Abstract Introduction Immunotherapy approaches against glioblastoma (GBM) such as immune checkpoint inhibitors (ICI), dendritic cell vaccination, and CAR-T have failed to durably cure most patients. Studies analyzing tumors from ICI responders show that abundance of CD4 and CD8 T cells prior to initiation of therapy predicts responsiveness. Thus, targeting the microenvironment to promote CD4 activity may synergize with ICI. The glioblastoma microenvironment contains high proportions of monocyte-derived macrophages which have potent immunosuppressive capabilities. Characterizing and targeting these immunosuppressive programs is a promising strategy for designing effective combination immunotherapies for glioblastoma patients. Methods We analyzed human glioblastoma snRNAseq datasets to find signaling receptors associated with myeloid immunosuppression and employed murine models of glioblastoma for functional validation. Results By analyzing patient-derived -omics datasets, we identified an understudied TNF-superfamily receptor, Fn14, as being upregulated in glioblastoma-associated myeloid cells with immunosuppressive gene signatures. Using in vitro approaches, we found that Fn14 activation in the tumor microenvironment can drive cytokines implicated in GBM immunosuppression such as IL6, IL1B, and IL8. Thus, Fn14 may serve as a surface marker for microenvironment-remodeling myeloid cells recruited to the tumor from peripheral circulation. Furthermore, in murine models of glioblastoma, we discovered Fn14 KO among glioblastoma-associated myeloid cells increases conventional dendritic cell type 1 (cDC1) and Th1 CD4+ T cell abundance in the tumor microenvironment and significantly prolongs survival. Conclusion These results suggest that blocking Fn14 in glioblastoma-associated myeloid cells can enhance Th1 immunity. Thus, Fn14 may represent a promising dual therapy target. Funding Source NIH Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Infection with Mycobacterium tuberculosis can cause diverse lesions, such as necrotic pneumonia, which can contribute to tuberculosis progression and transmission between individuals. Despite advances in understanding the role of ATP-gated P2RX7 ion channels in the development of severe forms of the disease, the regulation of this important signaling pathway remains unclear. Herein, we show that the ectonucleotidase CD39 plays an essential regulatory role in tuberculosis progression by preventing lung tissue damage, bacterial dissemination, and excessive inflammatory responses. Mechanistically, through its enzymatic activity on the cellular surface, CD39 protects infected macrophages from undergoing necrotic death mediated by P2RX7 activation. Cell-intrinsic CD39 expression also hinders the establishment of other myeloid cells, such as neutrophils, in the infected lung. We proposed that, by protecting infected macrophages from P2RX7-mediated cell death and bacterial dissemination in the lung tissue, CD39 prevents the development of necrotic lesions. Altogether, these findings uncover a significant role for CD39 as an essential component of the molecular regulation underlying the development of severe tuberculosis.

Lung cancer is the leading cause of cancer-related deaths worldwide, and despite advances in treatment, immune suppression remains an obstacle to effective therapy. Effector CD4+ T cells (CD4+ Teff) are critical for antitumor immunity, but their function is often inhibited by regulatory T cells (Treg), which accumulate in lung tumors and mediate 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. Herein, we identify the extracellular adenosine 5-triphosphate receptor P2RX7 as a key regulator of Treg function in lung tumors. In a murine lung cancer model induced by Lewis lung carcinoma cells, we found that P2RX7 enhanced the suppressive capacity of tumor-infiltrating Tregs, promoting tumor growth. In T cell-specific P2RX7-knockout (P2RX7-KO) mice, reduced Treg infiltration was accompanied by increased CD4+ Teff accumulation and improved tumor control. Treg-specific P2RX7-KO mice exhibited reduced tumor growth, confirming a Treg-intrinsic role of P2RX7. Suppression assays revealed that tumor-infiltrating wild-type Tregs had greater suppressive activity compared with P2RX7-KO Tregs, which failed to inhibit type 1 and follicular helper T-like responses. This was associated with increased tumor-specific IgG production by lung B cells in P2RX7-KO mice. We also observed that wild-type Tregs expressed higher levels of the immunosuppressive molecule CTLA-4 when compared with P2RX7-KO Tregs. Thus, we conclude that P2RX7 expression on Tregs is essential for their suppressive function in lung cancer and targeting P2RX7 may constitute a strategy to improve lung cancer treatment by alleviating Treg-mediated immune suppression.

