Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.

PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.

Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases.

To investigate whether nerve growth factor (NGF) induced angiogenesis of bone marrow mesenchymal stem cells (MSCs) and the underlying mechanisms.

Background: Phase 3 studies of immune checkpoint inhibitors have not shown a survival benefit in prostate cancer, but some patients have a profound anticancer response. Patients and Methods: We evaluated the efficacy of the CTLA-4 targeted agent, ipilimumab, in metastatic prostate cancer patients who had an incomplete biochemical response to initial androgen deprivation therapy (ADT) alone. Ten patients were enrolled, each treated with ipilimumab 10 mg/kg (every 3 weeks for up to 4 doses) with maintenance ipilimumab every 12 weeks for non-progressing patients. The primary endpoint was proportion of patients with an undetectable PSA. The total sample size was 30 patients, but there was an interim analysis planned at 10 for futility. If none of the 10 patients achieved an undetectable PSA, the study would be halted. Results: The study was halted at the interim analysis as none of the 10 patients achieved the primary endpoint, but 30% of patients demonstrated a >50% reduction in PSA, with one patient achieving a >90% reduction in PSA. Peripheral blood mononuclear cells (PBMC) examined by mass cytometry showed that patients with clinical responses had an increase in effector memory T-cell subsets as well as an increase in T-cell expression of T-bet, suggesting induction of a Th1 response. Conclusions: This study provides further evidence that ipilimumab has activity in some patients with prostate cancer and provides further rationale for the development of future studies aimed at identifying a subset of patients with CPRC that are more likely to derive a benefit from treatment with ipilimumab. Implications for Practice: There is insufficient evidence to use ipilimumab in prostate cancer in routine practice. Trial Registration: ClinicalTrials.gov, NCT01498978. Registered 26 December 2011. https://www.clinicaltrials.gov/ct2/show/NCT01498978?term=julie+graff&rank=3.

Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8+ T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1.

Ribociclib, a CDK4/6 inhibitor, demonstrates preclinical antitumor activity in combination with taxanes. We evaluated the safety and efficacy of ribociclib plus docetaxel in a phase Ib/II study in metastatic castration-resistant prostate cancer (mCRPC).

Therapeutic options currently available for metastatic castration-resistant prostate cancer (mCRPC) do not extend median overall survival >6 months. Therefore, the development of novel and effective therapies for mCRPC represents an urgent medical need. T cell engagers (TCEs) have emerged as a promising approach for the treatment of mCRPC due to their targeted mechanism of action. However, challenges remain in the clinic due to the limited efficacy of TCEs observed thus far in solid tumors as well as the toxicities associated with cytokine release syndrome (CRS) due to the usage of high-affinity anti-CD3 moieties such as OKT3.

White blood cells (WBCs) are a key component of the mammalian immune system and play an essential role in surveillance, defense, and adaptation against foreign pathogens. Apart from their roles in the active combat of infection and the development of adaptive immunity, immune cells are also involved in tumor development and metastasis. Antibody-based therapeutics have been developed to regulate (i.e. selectively activate or inhibit immune function) and harness immune cells to fight malignancy. Alternatively, non-invasive tracking of WBC distribution can diagnose inflammation, infection, fevers of unknown origin (FUOs), and cancer. Magnetic Particle Imaging (MPI) is a non-invasive, non-radioactive, and sensitive medical imaging technique that uses safe superparamagnetic iron oxide nanoparticles (SPIOs) as tracers. MPI has previously been shown to track therapeutic stem cells for over 87 days with a ~200 cell detection limit. In the current work, we utilized antibody-conjugated SPIOs specific to neutrophils for in situ labeling, and non-invasive and radiation-free tracking of these inflammatory cells to sites of infection and inflammation in an in vivo murine model of lipopolysaccharide-induced myositis. MPI showed sensitive detection of inflammation with a contrast-to-noise ratio of ~8-13.

