Silva, Ildefonso

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 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 Description Immunotherapy combinations can improve patient outcomes, yet the interactions within the tumor microenvironment (TME) that drive therapeutic synergy are poorly understood. Tumor establishment drives monocyte recruitment and differentiation into tumor-associated macrophages (TAMs), which have essential roles in coordinating immune responses and are thus attractive targets for therapeutic modulation. In a murine model of combination anti-PD-1 and anti-PD-L1 checkpoint blockade, tumor control was associated with increased infiltration of CD8+ T cells and M1-like repolarization of TAMs. Live-cell imaging of the tumor microenvironment revealed stable contacts between tumor-infiltrating CD8+ T cells and TAMs, in which the extent of the contact interfaces increased with combination immunotherapy. Treatment with anti-PD-L1 was able to increase macrophage expression of pro-inflammatory factors and phagocytic activity, suggesting a role for TAMs to reactivate CD8+ T cells in the TME. However, co-treatment with anti-PD-1 was ultimately necessary for tumor control, indicating the need for combination targeting of the TME. Funding Sources Mayo Clinic David and Margaret Grohne Cancer Immunotherapy Pilot Award Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

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 PD-1- and PD-L1-targeted immune checkpoint blockade therapy has shown great clinical potential. Unfortunately, this therapy has been ineffective in a majority of patients bearing solid tumors due to the highly immunosuppressive nature of the tumor microenvironment. As macrophages compose the most abundant myeloid cell subset within the tumor, current macrophage-targeted treatments aim to deplete macrophages within the tumor to reduce immune suppression. However, our goal is to determine whether αPD-1 and αPD-L1 therapy can harness macrophage potential by shifting tumor associated macrophages (TAMs) from a suppressive M2 to a pro-inflammatory M1 phenotype. Our preliminary data has shown that αPD-1/αPD-L1 treatment of mice bearing B16 murine melanoma leads to controlled tumor growth and this was accompanied by a higher M1:M2 ratio and CD8 T-cell abundance. Therefore, we hypothesized that αPD-1/αPD-L1 therapy may drive TAMs towards a pro-inflammatory phenotype and to become better activators of CD8 T-cells. By analyzing antigen presentation machinery on TAMs, we show that in the presence of tumor cells, αPD-L1 blockade upregulates expression of MHC1 and MHC2 on TAMs. Unexpectedly, direct αPD-L1 blockade on TAMs leads to reduced CD80 surface due to mechanisms still to be elucidated. Further, we analyzed co-cultures of αPD-L-treated TAMs loaded with ovalbumin and naïve OT1 cells, which led to greater proliferation of naïve CD8 T-cells compared to their isotype control counterparts. Our observations thus far support the ability of αPD-1/αPD-L1 therapy to induce an M1-phenotype shift among macrophages and also αPD-L1 blockade alone can drive TAMs to become better CD8 T-cell activators. Mayo Clinic David and Margaret Grohne Cancer Immunotherapy and Mayo Clinic Immune Monitoring Core Pilot Awards

Abstract Aging dramatically impacts T cells, contributing to diminished and delayed responses to infection. While the development and maintenance of lymphocytes certainly undergo intrinsic changes with age, the contribution of cell-extrinsic forces, such as the aging tissue microenvironment, has been difficult to uncouple. Macroscopic changes to the lymph nodes have been well described, but the impact of aging on lymph node stromal subsets is less understood. Here, we visualized naïve T cell migration in the aged murine lymph node using live-cell 2-photon microscopy and determined that T cell motility was impaired in regions of increased fibrotic deposition. Using fluorescent reporters for Ccl19 and Cxcl12, homeostatic chemokines important for stromal:lymphocyte crosstalk, we also found that the distribution of stromal cells expressing these chemokines was altered with age, and that migrating naïve T cells were excluded from regions of chemokine expression. Flow cytometric analyses of fibroblastic reticular cells (FRCs) revealed decreased proportions of FRCs within the mesenteric lymph nodes and decreased putative FRC progenitors in the skin-draining lymph nodes. FRC progenitors at both sites had reduced surface expression of lymphotoxin-beta receptor, an important receptor for FRC differentiation and maintenance. We hypothesize that age-associated changes to FRC maturation contribute to increased lymph node fibrosis and altered localization of homeostatic factors. Current studies have indicated that in vitro cultured lymph node fibroblasts are sensitive to TNF-alpha and respond by increasing expression of alpha-smooth muscle actin, suggesting a mechanism by which aging remodels the lymph node microenvironment.