Neoantigen Cancer Vaccines and Different Immune Checkpoint Therapies Each Utilize Both Converging and Distinct Mechanisms that in Combination Enable Synergistic Therapeutic Efficacy.

The goal of therapeutic cancer vaccines and immune checkpoint therapy (ICT) is to eliminate cancer by expanding and/or sustaining T cells with anti-tumor capabilities. However, whether cancer vaccines and ICT enhance anti-tumor immunity by distinct or overlapping mechanisms remains unclear. Here, we compared effective therapeutic tumor-specific mutant neoantigen (NeoAg) cancer vaccines with anti-CTLA-4 and/or anti-PD-1 ICT in preclinical models. Both NeoAg vaccines and ICT induce expansion of intratumoral NeoAg-specific CD8 T cells, though the degree of expansion and acquisition of effector activity was much more substantial following NeoAg vaccination. Further, we found that NeoAg vaccines are particularly adept at inducing proliferating and stem-like NeoAg-specific CD8 T cells. Single cell T cell receptor (TCR) sequencing revealed that TCR clonotype expansion and diversity of NeoAg-specific CD8 T cells relates to their phenotype and functional state associated with specific immunotherapies employed. Effective NeoAg vaccines and ICT required both CD8 and CD4 T cells. While NeoAg vaccines and anti-PD-1 affected the CD4 T cell compartment, it was to less of an extent than observed with anti-CTLA-4, which notably induced ICOS+Bhlhe40+ Th1-like CD4 T cells and, when combined with anti-PD-1, a small subset of Th2-like CD4 T cells. Although effective NeoAg vaccines or ICT expanded intratumoral M1-like iNOS+ macrophages, NeoAg vaccines expanded rather than suppressed (as observed with ICT) M2-like CX3CR1+CD206+ macrophages, associated with the vaccine adjuvant. Further, combining NeoAg vaccination with ICT induced superior efficacy compared to either therapy in isolation, highlighting the utility of combining these modalities to eliminate cancer.

CD5 expression by dendritic cells directs T cell immunity and sustains immunotherapy responses.

The induction of proinflammatory T cells by dendritic cell (DC) subtypes is critical for antitumor responses and effective immune checkpoint blockade (ICB) therapy. Here, we show that human CD1c+CD5+ DCs are reduced in melanoma-affected lymph nodes, with CD5 expression on DCs correlating with patient survival. Activating CD5 on DCs enhanced T cell priming and improved survival after ICB therapy. CD5+ DC numbers increased during ICB therapy, and low interleukin-6 (IL-6) concentrations promoted their de novo differentiation. Mechanistically, CD5 expression by DCs was required to generate optimally protective CD5hi T helper and CD8+ T cells; further, deletion of CD5 from T cells dampened tumor elimination in response to ICB therapy in vivo. Thus, CD5+ DCs are an essential component of optimal ICB therapy.

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.

Cancer Immunoediting in the Era of Immuno-oncology.

Basic science breakthroughs in T-cell biology and immune-tumor cell interactions ushered in a new era of cancer immunotherapy. Twenty years ago, cancer immunoediting was proposed as a framework to understand the dynamic process by which the immune system can both control and shape cancer and in its most complex form occurs through three phases termed elimination, equilibrium, and escape. During cancer progression through these phases, tumors undergo immunoediting, rendering them less immunogenic and more capable of establishing an immunosuppressive microenvironment. Therefore, cancer immunoediting integrates the complex immune-tumor cell interactions occurring in the tumor microenvironment and sculpts immunogenicity beyond shaping antigenicity. However, with the success of cancer immunotherapy resulting in durable clinical responses in the last decade and subsequent emergence of immuno-oncology as a clinical subspecialty, the phrase "cancer immunoediting" has recently, at times, been inappropriately restricted to describing neoantigen loss by immunoselection. This focus has obscured other mechanisms by which cancer immunoediting modifies tumor immunogenicity. Although establishment of the concept of cancer immunoediting and definitive experimental evidence supporting its existence was initially obtained from preclinical models in the absence of immunotherapy, cancer immunoediting is a continual process that also occurs during immunotherapy in human patients with cancer. Herein, we discuss the known mechanisms of cancer immunoediting obtained from preclinical and clinical data with an emphasis on how a greater understanding of cancer immunoediting may provide insights into immunotherapy resistance and how this resistance can be overcome.

BHLHE40 Regulates the T-Cell Effector Function Required for Tumor Microenvironment Remodeling and Immune Checkpoint Therapy Efficacy.

