Citations

Abstract Description The presence of Foxp3+CD4+ regulatory T cells (Tregs) hinders the protective antitumor T cell responses to lung cancer. The accumulation of Tregs is linked to lung cancer progression, resulting in poorer survival rates. However, the mechanisms behind this accumulation are not fully understood. Tregs express elevated levels of the extracellular ATP (eATP) receptor P2RX7 in both human and mouse lung cancer. Here, we show that P2RX7 is crucial for the accumulation of Tregs in lung tumor, promoting tumor growth in a murine lung cancer model. We observed, in T cell-specific P2RX7-KO mice (CD4-cre P2rx7fl/fl), reduced Treg infiltration and a concomitant increase in tumor CD4+ effector T cells, leading to better tumor control. In contrast, P2RX7-KO Tregs accumulated in mediastinal lymph nodes, suggesting that blocking P2RX7-KO signaling impairs the Tregs migratory ability. P2RX7+ Tregs had increased expression of T-bet and KLRG1, which are associated with greater suppressive function. Finally, Treg-specific P2RX7-KO mice (Foxp3-Cre-ERT2 P2rx7fl/fl) had reduced tumor growth and Treg tumor accumulation, indicating that P2RX7 controls lung tumor Tregs in a cell-intrinsic way. These results suggest that P2RX7 promotes lung tumor Treg infiltration and subsequent loss of cancer control. They also suggest that P2RX7 blockade can be used as a therapy to improve lung cancer control, which we are currently exploring in proof-of-concept experiments. Funding Sources Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Purpose This study investigates a novel optimization framework that integrates the newly developed MCF MKM relative biological effectiveness (RBE) model into intensity modulated particle therapy (IMPT) planning for carbon ion therapy. Unlike conventional RBE models such as MKM or LEM, the MCF MKM provides detailed microdosimetric information, which improves predictive accuracy of cell survival but elevates both memory and time requirement in the optimization process. Furthermore, to the implicit nature of the MCF MKM, the gradient information, which is essential in inverse planning, is hardly accessible. In response, a randomized gradient‐free optimization method (RGFM) was developed to mitigate the computational challenge and achieve improved RBE dose distributions. Materials and Methods An RBE dose optimization problem was formulated based on the discretized MCF MKM, known as the Abridged Microdosimetry Distribution Model (AMDM). The RGFM was derived to find feasible and high‐quality settings of monitor unit for IMPT plans without the need for explicit gradient calculations. To verify the clinical plausibility of the proposed method, it was tested on two clinical scenarios—prostate and brain cases in comparison with benchmark plans derived solely from physical dose optimization. Computational times and dose‐volume histograms (DVHs) for both physical and RBE doses were evaluated. Results The RGFM algorithm successfully optimized the MCF MKM‐based IMPT plans. Compared to the benchmark, the RBE dose distributions showed improved coverage of the target and reduced hot spots. Such improvements were observed to coincide with trade‐offs in physical dose distributions. The complexity of the MCF MKM extended the computational time, yet high‐quality solutions were attainable in a clinically reasonable timeframe. Multi‐field optimization (MFO) further enhanced target coverage and critical organ sparing over single‐field optimization (SFO). Conclusions This proof‐of‐principle study confirmed the feasibility of using a randomized gradient‐free method to optimize RBE dose based on the MCF MKM in IMPT planning for carbon ion therapy. The approach provides a pathway for clinical adoption of advanced RBE models, potentially leading to more biologically informed and effective carbon ion therapy treatments. Future work involves refining algorithms, incorporating parallel computing, and performing additional case studies to improve computational efficiency and generalizability.

