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.

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.

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.

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.

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.

Posttranscriptional gene regulation of IL-17 by the RNA-binding protein HuR is required for initiation of experimental autoimmune encephalomyelitis.

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17-producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3' untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4(+) Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17(+) cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3' untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4(+) T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

Conditional knockout of the RNA-binding protein HuR in CD4⁺ T cells reveals a gene dosage effect on cytokine production.

The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuR(fl/+)) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuR(fl/fl)) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles.

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy (ACT), have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA sequencing (RNA-seq) datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

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.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles.

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy, have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA-seq datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

Coordinate regulation of GATA-3 and Th2 cytokine gene expression by the RNA-binding protein HuR.

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate-rich elements (AREs) present in the 3' untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3'UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

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.

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer.

BACKGROUND: The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs). METHODS: The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. RESULTS: We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. CONCLUSION: This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.

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.