CD8+ T cell targeting of tumor antigens presented by HLA-E.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E-restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E-restricted CD8+ T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E-restricted CD8+ T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E+ prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8+ T cell-based immunotherapies.

Natural Killer Cell Dysfunction In Human Bladder Cancer Is Caused By Tissue-Specific Suppression of SLAMF6 Signaling.

NK cells are innate lymphocytes critical for surveillance of viruses and tumors, however the mechanisms underlying NK cell dysfunction in cancer are incompletely understood. We assessed the effector function of NK cells from bladder cancer patients and found severe dysfunction in NK cells derived from tumors versus peripheral blood. While both peripheral and tumor-infiltrating NK cells exhibited conserved patterns of inhibitory receptor over-expression, this did not explain the observed defects in NK surveillance in bladder tumors. Rather, TME-specific TGF-ß and metabolic perturbations such as hypoxia directly suppressed NK cell function. Specifically, an oxygen-dependent reduction in signaling through SLAMF6 was mechanistically responsible for poor NK cell function, as tumor-infiltrating NK cells cultured ex vivo under normoxic conditions exhibited complete restoration of function, while deletion of SLAMF6 abrogated NK cell cytolytic function even under normoxic conditions. Collectively, this work highlights the role of tissue-specific factors in dictating NK cell function, and implicates SLAMF6 signaling as a rational target for immuno-modulation to improve NK cell function in bladder cancer.

Society for Immunotherapy of Cancer (SITC) recommendations on intratumoral immunotherapy clinical trials (IICT): from premalignant to metastatic disease.

BACKGROUND: Intratumorally delivered immunotherapies have the potential to favorably alter the local tumor microenvironment and may stimulate systemic host immunity, offering an alternative or adjunct to other local and systemic treatments. Despite their potential, these therapies have had limited success in late-phase trials for advanced cancer resulting in few formal approvals. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to determine how to design clinical trials with the greatest chance of demonstrating the benefits of intratumoral immunotherapy for patients with cancers across all stages of pathogenesis. METHODS: An Intratumoral Immunotherapy Clinical Trials Expert Panel composed of international key stakeholders from academia and industry was assembled. A multiple choice/free response survey was distributed to the panel, and the results of this survey were discussed during a half-day consensus meeting. Key discussion points are summarized in the following manuscript. RESULTS: The panel determined unique clinical trial designs tailored to different stages of cancer development-from premalignant to unresectable/metastatic-that can maximize the chance of capturing the effect of intratumoral immunotherapies. Design elements discussed included study type, patient stratification and exclusion criteria, indications of randomization, study arm determination, endpoints, biological sample collection, and response assessment with biomarkers and imaging. Populations to prioritize for the study of intratumoral immunotherapy, including stage, type of cancer and line of treatment, were also discussed along with common barriers to the development of these local treatments. CONCLUSIONS: The SITC Intratumoral Immunotherapy Clinical Trials Expert Panel has identified key considerations for the design and implementation of studies that have the greatest potential to capture the effect of intratumorally delivered immunotherapies. With more effective and standardized trial designs, the potential of intratumoral immunotherapy can be realized and lead to regulatory approvals that will extend the benefit of these local treatments to the patients who need them the most.

Structural basis for self-discrimination by neoantigen-specific TCRs.

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

Gemcitabine and cisplatin plus nivolumab as organ-sparing treatment for muscle-invasive bladder cancer: a phase 2 trial.

Cystectomy is a standard treatment for muscle-invasive bladder cancer (MIBC), but it is life-altering. We initiated a phase 2 study in which patients with MIBC received four cycles of gemcitabine, cisplatin, plus nivolumab followed by clinical restaging. Patients achieving a clinical complete response (cCR) could proceed without cystectomy. The co-primary objectives were to assess the cCR rate and the positive predictive value of cCR for a composite outcome: 2-year metastasis-free survival in patients forgoing immediate cystectomy or

Genitourinary cancer neoadjuvant therapies: current and future approaches.

Neoadjuvant therapies can improve tolerability, reduce tumor volume to facilitate surgery, and assess subsequent treatment response. Therefore, there is much enthusiasm for expanding the benefits of cancer therapies to the neoadjuvant setting to reduce recurrence and improve survival in patients with localized or locally advanced genitourinary (GU) cancer. This approach is clinically pertinent because these treatments are administered primarily to treatment-naive patients and can elicit the greatest drug response. In addition, the results are not impacted by other anticancer treatments. While neoadjuvant therapies have been the standard treatment for bladder cancer in the past, they are presently restricted to clinical trials for renal and prostate cancer (PCa); however, changes are imminent. Precision neoadjuvant therapies will be ushered in by biomarker-stratified neoadjuvant trials with appropriate survival endpoints and comprehensive correlative and imaging studies. This review discusses neoadjuvant studies in GU malignancies and how they inform future study design considerations.