Abstract Description CD8 T cell programming creates a pool of naïve, effector, memory, or exhausted subsets, however the regulation of these trajectories is not expansively elucidated and has serious implications for cancer immunity and immune checkpoint inhibitor (ICI) therapy. We found a significant decrease of Dual Specificity Phosphatase 2 (DUSP2) in CX3CR1+ CD8s, a highly resilient subset, from ICI responders. Thus, we hypothesized that DUSP2 negatively regulates CD8 T cells in responses to ICI therapy. DUSP2 knockdown (KD) statistically improved CD8 cytotoxicity and expression of NKG7, a vital effector molecule. DUSP2 KD also increased SMARCA4 expression, a BRG/BRM-associated factor (BAF) complex ATPase, and increased open chromatin regions of BAF components. BAF complexes are chromatin remodelers implicated in CD8 differentiation pathways. In line with this, we found CD8s from DUSP2 conditional knockout (cKO) mice demonstrated a trend in defective memory generation. DUSP2 cKO CD8s also trended towards decreased PD-1 and increased CX3CR1 expression, demonstrating a more effector like phenotype. DUSP2 cKO mice did not show a significant difference in tumor protection in-vivo, presumably due to skewing of CD8 fates via BAF. Promisingly, DUSP2 KD increased ex-vivo cytotoxic functionality in about 50 percent of tested cancer patient PBMCs. Adjuvant BAF inhibition with DUSP2 KO versus KD in a specific CD8 subset may enhance responses further, providing a novel avenue for improving ICI efficacy. Funding Sources Supported by NCI 1F31CA287495-01 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Abstract Description Sepsis, the life-threatening organ dysfunction stemming from the severe inflammatory response to systemic microbial infection, can affect humans of any age. One consequence of sepsis is the development of prolonged immunoparalysis that contributes to increased susceptibility to infection. With vaccination being an important means for protecting humans from infection throughout their lifetime, it is surprising that there has been no study performed to date assessing vaccine efficacy in sepsis survivors. We found neonatal and adult mice given an intramuscularly delivered inactivated multivalent influenza vaccine 30 days after sepsis induction had reduced total and class switched Ab titers, compared to non-septic controls. The reduced humoral response was due, in part, to impaired CD4 T cell and B cell responses to the vaccine. Reduced Ab titers were similarly seen in post-septic mice given an intranasal live-attenuated influenza vaccine. ELISpot data demonstrating reduced numbers of antibody secreting cells in the spleens of vaccinated septic mice further support the ELISA results. Moreover, mice vaccinated prior to sepsis induction also showed reduced antibody titers. Importantly, vaccinated septic mice had reduced protection to a lethal influenza challenge, demonstrated by increased mortality and viral burden in the lungs. These data collectively show how sepsis can lead to increased susceptibility to pathogens normally neutralized by vaccine-induced immunity. Funding Sources Supported by NIH/NIAID R35 GM140881; 5R01 GM139046; 1T32TR004385 Topic Categories Translational and Interventional Immunology (TI)

Abstract Description Many cancers, including glioblastoma (GBM), exhibit a male-biased incidence and outcome disparity, partially driven by sex hormones. While androgens are known to suppress anti-tumor T cell immunity in certain solid tumors, we found that androgens instead enhance anti-tumor immunity in brain tumors. Upon castration, tumor growth was accelerated in brain tumor models, while the opposite effect was observed for tumors located outside the brain. Decreased T cell function was observed in both tumors and peripheral lymphoid organs in castrated mice, indicated by reduced anti-tumor cytokines and increased T cell exhaustion. Mechanistically, this systemic immunosuppression was driven by hyperactivation of the hypothalamus-pituitary-adrenal (HPA) axis in castrated mice with brain tumors. Blocking glucocorticoid receptors reversed the accelerated tumor growth in these mice, confirming the role of HPA axis dysregulation. This mechanism was brain-specific rather than GBM-specific, as hyperactivation of the HPA axis was also observed with intracranial implantation of non-GBM tumors. Furthermore, elevated production of proinflammatory cytokines (IL-1β, TNF) in the brain of castrated mice was identified as a driver of HPA axis activation. Taken together, these findings reveal unique endocrine-mediated mechanisms by which brain tumor modulate systemic immunity in the androgen-deprived setting and highlight the importance of organ-specific effects of anti-tumor immunity. Funding Sources NIH P01CA245705; NIH R35NS127083; American Brain Tumor Association; Cleveland Clinic; Case comprehensive cancer center Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Interactions between ST6GAL1-produced sialic acids and Siglec-2 (CD22) result in immune inactivation. CD22 is a well-characterized regulator of B cell function. We demonstrate that conventional type-2 dendritic cells (cDC2s) and hybrid macrophages express CD22 across the spleen and tumor microenvironment. To understand the function of CD22 on antigen-presenting cells, we generated novel CD22-floxed mice and crossed them with a) Zbtb46-Cre ones (those lacking CD22 on cDC2s and hybrid macrophages) as well as b) CX3CR1-Cre mice (without CD22 on hybrid macrophages nor microglia). From the former cross, we generated subcutaneous flank tumor and colitis-associated colorectal cancer models. CD22 expression on myeloid cells did not impact anti-tumor responses when tumors expressed low CD22 ligand levels. From here, we assess the impact of tumor cell CD22 ligand overexpression on tumor responses. Also, to evaluate CD8 T cell response to CD22, we challenged mice with Theiler’s murine encephalomyelitis virus (TMEV), modeling multiple sclerosis via CD8 T cell hyperactivation. CD22 did not mediate CD8 activation nor infiltration in brains of the latter cross. Thus, CD22 may be functionally redundant across myeloid and B cells. Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Abstract Description distinct forms of long-COVID that can last more than three years, are associated with persistent activation of T cells. We and others demonstrated that CD8 T cells that respond to influenza infection can spontaneously cause pulmonary fibrosis lasting a lifetime in aged mice. This model recapitulates key features of epithelial and fibroblast dysplasia observed in patients that require lung transplant due to COVID-19 related pulmonary fibrosis. Our data suggests understanding what distinguishes age-associated pro-fibrotic from quiescent T cell responses in the lung, could aid rational development of alternative therapeutics. Here, using intravital cell compartment labeling and a functional high-dimensional flow cytometry screen, we identified a subset of parenchymal CD8 T cells with age-exacerbated responses to many cytokines sustained in aged mouse lungs three months after infection. Albeit much lower-grade, these indiscriminate bystander responses elicit CD8 T cell functions usually restricted by the required presence of their extremely specific foreign antigen. Our supporting data from independent models, suggest a potential role for steady-state, but not recruited alveolar macrophages in limiting bystander susceptibility in a subset of parenchymal CD8 T cells. Our data implicate cellular and molecular mechanisms that link age and early infection severity with long-term CD8 T cell-dependent fibrotic responses to pandemic-relevant respiratory viruses. Funding Sources American Lung Association Grant: ERP-1251983 Topic Categories Viral Immunology (VIR)

