PurposeGiven the unmet need for novel immunotherapy in soft tissue sarcoma (STS), we sought to characterize the phenotype and function of intratumoral natural killer (NK) and T cells to identify novel strategies to augment tumor-infiltrating lymphocyte (TIL) function.Experimental designUsing prospectively collected specimens from dogs and humans with sarcomas, archived specimens, and The Cancer Genome Atlas (TCGA) data, we evaluated blood and tumor NK and T cell phenotype and function and correlated those with outcome. We then assessed the effects of interleukin 15 (IL-15) stimulation on both NK and T cell activation and TIGIT upregulation. Finally, we evaluated cytotoxic effects of IL-15 combined with TIGIT blockade using a novel anti-TIGIT antibody.ResultsTILs were strongly associated with survival outcome in both archived tissue and TCGA, but higher TIL content was also associated with higher TIGIT expression. Compared with blood, intratumoral NK and T cells showed significantly higher expression of both activation and exhaustion markers, in particular TIGIT. Ex vivo stimulation of blood and tumor NK and T cells from patients with STS with IL-15 further increased both activation and exhaustion markers, including TIGIT. Dogs with metastatic osteosarcoma receiving inhaled IL-15 also exhibited upregulation of activation markers and TIGIT. Ex vivo, combined IL-15 and TIGIT blockade using STS blood and tumor specimens significantly increased cytotoxicity against STS targets.ConclusionIntratumoral NK and T cells are prognostic in STS, but their activation is marked by significant upregulation of TIGIT. Our data suggest that combined IL-15 and TIGIT blockade may be a promising clinical strategy in STS.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by the occurrence of acute and chronic graft-versus-host disease (GVHD). We have recently demonstrated that obesity results in exacerbated acute gastrointestinal GVHD in both mouse models and clinical outcomes due to increased pro-inflammatory cytokine responses and microbiota alterations. We therefore wanted to delineate the role of the various parameters in obesity, adiposity, effects of high-fat (HF) diet, and the role of microbiome on GVHD pathogenesis, by taking advantage of a mouse strain resistant to diet-induced obesity (DIO). Female BALB/c mice are resistant to DIO phenotype with approximately 50% becoming DIO under HF diets. The DIO-susceptible recipients rapidly succumb to acute gut GVHD, whereas the DIO-resistant recipient littermates, which do not become obese, are partially protected from GVHD, indicating that being on HF diet alone contributes to but is not the primary driver of GVHD. Microbiome assessment revealed restricted diversity in both cohorts of mice, but coprophagy normalizes the microbiota in mice housed together. We then individually housed DIO-resistant, DIO-susceptible, and lean control mice. Notably, each of the individually housed groups demonstrates marked restricted diversity that has been shown to occur from the stress of single housing. Despite the restricted microbiome diversity, the GVHD pathogenesis profile remains consistent in the group-housed mice, with the lean control single-housed mice exhibiting no acute GVHD and DIO-resistant recipients showing again partial protection. These results demonstrate that the deleterious effects of obesity on acute gut GVHD are critically dependent on adiposity with the HF diet also playing a lesser role, and the microbiome alterations with obesity instead appear to fuel ongoing acute GVHD processes.

Despite obesity reaching pandemic proportions, its impact on antigen-specific T cell responses is still unclear. We have recently demonstrated that obesity results in increased expression of PD-1 on T cells, and checkpoint blockade targeting PD-1/PD-L1 surprisingly resulted in greater clinical efficacy in cancer therapy. Adverse events associated with this therapy center around autoimmune reactions. In this study, we examined the impact of obesity on T cell priming and on autoimmune pathogenesis using the mouse model experimental autoimmune encephalomyelitis (EAE), which is mediated by autoreactive myelin-specific T cells generated after immunization. We observed that diet-induced obese (DIO) mice had a markedly delayed EAE onset and developed milder clinical symptoms compared to mice on control diet (CD). This delay was associated with impaired generation of myelin-specific T cell numbers and concurrently correlated with increased PD-L1 upregulation on antigen-presenting cells in secondary lymphoid organs. PD-1 blockade during the priming stage of EAE restored disease onset and severity and increased numbers of pathogenic CD4+ T cells in the central nervous system (CNS) of DIO mice to similar levels to those of CD mice. Administration of anti-PD-1 after onset of clinical symptoms did not increase EAE pathogenesis demonstrating that initial priming is the critical juncture affected by obesity. These findings demonstrate that obesity impairs antigen-specific T cell priming, but this can be reversed with PD-1 blockade. Our results further suggest that PD-1 blockade may increase the risk of autoimmune toxicities, particularly in obesity.

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IntroductionThe incidence of obesity, a condition characterized by systemic chronic inflammation, has reached pandemic proportions and is a poor prognostic factor in many pathologic states. However, its role on immune parameters has been diverse and at times contradictory. We have previously demonstrated that obesity can result in what has been called the “obesity paradox” which results in increased T cell exhaustion, but also greater efficacy of immune checkpoint blockade in cancer treatment.MethodsThe role of obesity, particularly in the context of aging, has not been robustly explored using preclinical models. We therefore evaluated how age impacts the immune environment on T cell development and function using diet-induced obese (DIO) mice.ResultsWe observed that DIO mice initially displayed greater thymopoiesis but then developed greater thymic involution over time compared to their lean counterparts. Both aging and obesity resulted in increased T cell memory conversion combined with increased expression of T cell exhaustion markers and Treg expansion. This increased T cell immunosuppression with age then resulted in a loss of anti-tumor efficacy by immune checkpoint inhibitors (ICIs) in older DIO mice compared to the younger DIO counterparts.DiscussionThese results suggest that both aging and obesity contribute to T cell dysfunction resulting in increased thymic involution. This combined with increased T cell exhaustion and immunosuppressive parameters affects immunotherapy efficacy reducing the advantage of obesity in cancer immunotherapy responses.