Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

Purpose: Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS. Experimental Design: Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression. Results: Sorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival. Conclusions: Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.

Activation status dictates the function of unlicensed natural killer cells in mice and humans.

Natural killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class 1 molecules, resulting in differential responses upon activation in a process called "licensing" or "arming." NK cells expressing receptors that bind self-MHC are considered licensed due to an augmented effector lytic function capability compared with unlicensed subsets. However, we demonstrated that unlicensed NK subsets instead positively regulate the adaptive T-cell response during viral infections that are related to localization and cytokine production. In this study, the differential effects of the two types of NK subsets were contingent on the environment in viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) murine cytomegalovirus (MCMC) led to a loss of licensing-associated differences, as compared with mice with low-dose (LD) infection: the unlicensed NK subset no longer localized in lymph nodes (LNs), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled the phenotypes of both human and mouse NK cells in an HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to the effects of subset depletion in T-cell replete models, the licensed NK cell subsets still dominated antiviral responses after HSCT. Overall, our results highlight the intricate tuning of NK cells and how it affects overall immune responses with regard to licensing patterns and their dependency on the level of stimulation and activation status.

Increased efficacy of dual proinflammatory cytokine blockade on acute GVHD while maintaining GVT effects.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a potential curative option for treating a variety of hematologic diseases, but acute and chronic graft-versus-host disease (GVHD) remain major barriers limiting efficacy. Acute gut GVHD occurs with marked increases in proinflammatory cytokines (including TNF and IL-6), which we recently demonstrated was exacerbated in obesity resulting in severe gastrointestinal pathology. Given the pleiotropic and overlapping effects of these 2 cytokines, we assessed the impact of dual TNF and IL-6R blockade on GVHD as well as graft-versus tumor (GVT) effects in different mouse GVHD models. Early administration of combined blockade resulted in greater protection and survival from acute gut GVHD compared with single blockade regimens and even development of later chronic skin GVHD. Importantly, double cytokine blockade preserved GVT effects reinforcing that GVT and GVHD can be delineated and may result in greater efficacy in allo-HSCT.

PD-1 Blockade Reverses Obesity-Mediated T Cell Priming Impairment.

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.

Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute and chronic graft-versus-host disease (GVHD). The impact of obesity on allo-HSCT outcomes is poorly understood. Here, we report that obesity had a negative and selective impact on acute gut GVHD after allo-HSCT in mice with diet-induced obesity (DIO). These animals exhibited increased gut permeability, endotoxin translocation across the gut, and radiation-induced gastrointestinal damage after allo-HSCT. After allo-HSCT, both male and female DIO mouse recipients showed increased proinflammatory cytokine production and expression of the GVHD marker ST2 (IL-33R) and MHC class II molecules; they also exhibited decreased survival associated with acute severe gut GVHD. This rapid-onset, obesity-associated gut GVHD depended on donor CD4+ T cells and occurred even with a minor MHC mismatch between donor and recipient animals. Retrospective analysis of clinical cohorts receiving allo-HSCT transplants from unrelated donors revealed that recipients with a high body mass index (BMI, >30) had reduced survival and higher serum ST2 concentrations compared with nonobese transplant recipients. Assessment of both DIO mice and allo-HSCT recipients with a high BMI revealed reduced gut microbiota diversity and decreased Clostridiaceae abundance. Prophylactic antibiotic treatment protected DIO mouse recipients from endotoxin translocation across the gut and increased inflammatory cytokine production, as well as gut pathology and mortality, but did not protect against later development of chronic skin GVHD. These results suggest that obesity-induced alterations of the gut microbiota may affect GVHD after allo-HSCT in DIO mice, which could be ameliorated by prophylactic antibiotic treatment.

Analysis of tumor-infiltrating NK and T cells highlights IL-15 stimulation and TIGIT blockade as a combination immunotherapy strategy for soft tissue sarcomas.

PURPOSE: Given 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 DESIGN: Using 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. RESULTS: TILs 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. CONCLUSION: Intratumoral 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.

Minimal PD-1 expression in mouse and human NK cells under diverse conditions.

PD-1 expression is a hallmark of both early antigen-specific T cell activation and later chronic stimulation, suggesting key roles in both naive T cell priming and memory T cell responses. Although significant similarities exist between T cells and NK cells, there are critical differences in their biology and functions reflecting their respective adaptive and innate immune effector functions. Expression of PD-1 on NK cells is controversial despite rapid incorporation into clinical cancer trials. Our objective was to stringently and comprehensively assess expression of PD-1 on both mouse and human NK cells under multiple conditions and using a variety of readouts. We evaluated NK cells from primary human tumor samples, after ex vivo culturing, and from multiple mouse tumor and viral models using flow cytometry, quantitative reverse-transcriptase PCR (qRT-PCR), and RNA-Seq for PD-1 expression. We demonstrate that, under multiple conditions, human and mouse NK cells consistently lack PD-1 expression despite the marked upregulation of other activation/regulatory markers, such as TIGIT. This was in marked contrast to T cells, which were far more prominent within all tumors and expressed PD-1. These data have important implications when attempting to discern NK from T cell effects and to determine whether PD-1 targeting can be expected to have direct effects on NK cell functions.

Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade.

The recent successes of immunotherapy have shifted the paradigm in cancer treatment, but because only a percentage of patients are responsive to immunotherapy, it is imperative to identify factors impacting outcome. Obesity is reaching pandemic proportions and is a major risk factor for certain malignancies, but the impact of obesity on immune responses, in general and in cancer immunotherapy, is poorly understood. Here, we demonstrate, across multiple species and tumor models, that obesity results in increased immune aging, tumor progression and PD-1-mediated T cell dysfunction which is driven, at least in part, by leptin. However, obesity is also associated with increased efficacy of PD-1/PD-L1 blockade in both tumor-bearing mice and clinical cancer patients. These findings advance our understanding of obesity-induced immune dysfunction and its consequences in cancer and highlight obesity as a biomarker for some cancer immunotherapies. These data indicate a paradoxical impact of obesity on cancer. There is heightened immune dysfunction and tumor progression but also greater anti-tumor efficacy and survival after checkpoint blockade which directly targets some of the pathways activated in obesity.

Identification of QTLs for capsaicinoids, fruit quality, and plant architecture-related traits in an interspecific Capsicum RIL population.

Quantitative trait loci (QTL) analyses in pepper are common for horticultural, disease resistance, and fruit quality traits; although none of the studies to date have used sequence-based markers associated with genes. In this study we measured plant architectural, phenological, and fruit quality traits in a pepper mapping population consisting of 92 recombinant inbred lines derived from a cross between Capsicum frutescens acc. 2814-6 and C. annuum var. NuMexRNAKY. Phenotypic measurements were correlated to loci in a high-density EST-based genetic map. In total, 96 QTL were identified for 38 traits, including 12 QTL associated with capsaicinoid levels. Twenty-one loci showed correlation among seemingly unrelated phenotypic categories, highlighting tight linkage or shared genetics between previously unassociated traits in pepper.