Abstract Description Cholera toxin (CT), an exotoxin produced by Vibrio cholerae, causes severe diarrheal disease and contributes to the third leading cause of death in children under 5. Beyond its pathogenicity, CT possesses potent mucosal adjuvant properties, inducing type 2 immunity in the gastrointestinal tract. However, the immune response to acute oral CT exposure remains poorly characterized. To address this, we intragastrically administered a single nonlethal dose of CT to mice and assessed physiological and immune responses over 24 hours. In C57BL/6 mice, CT induced severe hypothermia, while BALB/c mice exhibited milder effects, with males of both strains showing heightened sensitivity. All treated animals developed diarrhea, and C57BL/6 mice experienced intestinal edema lasting up to 24 hours. Transcriptomic analysis of the small intestine revealed acute upregulation of inflammatory mediators such as IL-1β and IL-33. Immune profiling by full-spectrum flow cytometry showed CT-induced changes in the lamina propria compartment, including increased ILC3, reduced Treg RORγT+ and CD11b+CD103+ dendritic cells. In the epithelial layer, CT slightly elevated neutrophils, eosinophils, monocytes, and ILC3 while reducing CD3+ T lymphocytes and ILC1/ NK cells. These findings emphasize the broader action of CT in pathogenesis and immunity, shedding light on how immune sensing of bacterial toxins can drive protective defenses and adjuvant activity. Funding Sources Supported by Hypothesis Fund Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description The lifetime risk for colorectal cancer in the United States is approximately 4%. Individuals with Inflammatory Bowel Disease, including Ulcerative Colitis and Crohn’s Disease, have a substantially increased risk of developing colorectal cancer. The mechanisms for accelerated tumorigenesis due to enhanced inflammation are not fully characterized. Siglecs (sialic acid immunoglobulin lectin-like proteins) are a family of inhibitory receptors that are negative regulators of the immune response. Siglec-E is an inhibitory receptor that is expressed by innate immune cells, including monocytes, macrophages, neutrophils, and dendritic cells. Interestingly, mice deficient in Siglec-E have accelerated the development of tumors and reduced survival in a spontaneous mouse model of colorectal cancer (TS4-cre LSL-KRasG12D APClox468/wt). Siglec-E knockout mice also have accelerated gut inflammation using the DSS colitis model as compared to WT mice. Using the DSS/AOM model of colitis-associated colorectal cancer, we found that Siglec-E KO mice develop more tumors as compared to wild-type controls. Thus, Siglec-E is an important regulator of gut homeostasis, and the loss of Siglec-E accelerates colon cancer in two murine models. Funding Sources Mayo Clinic Center for Biomedical Discovery NIH R01 CA243545-01A1 to VSS T32 AI170478 Mayo Clinic Graduate School of Biomedical Sciences Deans fellowship: Initiative for maximizing student development (IMSD) R25 GM055252-26 Doyon Foundation Topic Categories Mucosal and Regional Immunology (MUC)

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)