Sotigalimab is an agonistic anti-CD40 mAb that can modulate antitumor immune responses. In a phase II clinical trial of sotigalimab combined with neoadjuvant chemoradiation (CRT) in locally advanced esophageal/gastroesophageal junction (E/GEJ) cancer with the primary outcome of efficacy as measured by pathologic complete response (pCR) rate, the combination induced pCR in 38% of treated patients. We investigated the mechanism of action of sotigalimab in samples obtained from this clinical trial. Tumor biopsies and peripheral blood samples were collected at baseline, following an initial dose of sotigalimab, and at the time of surgery after CRT completion from six patients. High dimensional single-cell techniques were used, including combined single-cell RNA-sequencing and proteomics (CITEseq) and multiplexed ion beam imaging, to analyze immune responses. Sotigalimab dramatically remodeled the immune compartment in the periphery and within the tumor microenvironment (TME), increasing expression of molecules related to antigen processing and presentation and altering metabolic pathways in myeloid cells. Concomitant with these changes in myeloid cells, sotigalimab treatment primed new T cell clonotypes and increased the density and activation of T cells with enhanced cytotoxic function. Sotigalimab treatment also induced a decrease in the frequency of Tregs in the TME. These findings indicate that a single dose of sotigalimab leads to enhanced antigen presentation that can activate T cells and induce new T cell clones. This restructuring of the TME provides elements which are critical to the development of effective antitumor immune responses and improved clinical outcomes.

Human endometrial stem cells (EnSCs) have the potential to be 'off the shelf' clinical reagents for the treatment of heart failure. Here, using an immunocompetent rat model of myocardial infarction (MI), we provide evidence that the functional benefits of EnSC transplantation are principally and possibly exclusively through a paracrine effect. Human EnSCs were delivered by intramyocardial injection into rats 30 min. after coronary ligation. EnSC therapy significantly preserved viable myocardium in the infarct zone and improved cardiac function at 28 days. Despite increased viable myocardium and vascular density, there was scant evidence of differentiation of EnSCs into any cardiovascular cell type. Cultured human EnSCs expressed a distinctive profile of cytokines that enhanced the survival, proliferation and function of endothelial cells in vitro. When injected into the peri-infarct zone, human EnSCs activated AKT, ERK1/2 and STAT3 and inhibited the p38 signalling pathway. EnSC therapy decreased apoptosis and promoted cell proliferation and c-kit+ cell recruitment in vivo. Myocardial protection and enhanced post-infarction regeneration by EnSCs is mediated primarily by paracrine effects conferred by secreted cytokines that activate survival pathways and recruit endogenous progenitor stem cells. Menstrual blood provides a potentially limitless source of biologically competent 'off the shelf' EnSCs for allogeneic myocardial regenerative medicine.

Sipuleucel-T is a US Food and Drug Administration-approved immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Its mechanism of action is not fully understood. This prospective trial evaluated the direct immune effects of systemically administered sipuleucel-T on prostatic cancer tissue in the preoperative setting.

CTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.

Although DNA methylation is a key regulator of gene expression, the comprehensive methylation landscape of metastatic cancer has never been defined. Through whole-genome bisulfite sequencing paired with deep whole-genome and transcriptome sequencing of 100 castration-resistant prostate metastases, we discovered alterations affecting driver genes that were detectable only with integrated whole-genome approaches. Notably, we observed that 22% of tumors exhibited a novel epigenomic subtype associated with hypermethylation and somatic mutations in TET2, DNMT3B, IDH1 and BRAF. We also identified intergenic regions where methylation is associated with RNA expression of the oncogenic driver genes AR, MYC and ERG. Finally, we showed that differential methylation during progression preferentially occurs at somatic mutational hotspots and putative regulatory regions. This study is a large integrated study of whole-genome, whole-methylome and whole-transcriptome sequencing in metastatic cancer that provides a comprehensive overview of the important regulatory role of methylation in metastatic castration-resistant prostate cancer.

Immune checkpoint inhibitors (ICI) can achieve durable responses in a subset of patients with locally advanced or metastatic urothelial carcinoma (aUC). The use of tumor genomic profiling in clinical practice may help suggest biomarkers to identify patients most likely to benefit from ICI.

