Silva, Henrique Borges da

Abstract Description Developing a universal influenza vaccine requires understanding how lung-resident memory CD4+ T cells are formed. These cells remain in the lung for a long time and strengthen the immune response to secondary infections. Tissue-resident helper cells (TRH), a subset of memory T cells, share characteristics with follicular helper cells (TFH) and boost the IgA and IgG production by lung-resident B cells. TRH cells express elevated levels of the extracellular ATP (eATP) sensor P2RX7. Here we demonstrate that P2RX7 ablation on T cells impairs the establishment of flu-specific TRH cells. P2RX7-induced decrease of TRH cells negatively impacted the protection against homologous challenge, since T cell-specific P2RX7-KO mice exhibit worse control of viral load than WT mice. P2RX7-KO mice also showed a reduction in flu-specific lung-resident germinal center-like B cells and lower levels of IgA and IgG in the lungs. P2RX7-KO did not induce major changes in the numbers of TFH-induced responses in the draining lymph nodes. Our single-cell RNAseq showed reduced expression of mitochondrial genes in P2RX7-KO TRH cells, which reflects decreased mitochondrial content presence in these cells. We observed an aberrant metabolic pathway usage by P2RX7-KO TRH cells, which beyond having less mitochondria also cease to utilize glycolytic metabolism to support protein synthesis. Our findings suggest the eATP-P2RX7 axis is required to establish TRH cells that protect against influenza. Funding Sources Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Persistent exposure to antigens and immunosuppressive signals in tumors or chronic infections drives CD8+ T cell exhaustion (Tex), a programmed state of T cell dysfunction. While efforts have been made to combat Tex through immunotherapies, they often fail to induce lasting protective effects. Exhausted CD8+ T cells consist of various subsets with distinct transcriptional, functional, and metabolic features. Among these subsets, progenitor exhausted CD8+ T cells (Tprog) are important: they are metabolically quiescent, retain stemness capacity and respond better to checkpoint blockade therapy (e.g., anti-PD1). Here, we report that, in response to chronic viral infection (LCMV Clone 13), P2RX7 is crucial for the establishment of Tprog cells. P2RX7-KO CD8+ T cells had decreased numbers of Tprog cells. P2RX7-KO Tprog cells lose metabolic quiescence, displaying increased glycolytic function and loss of mitochondrial respiration dependence. Our meta-analyses of gene (scRNAseq) and protein (LegendScreen assay) expression suggest that P2RX7-KO Tprog cells undergo abnormal exhaustion differentiation paths, and express reduced levels of integrin beta-1 (ITGB1), a molecule associated with lymphoid residency of Tprog cells. Finally, we found that P2RX7 is crucial for exhausted CD8+ T cells to respond to anti-PD1 in vivo. These data open the possibility of using P2RX7 agonism to boost the efficacy of existing checkpoint blockade immunotherapies aimed to revitalize CD8+ T cell responses. Funding Sources NIH Grants K99/R00A139381 and R01A170649 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Description Immune responses to allergens vary depending on the route of sensitization, but the mechanisms behind these differences are not clear. Damage signals from the microenvironment dictate the immune response magnitude and may regulate route of sensitization differences. Extracellular ATP (eATP) is a danger signal released by inflamed tissues and sensed by immune cells via P2RX7. The role of P2RX7 in CD4+ Th2 cell responses to allergen is unclear, with past reports suggesting opposite effects of P2RX7 knockout (KO). Here, we tracked the role of P2RX7 on CD4+ T cell-mediated lung allergy in response to different routes of sensitization. Comparing Papain + Ovalbumin (OVA) intranasal versus subcutaneous sensitization, we found increased lung type 2 inflammation and lung CD4+CD69+GATA3+ T cells in response to the subcutaneous route, compared to intranasal route. P2RX7-KO led to decreased lung CD4+ Th2 cell responses and reduced inflammation in mice exposed to subcutaneous, but not intranasal sensitization. Subcutaneous OVA + Papain led to a stronger eosinophilic response, while both eosinophils and neutrophils were elicited by intranasal route. Our results indicate that sensing of eATP through P2RX7 exacerbate the intensity of subcutaneous allergen-induced Th2 responses. We are currently comparing the Th2 responses induced by subcutaneous versus intranasal allergen sensitization, and how P2RX7 oppositely regulate such responses. Funding Sources NIH K99/R00A139381 NIH R01A170649 Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract The effectiveness of T cell–based immunotherapy depends on durable T-cell responses that can efficiently eliminate tumor cells. NKG7 was discovered three decades ago as a protein associated with lytic granules. However, only studies published over the past 5 years have contributed substantially to our understanding of NKG7 in T-cell biology. NKG7 has been recognized as an important T-cell functional marker in responses to immune checkpoint inhibitor therapy and in the prognosis of certain cancers. Besides its role in the generation, trafficking, and release of lytic granules, which is critical for efficient T-cell cytotoxicity against tumor cells, NKG7 has been identified as a key negative regulator of mTORC1 activity. By restraining mTORC1 activity, NKG7 promotes T-cell longevity and memory generation after infection. Importantly, NKG7 upregulation has demonstrated therapeutic potential in preclinical T-cell therapy for cancer. Collectively, NKG7 is emerging as a promising biomarker and therapeutic addition to T cell–based immunotherapies.

