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To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250??$\mu$g/ip day 0) and CTLA4-Ig (200??$\mu$g/ip day 2), we administered low-dose IL-2 (2000??IU/day) starting on posttransplant day 14 for 3??weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100??days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGF$\beta$ pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules.

BACKGROUND Current immunotherapies still have limited successful rates among cancers. It is now recognized that T cell functional state in the tumor microenvironment (TME) is a key determinant for effective antitumor immunity and immunotherapy. In addition to exhaustion, cellular senescence in tumor-infiltrating T cells (TILs) has recently been identified as an important T cell dysfunctional state induced by various malignant tumors. Therefore, a better understanding of the molecular mechanism responsible for T cell senescence in the TME and development of novel strategies to prevent effector T cell senescence are urgently needed for cancer immunotherapy. METHODS Senescent T cell populations in the TMEs in mouse lung cancer, breast cancer, and melanoma tumor models were evaluated. Furthermore, T cell senescence induced by mouse tumor and regulatory T (Treg) cells in vitro was determined with multiple markers and assays, including real-time PCR, flow cytometry, and histochemistry staining. Loss-of-function strategies with pharmacological inhibitors and the knockout mouse model were used to identify the potential molecules and pathways involved in T cell senescence. In addition, melanoma mouse tumor immunotherapy models were performed to explore the synergistical efficacy of antitumor immunity via prevention of tumor-specific T cell senescence combined with anti-programmed death-ligand 1 (anti-PD-L1) checkpoint blockade therapy. RESULTS We report that both mouse malignant tumor cells and Treg cells can induce responder T cell senescence, similar as shown in human Treg and tumor cells. Accumulated senescent T cells also exist in the TME in tumor models of lung cancer, breast cancer and melanoma. Induction of ataxia-telangiectasia mutated protein (ATM)-associated DNA damage is the cause for T cell senescence induced by both mouse tumor cells and Treg cells, which is also regulated by mitogen-activated protein kinase (MAPK) signaling. Furthermore, blockages of ATM-associated DNA damage and/or MAPK signaling pathways in T cells can prevent T cell senescence mediated by tumor cells and Treg cells in vitro and enhance antitumor immunity and immunotherapy in vivo in adoptive transfer T cell therapy melanoma models. Importantly, prevention of tumor-specific T cell senescence via ATM and/or MAPK signaling inhibition combined with anti-PD-L1 checkpoint blockade can synergistically enhance antitumor immunity and immunotherapy in vivo. CONCLUSIONS These studies prove the novel concept that targeting both effector T cell senescence and exhaustion is an effective strategy and can synergistically enhance cancer immunotherapy.

BACKGROUND AND AIMS Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.