Antitumor immunity as the basis for durable disease-free treatment-free survival in patients with metastatic urothelial cancer.

Cisplatin-based chemotherapy has been associated with durable disease control in a small subset of patients with metastatic urothelial cancer. However, the mechanistic basis for this phenomenon has remained elusive. Antitumor immunity may underlie these exceptional responders. In a phase II trial evaluating a phased schedule of gemcitabine and cisplatin followed by gemcitabine and cisplatin with ipilimumab for metastatic urothelial cancer, 4 of 36 patients achieved durable disease-free treatment-free survival (DDFTFS) and remain in remission over 5 years after enrolment on the study. We sought to identify the genomic and immunological mechanisms associated with functional cures of such patients. Whole exome sequencing was performed on pretreatment archival tumor tissue. Neoantigen prediction and ranking were performed using a novel pipeline. For a subset of patients with available biospecimens, selected peptides were tested for neoantigen-specific T cell reactivity in peripheral blood CD4+ and CD8+ T cells cultured with autologous antigen-presenting cells at baseline, postchemotherapy, and postchemotherapy and ipilimumab timepoints. Multiplex assays of serum protein analytes were also assessed at each time point. Serum proteomic analysis revealed that pretreatment, patients achieving DDFTFS demonstrated an immune activated phenotype with elevations in TH1 adaptive immunity, costimulatory molecules, and immune checkpoint markers. After combination cisplatin-based chemotherapy and ipilimumab treatment, DDFTFS patients again displayed enrichment for markers of adaptive immunity, as well as T cell cytotoxicity. CD27 was uniquely enriched in DDFTFS patients at all timepoints. Neoantigen reactivity was not detected in any patient at baseline or post two cycles of chemotherapy. Both CD4+ and CD8+ neoantigen-specific T cell reactivity was detected in two of two DDFTFS patients in comparison to zero of five non-DDFTFS patients after combination cisplatin-based chemotherapy and ipilimumab treatment. Antitumor immunity may underlie functional cures achieved in patients with metastatic urothelial cancer treated with cisplatin-based chemotherapy and immune checkpoint blockade. Probing the mechanistic basis for DDFTFS may facilitate the identification of biomarkers, therapeutic components, and optimal treatment sequences necessary to extend this ultimate goal to a larger subset of patients.

Immune escape and resistance to immunotherapy in mismatch repair deficient tumors.

Up to 30% of colorectal, endometrial and gastric cancers have a deficiency in mismatch repair (MMR) protein expression due to either germline or epigenetic inactivation. Patients with Lynch Syndrome who inherit an inactive MMR allele have an up to 80% risk for developing a mismatch repair deficient (MMRd) cancer. Due to an inability to repair DNA, MMRd tumors present with genomic instability in microsatellite regions (MS). Tumors with high MS instability (MSI-H) are characterized by an increased frequency of insertion/deletions (indels) that can encode novel neoantigens if they occur in coding regions. The high tumor antigen burden for MMRd cancers is accompanied by an inflamed tumor microenvironment (TME) that contributes to the clinical effectiveness of anti-PD-1 therapy in this patient population. However, between 40 and 70% of MMRd cancer patients do not respond to treatment with PD-1 blockade, suggesting that tumor-intrinsic and -extrinsic resistance mechanisms may affect the success of checkpoint blockade. Immune evasion mechanisms that occur during early tumorigenesis and persist through cancer development may provide a window into resistance pathways that limit the effectiveness of anti-PD-1 therapy. Here, we review the mechanisms of immune escape in MMRd tumors during development and checkpoint blockade treatment, including T cell dysregulation and myeloid cell-mediated immunosuppression in the TME. Finally, we discuss the development of new therapeutic approaches to tackle resistance in MMRd tumors, including cancer vaccines, therapies targeting immunosuppressive myeloid programs, and immune checkpoint combination strategies.

Cancer immunity derailed: PGE2 misconducts cDC1s.

Type 1 conventional dendritic cells (cDC1s) are critical for CD8+ T cell-mediated tumor control. In this issue of Immunity, Bayerl et al.1 expose a mechanism leading to cancer progression where prostaglandin E2 induces dysfunctional cDC1s, which cannot coordinate CD8+ T cell migration and expansion.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer.