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell-dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti-PD-1 and anti-CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen-specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential ICT-induced immune cell remodeling. The BHLHE40-dependent gene expression changes indicated dysregulated metabolism, NF-κB signaling, and IFNγ response within certain subpopulations of CD4+ and CD8+ T cells. Intratumoral CD4+ and CD8+ T cells from BHLHE40-deficient mice exhibited higher expression of the inhibitory receptor gene Tigit and displayed alterations in expression of genes encoding chemokines/chemokine receptors and granzyme family members. Mice lacking BHLHE40 had reduced ICT-driven IFNγ production by CD4+ and CD8+ T cells and defects in ICT-induced remodeling of macrophages from a CX3CR1+CD206+ subpopulation to an iNOS+ subpopulation that is typically observed during effective ICT. Although both anti-PD-1 and anti-CTLA-4 ICT in BHLHE40-deficient mice led to the same outcome-tumor outgrowth-several BHLHE40-dependent alterations were specific to the ICT that was used. Our results reveal a crucial role for BHLHE40 in effective ICT and suggest that BHLHE40 may be a predictive or prognostic biomarker for ICT efficacy and a potential therapeutic target.

TREM2 Modulation Remodels the Tumor Myeloid Landscape Enhancing Anti-PD-1 Immunotherapy.

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer's disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy.

MHC-II neoantigens shape tumour immunity and response to immunotherapy.

The ability of the immune system to eliminate and shape the immunogenicity of tumours defines the process of cancer immunoediting1. Immunotherapies such as those that target immune checkpoint molecules can be used to augment immune-mediated elimination of tumours and have resulted in durable responses in patients with cancer that did not respond to previous treatments. However, only a subset of patients benefit from immunotherapy and more knowledge about what is required for successful treatment is needed2-4. Although the role of tumour neoantigen-specific CD8+ T cells in tumour rejection is well established5-9, the roles of other subsets of T cells have received less attention. Here we show that spontaneous and immunotherapy-induced anti-tumour responses require the activity of both tumour-antigen-specific CD8+ and CD4+ T cells, even in tumours that do not express major histocompatibility complex (MHC) class II molecules. In addition, the expression of MHC class II-restricted antigens by tumour cells is required at the site of successful rejection, indicating that activation of CD4+ T cells must also occur in the tumour microenvironment. These findings suggest that MHC class II-restricted neoantigens have a key function in the anti-tumour response that is nonoverlapping with that of MHC class I-restricted neoantigens and therefore needs to be considered when identifying patients who will most benefit from immunotherapy.

High-Dimensional Analysis Delineates Myeloid and Lymphoid Compartment Remodeling during Successful Immune-Checkpoint Cancer Therapy.

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.

Cancer Immunology and Immunotherapy: Taking a Place in Mainstream Oncology Keystone Symposia Meeting Summary.

The Keystone Symposia conference on Cancer Immunology and Immunotherapy: Taking a Place in Mainstream Oncology was held at the Fairmont Chateau in Whistler, British Columbia, Canada, on March 19-23, 2017. The conference brought together a sold-out audience of 654 scientists, clinicians, and others from both academia and industry to discuss the latest developments in cancer immunology and immunotherapy. This meeting report summarizes the main themes that emerged during the four-day conference. Cancer Immunol Res; 5(6); 434-8. ©2017 AACR.

Temporally Distinct PD-L1 Expression by Tumor and Host Cells Contributes to Immune Escape.

Antibody blockade of programmed death-1 (PD-1) or its ligand, PD-L1, has led to unprecedented therapeutic responses in certain tumor-bearing individuals, but PD-L1 expression's prognostic value in stratifying cancer patients for such treatment remains unclear. Reports conflict on the significance of correlations between PD-L1 on tumor cells and positive clinical outcomes to PD-1/PD-L1 blockade. We investigated this issue using genomically related, clonal subsets from the same methylcholanthrene-induced sarcoma: a highly immunogenic subset that is spontaneously eliminated in vivo by adaptive immunity and a less immunogenic subset that forms tumors in immunocompetent mice, but is sensitive to PD-1/PD-L1 blockade therapy. Using CRISPR/Cas9-induced loss-of-function approaches and overexpression gain-of-function techniques, we confirmed that PD-L1 on tumor cells is key to promoting tumor escape. In addition, the capacity of PD-L1 to suppress antitumor responses was inversely proportional to tumor cell antigenicity. PD-L1 expression on host cells, particularly tumor-associated macrophages (TAM), was also important for tumor immune escape. We demonstrated that induction of PD-L1 on tumor cells was IFNγ-dependent and transient, but PD-L1 induction on TAMs was of greater magnitude, only partially IFNγ dependent, and was stable over time. Thus, PD-L1 expression on either tumor cells or host immune cells could lead to tumor escape from immune control, indicating that total PD-L1 expression in the immediate tumor microenvironment may represent a more accurate biomarker for predicting response to PD-1/PD-L1 blockade therapy, compared with monitoring PD-L1 expression on tumor cells alone. Cancer Immunol Res; 5(2); 106-17. ©2017 AACR.

