope labeling with deuterated water to quantify memory CD8+ T cell survival in healthy individuals and patients with chronic viral infections.RESULTSWe showed that an individual's iKIR-ligand genotype was a significant determinant of CD8+ T cell lifespan: in individuals with 2 iKIR-ligand gene pairs, memory CD8+ T cells survived, on average, for 125 days; in individuals with 4 iKIR-ligand gene pairs, the memory CD8+ T cell lifespan doubled to 250 days. Additionally, we showed that this survival advantage was independent of iKIR expression by the T cell of interest and, further, that the iKIR-ligand genotype altered the CD8+ and CD4+ T cell immune aging phenotype.CONCLUSIONSTogether, these data reveal an unexpectedly large effect of iKIR genotype on T cell survival.FUNDINGWellcome Trust; Medical Research Council; EU Horizon 2020; EU FP7; Leukemia and Lymphoma Research; National Institute of Health Research (NIHR) Imperial Biomedical Research Centre; Imperial College Research Fellowship; National Institutes of Health; Jefferiss Trust.

Sex-specific T cell exhaustion drives differential immune responses in glioblastoma

Abstract Sex differences in glioblastoma (GBM) incidence and outcome are well recognized; males show higher incidence and worse prognosis than females. Emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using orthotopic GBM animal models, we recapitulated these sex differences in immune-competent B6 mice but not in immune-deficient mouse strains (NSG and RAG1KO). By depleting CD8+ T cells, we further confirm that CD8+ T cells play a critical role in driving sex differences in survival. Flow cytometry analysis revealed that male CD8+ T cells from tumor expressed higher levels of exhaustion markers such as PD-1, CTLA-4 and Tox, with higher frequency of progenitor exhausted T cell subset whereas female tumor contained more effector-like CD8+ T cells expressing elevated cytokine production. Anti-PD-1 antibody treatment exclusively extended the survival of male mice via inducing decreased exhausted CD8+ T cell subsets and increased effector function and proliferation. Survival analysis on bone marrow chimera model and adoptive transfer model indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, as the sex of donor cells was a critical contributing factor. Finally, we confirmed these observations in GBM patient tumor samples with male tumors having a higher frequency of progenitor exhausted T cells with increased TOX expression. Taken together, these findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy responses. Supported by grants from NIH (R35 NS127083, P01 CA245705).

Low avidity CD8 +T cells are less exhausted than their high avidity counterparts and provide superior anti-tumor immunity

Abstract The interaction of T cell receptors (TCRs) on T cells with MHC I-peptide complexes on cancer cells elicits the cytotoxic activity of CD8 +T cells. Here we analyze how the strength of this interaction, or T cell avidity, shapes the exhaustion of CD8 +TILs and dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We used this method to study low and high avidity CD8 +TILs responding to the tumor neoantigen PDPR MUTof the murine sarcoma Meth A. Single cell sequencing reveals that high avidity CD8 +TILs are more terminally exhausted (51.41% vs 27.21%) and less effector-like (28.39% vs. 60.0%) than low avidity CD8 +TILs. We used flow cytometry to analyze PDPR MUT-specific CD8 +TILs with low or high avidity from 28 day-old Meth A tumors. There were significantly more TIM3 +PD-1 +cells (Paired t-test; P < 0.0001) and TOX +PD-1 +cells (Paired t-test; P < 0.05) in the high avidity CD8 +TILs than the low avidity CD8 +TILs. We adoptively transferred 1,000 PDPR MUT-specific low or high avidity CD8 +T cells, or control CD8 +T cells into tumor-bearing mice 10 days after tumor challenge. Low avidity T cells significantly improved the survival of mice (Mantel-Cox; P = 0.0044), while high avidity t cells did not do so (Mantel-Cox; P = 0.7081). These results establish a novel correlation between avidity, exhaustion and T cell-mediated tumor control in vivo.