Sipuleucel-T is a US Food and Drug Administration-approved autologous cellular immunotherapy that improves survival in patients with metastatic castration-resistant prostate cancer (mCRPC). We examined whether administering ipilimumab after sipuleucel-T could modify immune and/or clinical responses to this treatment.

Men with metastatic castration-resistant prostate cancer (mCRPC) have limited treatment options after progressing on hormonal therapy and chemotherapy. Here, we evaluate the safety and efficacy of atezolizumab (anti-PD-L1) + radium-223 dichloride (radium-223) in men with mCRPC.

Increasing evidence supports targeting the adenosine pathway in immuno-oncology with several clinical programs directed at adenosine A2 receptor (A2AR, A2BR), CD73 and CD39 in development. Through a cyclic-AMP-mediated intracellular cascade, adenosine shifts the cytokine and cellular profile of the tumor microenvironment away from cytotoxic T cell inflammation toward one of immune tolerance. A perpetuating cycle of tumor cell proliferation, tissue injury, dysregulated angiogenesis, and hypoxia promote adenosine accumulation via ATP catabolism. Adenosine receptor (eg, A2AR, A2BR) stimulation of both the innate and adaptive cellular precursors lead to immunosuppressive phenotypic differentiation. Preclinical work in various tumor models with adenosine receptor inhibition has demonstrated restoration of immune cell function and tumor regression. Given the broad activity but known limitations of anti-programmed cell death protein (PD1) therapy and other checkpoint inhibitors, ongoing studies have sought to augment the successful outcomes of anti-PD1 therapy with combinatorial approaches, particularly adenosine signaling blockade. Preliminary data have demonstrated an optimal safety profile and enhanced overall response rates in several early phase clinical trials with A2AR and more recently CD73 inhibitors. However, beneficial outcomes for both monotherapy and combinations have been mostly lower than expected based on preclinical studies, indicating a need for more nuanced patient selection or biomarker integration that might predict and optimize patient outcomes. In the context of known immuno-oncology biomarkers such as tumor mutational burden and interferon-associated gene expression, a comparison of adenosine-related gene signatures associated with clinical response indicates an underlying biology related to immunosuppression, angiogenesis, and T cell inflammation. Importantly, though, adenosine associated gene expression may point to a unique intratumoral phenotype independent from IFN-γ related pathways. Here, we discuss the cellular and molecular mechanisms of adenosine-mediated immunosuppression, preclinical investigation of adenosine signaling blockade, recent response data from clinical trials with A2AR, CD73, CD39 and PD1/L1 inhibitors, and ongoing development of predictive gene signatures to enhance combinatorial immune-based therapies.

Intratumoral heterogeneity (ITH)-defined as genetic and cellular diversity within a tumor-is linked to failure of immunotherapy and an inferior anti-tumor immune response. The underlying mechanism of this association is unknown. To address this question, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population, labeled with YFP and RFP tags respectively to enable precise spatial tracking. The resulting mixed-population tumors exhibited distinct regions comprised of YFP+ (hot) cells, RFP+ (cold) cells, or a mixture. We found that tumor regions occupied by hot tumor cells (YFP+) harbored more total T cells and a higher frequency of Th1 cells and IFNγ+ CD8 T cells compared to regions occupied by cold tumor cells (RFP+), whereas immunosuppressive macrophages showed the opposite spatial pattern. We identified the chemokine CX3CL1, produced at higher levels by our cold tumors, as a mediator of intratumoral macrophage accumulation, particularly immunosuppressive CD206Hi macrophages. Furthermore, we examined the response of heterogeneous tumors to a therapeutic combination of PD-1 blockade and CD40 agonist on a region-by-region basis. While the combination successfully increases Th1 abundance in "cold" tumor regions, it fails to bring overall T cell activity to the same level as seen in "hot" regions. The presence of the "cold" cells thus ultimately leads to a failure of the therapy to induce tumor rejection. Collectively, our results demonstrate that the organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.