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. NIH K99/R00A139381 NIH R01A170649 Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

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. Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

BackgroundThe development of ulcerative colitis (UC) is associated with inflammatory responses driven by effector CD4+T cells, including type 3 (Th17) cells and atypical pathogenic type 2 (Th2-like) cells. UC is also linked to accumulation of neutrophils that can amplify intestinal damage. The mechanisms behind the accumulation of colitogenic CD4+ T cells are not fully understood, particularly regarding how regulators of intracellular versus extracellular metabolites can drive such responses.Main findingsHere, we found that Pannexin-1 (PANX1) hemichannels, which promote ATP export to the extracellular environment, are crucial for the development of colitis. We found that PANX1, which is upregulated in UC patients, is required for the induction of colitis in multiple experimental models. The role of PANX1 is effector T cell-specific and is correlated with the accumulation of TNF-α producing pathogenic Th2-like cells. Effector conversion of CD4+ T cells into Th2-like cells depends on PANX1. Finally, PANX1-mediated pathogenic CD4+ T cell responses correlate with the accumulation of neutrophils during colitis.ConclusionsTogether, our results suggest that PANX1 promotes colitis-associated pathogenic Th2-like responses and a possible link between these cells and colitis neutrophilia.

Background The incidence of lung allergies is reduced in countries with higher prevalence of infection and environmental exposure to microbes. However, enteric bacterial infections do not always correlate with lower incidence of allergic disorders and how lung immunity to allergens can be regulated by gut exposure to pathogens and their toxins is not fully understood. Objective We used mouse models of enterotoxigenic Escherichia coli (ETEC) infection and lung allergy to examine how gut exposure to bacteria, or their related toxins, affects allergic lung inflammation. Methods Naïve C57BL/6 mice were infected with enterotoxigenic Escherichia coli (ETEC) or orally treated with the ETEC LT toxin, to mimic enteric bacterial infections. After two weeks, these mice were treated intranasally with Ovalbumin (OVA) and Papain or IL-33, followed by challenge with OVA, to induce allergic lung inflammation that was assessed using multiple readouts. Results Gut exposure to ETEC significantly inhibited allergic lung inflammation in a LT-dependent manner, as demonstrated by reduced tissue inflammation, less accumulation of type 2 cytokines, and reduced lung numbers of type 2 immune cells such as type 2 innate lymphoid cells (ILC2) and eosinophils. The anti-allergic capacity of LT was associated with reduced ability of lung ILC2s to recognize IL-33. Counterintuitively, deletion of either IL-33 or ILC2s significantly reverted the LT protective effect, suggesting the LT-mediated protection may occur through gut release and local sensing of IL-33. Conclusions Exposure to ETEC protects hosts against allergic lung inflammation through a negative feedback loop regulated by gut IL-33 release and sensing, suggesting a possible new immunological mechanism for reduced lung allergy incidence observed in areas with enteric bacterial infections. Key Messages ### Competing Interest Statement The authors have declared no competing interest. * CEUA : Comissão de Ética no Uso de Animais (Ethics Committee for Animal Use, USP) DTT : Dithiothreitol EDTA : Ethylenediaminetetraacetic acid ETEC : Enterotoxigenic Escherichia coli FACS : Fluorescence-activated cell sorting GM1 : Monosialotetrahexosylganglioside gMFI : Geometric mean fluorescence intensity H&E : Hematoxylin and eosin staining IACUC : Institutional Animal Care and Use Committee IL-33KO : Interleukin-33 knockout ILC2KO : Group 2 innate lymphoid cell–deficient mice (Il7r-Cre × Rorα flox/flox) KO : Knockout LD : Live/Dead dye LT : Heat-labile toxin (enterotoxin from E. coli ) LTB : Heat-labile toxin subunit B LTK63 : Non-toxic mutant of heat-labile toxin LT-KO ETEC : Plasmid-cured non-toxinogenic ETEC (LT−) Pap : Papain PAS : Periodic acid–Schiff staining PMA : Phorbol myristate acetate SCIREQ : Scientific Respiratory Equipment Inc. SD : Standard deviation siLP : small intestine lamina propria WT : Wild type São Paulo Research Foundation (FAPESP), 2021/06881-5, 2021/15185-2, 2019/13916-0, 2015/25364-0 National Council for Scientific and Technological Development (CNPq), 315712/2023-6 National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID), AI170649

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. Mayo Clinic and National Institutes of Health (NIH - H.B.d.S.: R01 AI170649) Mucosal and Regional Immunology (MUC)