Sphingosine 1-phosphate / Acetyl CoA Carboxylase1 Axis regulates the Differentiation and Function of Tumor-associated Myeloid-Derived Suppressor Cells

Abstract The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) poses a serious obstacle to cancer treatment. The bioactive lysophospholipid sphingosine 1-phosphate (S1P), which is produced by sphingosine kinases (SphK1 or SphK2), regulates tumor and T cell survival and activity. Thus, it has been targeted to improve the outcome of anti-cancer therapeutics. However, S1P has not yet been investigated in terms of how it regulates myeloid cell activity. The present study demonstrates that deletion of SphK2-mediated S1P in MDSCs favorably modifies the tumor microenvironment and renders infiltrating myeloid cells with antigen presenting phenotype, leading to increased ability to control tumor growth. Furthermore, in preclinical prostate and melanoma cancer models, the combination of PD1 antibody and SphK2 pharmacological inhibitor (ABC294640) dramatically reduced tumor growth and increased the survival of tumor bearing mice. Mechanistically, the S1P pulldown and subsequent MS-based proteomics analysis revealed that S1P binds to various proteins involved in metabolic and ribosomal biogenesis pathways. Notably, both the isoforms of Acetyl CoA Carboxylase, the rate-limiting fatty acid metabolic enzyme, binds to S1P. Furthermore, investigations using deletion, overexpression, and site-directed mutagenesis demonstrated that ACC1 activity is necessary to preserve the immunogenic capacity of SphK2 KO MDSCs. This study highlights the clinical value of manipulating MDSC suppressive activity by targeting the S1P/ACC1 axis to boost T cell-mediated tumor control and highlights the crucial function that S1P plays as an immunoregulatory mediator in MDSCs. Supported grants from NIH (R01 CA250458, R01 CA236379)

Elucidating the role played by the NKG2 family of receptors in natural killer cell responses to JC polyomavirus

Abstract JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression due to monoclonal antibody treatments for cancer, autoimmune diseases and transplantation, or due to uncontrolled HIV infection. There is currently no effective treatment against PML and novel immunotherapies are urgently needed. Natural killer (NK) cells play critical roles in defense against viral infections, yet NK cell responses to JCPyV remain largely unexplored. Here, we first compared NK and T cell responses against JCPyV VP1 peptide pools by intracellular cytokine staining. In about 40% of healthy donors, we detected robust CD107a upregulation and IFNγ production by NK cells, extending beyond T cell responses. Next, using a novel flow cytometry-based killing assay, we showed that co-culture of NK cells and JCPyV-infected SVG-A cells led to a 65% reduction in infection, on average. To identify receptor-ligand interactions modulating NK cell-mediated responses to JCPyV, we performed co-cultures in the presence of select blocking antibodies. We observed enhanced expression of ligands for the activating NKG2D on infected cells and accordingly, NKG2D blockade resulted in decreased NK cell degranulation. In contrast, blockade of inhibitory NKG2A enhanced NK cell response to JCPyV-infected targets. Moreover, we identified JCPyV-derived peptides that elicit dominant NK cell responses and can stabilize HLA-E, the ligand for NKG2A and the activating NKG2C. Altogether, these findings suggest that modulation of NKG2D and NKG2A/C signaling may play key roles in NK cell-mediated control of JCPyV and may be promising immunotherapeutic targets to boost NK cell anti-JCPyV activity. Supported by grant from NIH (R01 NS116278)

Severe COVID-19 patients have impaired plasmacytoid dendritic cell-mediated control of SARS-CoV-2

Type I and III interferons (IFN-I/λ) are important antiviral mediators against SARS-CoV-2 infection. Here, we demonstrate that plasmacytoid dendritic cells (pDC) are the predominant IFN-I/λ source following their sensing of SARS-CoV-2-infected cells. Mechanistically, this short-range sensing by pDCs requires sustained integrin-mediated cell adhesion with infected cells. In turn, pDCs restrict viral spread by an IFN-I/λ response directed toward SARS-CoV-2-infected cells. This specialized function enables pDCs to efficiently turn-off viral replication, likely via a local response at the contact site with infected cells. By exploring the pDC response in SARS-CoV-2 patients, we further demonstrate that pDC responsiveness inversely correlates with the severity of the disease. The pDC response is particularly impaired in severe COVID-19 patients. Overall, we propose that pDC activation is essential to control SARS-CoV-2-infection. Failure to develop this response could be important to understand severe cases of COVID-19.