Natural killer (NK) cells are commonly reduced in human tumors, enabling many to evade surveillance. Here, we sought to identify cues that alter NK cell activity in tumors. We found that, in human lung cancer, the presence of NK cells inversely correlated with that of monocyte-derived macrophages (mo-macs). In a murine model of lung adenocarcinoma, we show that engulfment of tumor debris by mo-macs triggers a pro-tumorigenic program governed by triggering receptor expressed on myeloid cells 2 (TREM2). Genetic deletion of Trem2 rescued NK cell accumulation and enabled an NK cell-mediated regression of lung tumors. TREM2+ mo-macs reduced NK cell activity by modulating interleukin (IL)-18/IL-18BP decoy interactions and IL-15 production. Notably, TREM2 blockade synergized with an NK cell-activating agent to further inhibit tumor growth. Altogether, our findings identify a new axis, in which TREM2+ mo-macs suppress NK cell accumulation and cytolytic activity. Dual targeting of macrophages and NK cells represents a new strategy to boost antitumor immunity.

Structural Basis for Self-Discrimination by Neoantigen-Specific TCRs.

Physical interactions between T cell receptors (TCRs) and mutation-derived tumour neoantigens (neoAg) presented by major histocompatibility class-I (MHC-I) enable sensitive and specific cytolysis of tumour cells. Adoptive transfer of neoAg-reactive T cells in patients is correlated with response to immunotherapy; however, the structural and cellular mechanisms of neoAg recognition remain poorly understood. We have identified multiple cognate neoAg:TCRs from B16F10, a common murine implantable tumour model of melanoma. We identified a high affinity TCR targeting H2-Db-restricted Hsf2K72N that conferred specific recognition of B16F10 in vitro and in vivo. Structural characterization of the peptide-MHC (pMHC) binary and pMHC:TCR ternary complexes yielded high-resolution crystal structures, revealing the formation of a solvent-exposed hydrophobic arch in H2-Db that enables multiple intermolecular contacts between pMHC and the TCR. These features of structural stability strikingly mimic that of a previously published influenza peptide-H2-Db complex and its corresponding TCR, suggesting that there are shared structural motifs between neoantigens and viral peptides that explain their shared immunogenicity.

Strategies to overcome DC dysregulation in the tumor microenvironment.

Dendritic cells (DCs) play a key role to modulate anti-cancer immunity in the tumor microenvironment (TME). They link innate to adaptive immunity by processing and presenting tumor antigens to T cells thereby initiating an anti-tumor response. However, subsets of DCs also induce immune-tolerance, leading to tumor immune escape. In this regard, the TME plays a major role in adversely affecting DC function. Better understanding of DC impairment mechanisms in the TME will lead to more efficient DC-targeting immunotherapy. Here, we review the different subtypes and functions of DCs in the TME, including conventional DCs, plasmacytoid DC and the newly proposed subset, mregDC. We further focus on how cancer cells modulate DCs to escape from the host's immune-surveillance. Immune checkpoint expression, small molecule mediators, metabolites, deprivation of pro-immunogenic and release of pro-tumorigenic cytokine secretion by tumors and tumor-attracted immuno-suppressive cells inhibit DC differentiation and function. Finally, we discuss the impact of established therapies on DCs, such as immune checkpoint blockade. Creative DC-targeted therapeutic strategies will be highlighted, including cancer vaccines and cell-based therapies.

Tumor-Infiltrating Myeloid Cells Confer De Novo Resistance to PD-L1 Blockade through EMT-Stromal and Tgfß-Dependent Mechanisms.

SIGNIFICANCE: Most patients with bladder cancer do not respond to ICB targeting of the PD-L1 signaling axis. Our modeling applied a de novo resistance signature to show that tumor-infiltrating myeloid cells promote poor treatment response in a TGFß-dependent mechanism.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.

Dark genome, bright ideas: Recent approaches to harness transposable elements in immunotherapies.

Transposable elements (TEs), which make up almost half of the human genome, often display altered expression in cancers. Here, we review recent progress in elucidating the role of TEs as mediators of immune responses in cancer and discuss how novel therapeutic strategies can harness TE immunogenicity for cancer immunotherapy.

Y RNAs are conserved endogenous RIG-I ligands across RNA virus infection and are targeted by HIV-1.

Pattern recognition receptors (PRRs) protect against microbial invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specifically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphosphate extremity. Further, we found that HIV-1 triggered a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a transcriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endogenous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.

Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.