Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.
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
Abstract The goal of cancer vaccines and immune checkpoint therapy (ICT) is to expand and sustain T cells with enhanced anti-tumor capabilities. Here, we asked whether these distinct immunotherapies utilize similar cellular and functional mechanisms. We used multiple approaches to compare effective therapeutic mutant neoantigen (NeoAg) cancer vaccines with αPD-1, αCTLA-4, or αPD-1 and αCTLA-4 ICT in preclinical BrafV600EPten−/−Cdkn2a−/− melanoma models. Synthetic long peptide (SLP) MHC-I NeoAg vaccines induced a more than 3-fold expansion of proliferating intratumoral NeoAg-specific CD8 T cells that were functional despite high expression of PD-1 and TIM-3. NeoAg vaccines required not only CD8, but also CD4 T cells for efficacy. αCTLA-4 and/or αPD-1 ICT also increased the frequency and effector capabilities of CD8 T cells but to much less of an extent than NeoAg vaccines. These included NeoAg-specific CD8 T cells that displayed reduced expression of PD-1 and TIM-3. αCTLA-4 treated tumors displayed robust induction of ICOS+ Th1-like CD4 T cells expressing Bhlhe40 that resembled Th1-like CD4 T cells described in αCTLA-4-treated patients and when αCTLA-4 was combined with αPD-1, a small subset of Th2-like CD4 T cells was observed. Remarkably, we noted divergent effects on certain subsets of intratumoral macrophage populations induced by NeoAg vaccines as compared to ICT. Although effective NeoAg vaccines expanded M1-like iNOS+ macrophages with ICT also following this same pattern, NeoAg vaccines expanded rather than suppressed (as observed with ICT) distinct subpopulations of M2-like CX3CR1+ CD206+ macrophages. CODEX spatial imaging using a 46-marker panel revealed tumors from mice treated with NeoAg vaccines displayed concentrations of CD8 T cells expressing functional markers and the proliferation marker Ki67, with mice treated with αCTLA-4 or αPD-1 displaying both Ki67+ conventional CD4 and CD8 T cells within interior regions of tumor. Interestingly, these regions contained prominent blood vessels, along with MHC-II+ dendritic cells and iNOS+ macrophages. Our work shows that NeoAg vaccines lead to distinct, as well as overlapping alterations when compared to ICT. These distinct alterations provided a rationale for combination therapies that we validated in both our melanoma model, as well as the MC38 tumor model, where delayed treatment with NeoAg vaccines combined with αCTLA-4 or αPD-1 yielded anti-tumor immunity that was superior to combination αPD-1 and αCTLA-4. These findings suggest that NeoAg vaccines combined with single-agent ICT may be an effective therapy with potentially less toxicity than αPD-1/αCTLA-4 combination ICT. Further, we hypothesize that immunotherapies that deplete CX3CR1+ CD206+ macrophages may synergize with NeoAg cancer vaccines, which we are currently assessing. Citation Format: Sunita Keshari, Alexander S. Shavkunov, Qi Miao, Akata Saha, Charmelle D. Williams, Josué E. Pineda, Kenneth Hu, Kristen E. Pauken, Ken Chen, Matthew M. Gubin. Overlapping and distinct mechanisms of effective neoantigen cancer vaccines and immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6738.
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 HuRfl/+) 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 HuRfl/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.
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.
Abstract Background: Despite the recent success in Immune checkpoint blockade (ICB) therapy, limited patients benefit due to immune-related adverse events or treatment resistance, especially in solid tumors. To overcome these hurdles, understanding the molecular mechanisms behind treatment response is essential. However, the current lack of immune gene transcriptional programs (GTPs) hampers data-driven immunological discovery. Constructing immunity-specific knowledgebases with rigorously curated gene sets (irGSs) and rich immunological language can unlock new insights, enabling interpretation of high-throughput immune microenvironment profiling studies and fostering personalized, effective cancer treatments. Methods: We collected 83 BulkRNAseq datasets from the ImmuneSigDB. These datasets contain 1826 samples challenged with infections, cytokines or immunological perturbations of different kinds and magnitude, possessing yet-to-be discovered immune functions that lie beneath the transcriptomic profiles. Using non-negative matrix factorization (NMF), we identified gene sets with coordinated expression. We used CITE-seq and selected scRNAseq to annotate the immunological functions of each gene set and validated the clinical utilities of these gene sets from different aspects using data from Cancer Genome Atlas Program (TCGA) pan-cancer, ICB cohorts and a 10X Genomics Visium FFPE Human Breast Cancer spatial slide. Results: We presented 19 lymphoid-data derived and 9 myeloid-data derived gene sets (irGSs), encompassing a diverse array of immune functions. Through extensive validation using BulkRNAseq, scRNAseq and spatial transcriptomics data, we revealed six irGSs-defined pan-cancer microenvironment subtypes in TCGA with significantly distinct survival patterns regardless of cancer types. Moreover, irGSs are associated with T cell exhaustion phenotypes, which in turn well predicted (classification accuracy of 72%) Nivolumab response using 104 treatment-naïve melanoma patients. Lastly, irGSs simultaneously isolated out tumor regions and highlighted immune infiltration with high specificity in breast cancer spatial transcriptomics H&E image. Conclusions: These pioneering gene sets, originally derived from non-cancerous experiments, hold tremendous promise for cancer research across diverse contexts. Similarities between cancerous and sepsis immune microenvironments underscore their wide applicability. By studying gene set activities, immunologists can gain profound insights into cancer survival drivers, unravel the intricacies of ICB treatment mechanisms, and potentially conquer therapeutic resistance. These translational utilities herald a transformative era in cancer immunotherapy and open new frontiers in the fight against cancer. Citation Format: Shan He, Vakul Mohanty, Matthew Gubin, Hind Rafei, Rafet Basar, Merve Dede, Xianli Jiang, Yukun Tan, Maura Gillison, Katayoun Rezvani, Weiyi Peng, Ken Chen. Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 873.
Abstract 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.
