Lima, Maria Regina D’Império

Abstract 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.

Abstract Description Vaccination is essential for preventing viral infections and promoting public health; however, its efficacy may vary due to factors such as the nature of the pathogen, vaccine type, and individual immune response. It is known that intranasal immunizations can enhance the immune response by stimulating tissue-resident memory T cells (TRMs), which promote early viral clearance at the infection site. Our study aims to optimize the generation and maintenance of TRMs in the lungs using purinergic signaling components as adjuvants. For this, we crossed B knockout mice with hACE2 mice, generating a B cell-deficient lineage capable of internalizing active SARS-CoV-2 (BKO-hACE2). C57BL/6 and BKO-hACE2 mice were immunized with inactivated SARS-CoV-2 receiving one intravenous dose followed by three intranasal booster doses. After 30 days, lungs were analyzed by flow cytometry and histopathology. Additionally, we evaluated the effects of ATP (0.5 mM to 10 mM) during immunizations, finding that lower doses (0.5 mM) increased the frequency of TRMs (CD4+ and CD8+), while higher doses reduced this population. This suggests that ATP may interact with the P2X7 receptor, promoting either proliferation or cell death. Although infection data did not provide conclusive evidence of T cell-mediated protection in the absence of antibodies, the potential of ATP as a possible adjuvant opens new avenues for enhancing immune responses in experimental models. Funding Sources FAPESP - 2023/15373-9 Topic Categories Viral Immunology (VIR)

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)

SUMMARY 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. GRAPHICAL ABSTRACT 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.

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 Recent studies suggest that the intestinal microenvironment regulate lung immune cells. Conversely, changes in the lung can modulate the gut immune system. Yet, mucosal-associated immune responses have been studied in a compartmentalized manner, as if there is no communication between different organs. Thus, the role of the gut-lung axis in mucosal immune responses is poorly understood. Using enterotoxigenic Escherichia coli (ETEC), we established a model of intestine-localized infection without translocation to any other tissue. In response to ETEC, type 2 innate lymphoid cells (ILC2s) and eosinophils accumulated in the intestinal lamina propria, even after bacterial clearance. In addition, oral ETEC transiently increased the numbers of ILC2 and eosinophils in the lung parenchyma. To test if oral ETEC promote lung immune responses to allergen, we exposed mice that recently cleared ETEC to intranasal papain/ovalbumin allergy induction model. Unexpectedly, ETEC-exposed mice were significantly resistant to allergen-induced lung inflammation, with reduced number of ILC2, Type 2 effector CD4+ T cells (Th2) and eosinophils. Paradoxically, oral ETEC-induced protection against lung allergen exposure was dependent on IL-33: IL-33KO mice developed partial lung inflammation in this model, and no protection induced by ETEC exposure was observed. Importantly, protection from allergy was achieved with ETEC heat-labile toxin (LT) oral exposure; we also found that the toxicity domain of LT is needed for this protection. Our results indicate that gastrointestinal bacterial infection mediators (toxins) negatively regulate the onset of allergic inflammation and may provide a novel immunological basis for the “hygiene hypothesis”. R01AI170649 FAPESP 2021/15185-2

Abstract CD4+ T cells are crucial to control pulmonary tuberculosis, but also favor uncontrolled inflammation in severe disease. Effective CD4+ T cell response to tuberculosis is related to tissue location: Lung-resident CD4+ T cells display higher effector function in comparison to circulating cells. Among the factors favoring T cell residency in non-lymphoid tissues, extracellular ATP sensing via P2RX7 is crucial for the generation of virus-specific resident memory CD8+ T cells. Yet, its role in CD4+ T cell non-lymphoid tissue residency has not been defined. Moreover, mycobacterial infection induces high extracellular ATP levels due to lung tissue damage, which is increased in response to hypervirulent strains. Here, we used P2rx7 ablation models to test the role of P2RX7 in CD4+T cell function in tuberculosis. In response to the hypervirulent Mycobacterium bovis MP287/03 strain, lung-resident WT CD4+ T cells express higher P2RX7 levels compared to circulating CD4+ T cells. Compared to WT mice, P2RX7-deficient (P2rx7−/−) mice display reduced numbers of antigen-specific (I-AbESAT-64-17) and IFN-γ-producing CD4+ T cells in the lung parenchyma. Using cell adoptive transfers, while recipient CD4-deficient mice transferred with CD4+ WT T cells are protected, mice that received CD4+P2rx7−/− T cells develop severe tuberculosis. Mechanistically, CD4+P2rx7−/− T cells have poorer accumulation and IFN-γ production in the lung parenchyma; moreover, they show reduced proliferation in situ. This defect in proliferation is exclusively observed in the lung, with no differences observed in draining lymph nodes. Together, our results show that P2RX7 promote the accumulation of protective CD4+ T cells in the lung during severe tuberculosis.