Colorectal cancer (CRC) remains a significant global health burden, affecting millions of people every year. Despite recent advances, immunotherapies still face limited success against CRC, partly due to our incomplete understanding about how CRC induces CD8+ T cell exhaustion and which intracellular metabolic pathways can be leveraged to favor CD8+ T cell responses to CRC. CD8+ T cells express membrane-bound channels that can export metabolites, such as Pannexin-1 (PANX1). Panx1 exports ATP to the extracellular environment and has been shown, in our previous work, to promote the survival of memory CD8+ T cells through upregulation of the AMPK pathway. Notably, high PANX1 expression is associated with CD8+ T cell infiltration and better prognosis in stage IV CRC patients. Here, we show that PANX1 is essential for the induction of protective CD8+ T cell responses to CRC. PANX1-KO mice fail to control MC38 CRC tumors, had decreased numbers of antigen-specific tumor CD8+ T cells and increased expression of terminal exhaustion markers such as Tim-3. The defect in tumor CD8+ T cell accumulation is cell-intrinsic, as revealed by WT/PANX1-KO co-adoptive transfer experiments. We also found that intratumoral CD8+ T cells with better ability to respond to immunotherapy had increased AMPK activation. We are currently investigating whether PANX1 is necessary for CD8+ T cell AMPK activation inside tumors, and how PANX1 affects CD8+ T cells response to immunotherapy in the context of CRC. NIH AI139381 (R00) Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Tuberculosis induces diverse lesions, such as necrotic pneumonia, contributing to disease progression and transmission. Despite advances in understanding the role of ATP-gated P2RX7 ion channels in developing severe forms of tuberculosis, the regulation of this important signaling pathway remains unclear. Herein, we show that the ectonucleotidase CD39 plays an essential regulatory role in TB progression by preventing lung tissue damage, bacterial dissemination, and excessive inflammatory responses. Mechanistically, through its enzymatic activity on the cellular surface, CD39 protects infected macrophages from undergoing necrotic death mediated by P2RX7 activation. We proposed that by protecting macrophages from P2RX7-mediated cell death and bacterial dissemination, CD39 prevents the development of necrotic lesions. Altogether, these findings uncover a significant role for CD39 as an essential component of the molecular regulation underlying the development of severe tuberculosis. ![Figure][1] Brief In tuberculosis, necrotic granuloma-like structures release extracellular ATP (eATP), which triggers P2RX7-mediated immune cell death. CD39 degrades eATP, preventing P2RX7 activation and promoting macrophage survival, thereby limiting inflammation, tissue damage, and bacterial dissemination. ### Competing Interest Statement The authors have declared no competing interest. São Paulo Research Foundation, https://ror.org/02ddkpn78, 2015/20432-8, 2019/24700-8, 2020/09043-8 National Council for Scientific and Technological Development, https://ror.org/03swz6y49, 408909/2018-8, 303810/2018-1, 308870/2023-0, 140666/2018-4 [1]: pending:yes

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.

Sensing of extracellular ATP (eATP) controls CD8 + T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin 1 (Panx1). Whether Panx1 controls CD8 + T cell immune responses in vivo , however, has not been previously addressed. Here, we report that T -cell -specific Panx1 is needed for CD8 + T cell responses to viral infections and cancer. We found that CD8specific Panx1 promotes both effector and memory CD8 + T cell responses. Panx1 favors initial effector CD8 + T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation, which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast, Panx1 promotes memory CD8 + T cell survival primarily through ATP export and subsequent P2RX7 engagement, leading to improved mitochondrial metabolism. In summary, Panx1mediated eATP export regulates effector and memory CD8 + T cells through distinct purinergic receptors and different metabolic and signaling pathways.

CD4+ T cells are key components of the immune response during lung infections and can mediate protection against tuberculosis (TB) or influenza. However, CD4+ T cells can also promote lung pathology during these infections, making it unclear how these cells control such discrepant effects. Using mouse models of hyper virulent TB and influenza, we observe that exaggerated accumulation of parenchymal CD4+ T cells promotes lung damage. Low numbers of lung CD4+ T cells, in contrast, are sufficient to protect against hypervirulent TB. In both situations, lung CD4+ T cell accumulation is mediated by CD4+ T cell-specific expression of the extracellular ATP (eATP) receptor P2RX7. P2RX7 upregulation in lung CD4+ T cells promotes expression of the chemokine receptor CXCR3, favoring parenchymal CD4+ T cell accumulation. Our findings suggest that direct sensing of lung eATP by CD4+T cells is critical to induce tissue CD4+T cell accumulation and pathology during lung infections.

Tissue-resident memory T cells (TRM cells) are vital for the promotion of barrier immunity. The lung, a tissue constantly exposed to foreign pathogenic or non-pathogenic antigens, is not devoid of these cells. Lung TRM cells have been considered major players in either the protection against respiratory viral infections or the pathogenesis of lung allergies. Establishment of lung TRM cells rely on intrinsic and extrinsic factors. Among the extrinsic regulators of lung TRM cells, the magnitude of the impact of factors such as the route of antigen entry or the antigen natural tropism for the lung is not entirely clear. In this perspective, we provide a summary of the literature covering this subject and present some preliminary results on this potential dichotomy between antigen location versus antigen type. Finally, we propose a hypothesis to synthesize the potential contributions of these two variables for lung TRM cell development.