The RNA-Binding Protein HuR Posttranscriptionally Regulates IL-2 Homeostasis and CD4+ Th2 Differentiation.

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3' untranslated region adenylate and uridylate-rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK-STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

The Role of Neoantigens in Naturally Occurring and Therapeutically Induced Immune Responses to Cancer.

Definitive experimental evidence from mouse cancer models and strong correlative clinical data gave rise to the Cancer Immunoediting concept that explains the dual host-protective and tumor-promoting actions of immunity on developing cancers. Tumor-specific neoantigens can serve as targets of spontaneously arising adaptive immunity to cancer and thereby determine the ultimate fate of developing tumors. Tumor-specific neoantigens can also function as optimal targets of cancer immunotherapy against established tumors. These antigens are derived from nonsynonymous mutations that occur during cellular transformation and, because they are foreign to the host genome, are not subject to central tolerance. In this review, we summarize the experimental evidence indicating that cancer neoantigens are the source of both spontaneously occurring and therapeutically induced immune responses against cancer. We also review the advances in genomics, bioinformatics, and cancer immunotherapy that have facilitated identification of neoantigens and have moved personalized cancer immunotherapies into clinical trials, with the promise of providing more specific, safer, more effective, and perhaps even more generalizable treatments to cancer patients than current immunotherapies.

Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.

Failure of T cells to protect against cancer is thought to result from lack of antigen recognition, chronic activation, and/or suppression by other cells. Using a mouse sarcoma model, we show that glucose consumption by tumors metabolically restricts T cells, leading to their dampened mTOR activity, glycolytic capacity, and IFN-γ production, thereby allowing tumor progression. We show that enhancing glycolysis in an antigenic "regressor" tumor is sufficient to override the protective ability of T cells to control tumor growth. We also show that checkpoint blockade antibodies against CTLA-4, PD-1, and PD-L1, which are used clinically, restore glucose in tumor microenvironment, permitting T cell glycolysis and IFN-γ production. Furthermore, we found that blocking PD-L1 directly on tumors dampens glycolysis by inhibiting mTOR activity and decreasing expression of glycolysis enzymes, reflecting a role for PD-L1 in tumor glucose utilization. Our results establish that tumor-imposed metabolic restrictions can mediate T cell hyporesponsiveness during cancer.

Tumor neoantigens: building a framework for personalized cancer immunotherapy.

It is now well established that the immune system can recognize developing cancers and that therapeutic manipulation of immunity can induce tumor regression. The capacity to manifest remarkably durable responses in some patients has been ascribed in part to T cells that can (a) kill tumor cells directly, (b) orchestrate diverse antitumor immune responses, (c) manifest long-lasting memory, and (d) display remarkable specificity for tumor-derived proteins. This specificity stems from fundamental differences between cancer cells and their normal counterparts in that the former develop protein-altering mutations and undergo epigenetic and genetic alterations, resulting in aberrant protein expression. These events can result in formation of tumor antigens. The identification of mutated and aberrantly expressed self-tumor antigens has historically been time consuming and laborious. While mutant antigens are usually expressed in a tumor-specific manner, aberrantly expressed antigens are often shared between cancers and, therefore, in the past, have been the major focus of therapeutic cancer vaccines. However, advances in next-generation sequencing and epitope prediction now permit the rapid identification of mutant tumor neoantigens. This review focuses on a discussion of mutant tumor neoantigens and their use in personalizing cancer immunotherapies.

Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits-including durable responses--to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

New insights into cancer immunoediting and its three component phases--elimination, equilibrium and escape.

The principles of cancer immunoediting have set the foundations for understanding the dual host-protective and tumour sculpting actions of immunity on cancer and establishing the basis for novel individualized cancer immunotherapies. During cancer immunoediting, the host immune system shapes tumour fate in three phases through the activation of innate and adaptive immune mechanisms. In the first phase, Elimination, transformed cells are destroyed by a competent immune system. Sporadic tumour cells that manage to survive immune destruction may then enter an Equilibrium phase where editing occurs. The Escape phase represents the third and final phase of the process, where immunologically sculpted tumours begin to grow progressively, become clinically apparent and establish an immunosuppressive tumour microenvironment. This review focuses on important recent developments that have enhanced our understanding of each phase of the cancer immunoediting process, summarizes the discovery of new predictive and prognostic biomarkers and discusses development of novel and objectively effective cancer immunotherapies.