The differentiation of naive CD8+ T cells into effector or memory populations requires dynamic remodeling of cellular metabolism and proteome composition. In a recent study published in Nature Immunology, Sinclair et al offer critical insights into the role of autophagy, particularly mitophagy, in regulating these processes during CD8+ T cell differentiation. Autophagy, a conserved catabolic mechanism, is traditionally associated with cellular homeostasis and survival during nutrient deprivation. In contrast, Sinclair et al reveal that, in the immune system, autophagy is not simply a survival mechanism but a fine-tuned regulator of CD8+ T cell metabolism and function, fine-tuning CD8+ T cell effector vs quiescence choices.

ABSTRACT Lung cancer is the leading cause of cancer-related deaths worldwide and, despite treatment advances, immune suppression remains an obstacle to effective therapy. Effector CD4 + T cells (CD4 + Teffs) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Tregs), which accumulate in lung tumors and perform suppressive functions through multiple mechanisms. This suppression leads to tumor progression and poor patient outcomes. However, the mechanisms underlying Treg-mediated suppression are not fully understood. Here, we identify the extracellular ATP receptor P2RX7 as a key regulator of Treg function in lung tumors. Using a murine lung cancer model induced by Lewis lung carcinoma cells, we demonstrate that P2RX7 enhances the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell-specific P2RX7-KO mice, reduced Treg infiltration was accompanied by increased CD4 + Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibit reduced tumor growth, confirming a cell-intrinsic role of P2RX7 in Tregs. Suppression assays revealed that tumor-infiltrating WT Tregs have greater suppressive activity compared to P2RX7-KO Tregs, which failed to inhibit type 1 and Tfh-like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that WT Tregs express higher levels of the immunosuppressive surface molecule CTLA-4 when compared to P2RX7-KO Tregs. In summary, we show that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer, and targeting of P2RX7 may constitute a novel strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

Abstract Durable T cell immunity against cancer depends on the continual replenishment of effector CD8+ T cells. Thymic output has been correlated with favorable prognosis in cancer patients across a range of ages, suggesting that the thymus is an important source for replenishing T cells capable of controlling cancer progression. However, the effector potential of thymic mature CD8+ T cells and their regulation have not been clearly defined. In this study, we identified the ability of thymic single positive CD8+ T cells to gain effector potential after thymic selection, but they are subject to the regulation of PD-1. We found a previously undisclosed role of PD-1 in limiting both the cytotoxic and exhaustion potential of thymic and peripheral CD8+ T cells. Our results show that although PD-1 inhibition facilitates the expansion of effector CD8+ T cells, effector CD8+ cells gradually lose their antitumor activity within tumor tissues due to advanced exhaustion in the absence of PD-1. Thus, although the preset effector potential in thymic mature CD8+ T cells allows them to rapidly respond to malignant cells in the periphery, PD-1, as a checkpoint, is embedded in the thymic mature CD8+ T cells after positive selection to balance their effector function from exaggeration and exhaustion. Therefore, we propose that a strategy capable of upholding the cytotoxic capacity and avoiding exhaustion of CD8+ T cells during the early stages of PD-1 inhibition therapy is needed to achieve durable antitumor immunity.

Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis [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 6867.

Abstract CD8+ T-cell responses are influenced by ion abundance, which can widely vary within the tumor microenvironment. In this issue, Collier and colleagues investigated how intracellular versus extracellular potassium ion (K+) regulates intratumoral CD8+ T cells. They show that, while excessive extracellular K+ induces exhaustion, intracellular K+ is needed for protection from dysfunction. This work shows additional evidence that the regulation of CD8+ T-cell responses depends on a fine balance between intracellular and extracellular metabolite levels. See related article by Collier et al., p. 36 (2) .