Cytotoxic innate lymphoid cells sense cancer cell-expressed interleukin-15 to suppress human and murine malignancies.

Malignancy can be suppressed by the immune system. However, the classes of immunosurveillance responses and their mode of tumor sensing remain incompletely understood. Here, we show that although clear cell renal cell carcinoma (ccRCC) was infiltrated by exhaustion-phenotype CD8+ T cells that negatively correlated with patient prognosis, chromophobe RCC (chRCC) had abundant infiltration of granzyme A-expressing intraepithelial type 1 innate lymphoid cells (ILC1s) that positively associated with patient survival. Interleukin-15 (IL-15) promoted ILC1 granzyme A expression and cytotoxicity, and IL-15 expression in chRCC tumor tissue positively tracked with the ILC1 response. An ILC1 gene signature also predicted survival of a subset of breast cancer patients in association with IL-15 expression. Notably, ILC1s directly interacted with cancer cells, and IL-15 produced by cancer cells supported the expansion and anti-tumor function of ILC1s in a murine breast cancer model. Thus, ILC1 sensing of cancer cell IL-15 defines an immunosurveillance mechanism of epithelial malignancies.

The landscape of immune dysregulation in Crohn's disease revealed through single-cell transcriptomic profiling in the ileum and colon.

Crohn's disease (CD) is a chronic gastrointestinal disease that is increasing in prevalence worldwide. CD is multifactorial, involving the complex interplay of genetic, immune, and environmental factors, necessitating a system-level understanding of its etiology. To characterize cell-type-specific transcriptional heterogeneity in active CD, we profiled 720,633 cells from the terminal ileum and colon of 71 donors with varying inflammation status. Our integrated datasets revealed organ- and compartment-specific responses to acute and chronic inflammation; most immune changes were in cell composition, whereas transcriptional changes dominated among epithelial and stromal cells. These changes correlated with endoscopic inflammation, but small and large intestines exhibited distinct responses, which were particularly apparent when focusing on IBD risk genes. Finally, we mapped markers of disease-associated myofibroblast activation and identified CHMP1A, TBX3, and RNF168 as regulators of fibrotic complications. Altogether, our results provide a roadmap for understanding cell-type- and organ-specific differences in CD and potential directions for therapeutic development.

ILC1 Confer Early Host Protection at Initial Sites of Viral Infection.

Infection is restrained by the concerted activation of tissue-resident and circulating immune cells. Whether tissue-resident lymphocytes confer early antiviral immunity at local sites of primary infection prior to the initiation of circulating responses is not well understood. Furthermore, the kinetics of initial antiviral responses at sites of infection remain unclear. Here, we show that tissue-resident type 1 innate lymphoid cells (ILC1) serve an essential early role in host immunity through rapid production of interferon (IFN)-γ following viral infection. Ablation of Zfp683-dependent liver ILC1 lead to increased viral load in the presence of intact adaptive and innate immune cells critical for mouse cytomegalovirus (MCMV) clearance. Swift production of interleukin (IL)-12 by tissue-resident XCR1+ conventional dendritic cells (cDC1) promoted ILC1 production of IFN-γ in a STAT4-dependent manner to limit early viral burden. Thus, ILC1 contribute an essential role in viral immunosurveillance at sites of initial infection in response to local cDC1-derived proinflammatory cytokines.

Mouse cytomegalovirus-experienced ILC1s acquire a memory response dependent on the viral glycoprotein m12.

Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.

Tumor-associated macrophages expressing the transcription factor IRF8 promote T cell exhaustion in cancer.

Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.

Programme of self-reactive innate-like T cell-mediated cancer immunity.

Cellular transformation induces phenotypically diverse populations of tumour-infiltrating T cells1-5, and immune checkpoint blockade therapies preferentially target T cells that recognize cancer cell neoantigens6,7. Yet, how other classes of tumour-infiltrating T cells contribute to cancer immunosurveillance remains elusive. Here, in a survey of T cells in mouse and human malignancies, we identified a population of αß T cell receptor (TCR)-positive FCER1G-expressing innate-like T cells with high cytotoxic potential8 (ILTCKs). These cells were broadly reactive to unmutated self-antigens, arose from distinct thymic progenitors following early encounter with cognate antigens, and were continuously replenished by thymic progenitors during tumour progression. Notably, expansion and effector differentiation of intratumoural ILTCKs depended on interleukin-15 (IL-15) expression in cancer cells, and inducible activation of IL-15 signalling in adoptively transferred ILTCK progenitors suppressed tumour growth. Thus, the antigen receptor self-reactivity, unique ontogeny, and distinct cancer cell-sensing mechanism distinguish ILTCKs from conventional cytotoxic T cells, and define a new class of tumour-elicited immune response.

Unsupervised and supervised AI on molecular dynamics simulations reveals complex characteristics of HLA-A2-peptide immunogenicity.

Immunologic recognition of peptide antigens bound to class I major histocompatibility complex (MHC) molecules is essential to both novel immunotherapeutic development and human health at large. Current methods for predicting antigen peptide immunogenicity rely primarily on simple sequence representations, which allow for some understanding of immunogenic features but provide inadequate consideration of the full scale of molecular mechanisms tied to peptide recognition. We here characterize contributions that unsupervised and supervised artificial intelligence (AI) methods can make toward understanding and predicting MHC(HLA-A2)-peptide complex immunogenicity when applied to large ensembles of molecular dynamics simulations. We first show that an unsupervised AI method allows us to identify subtle features that drive immunogenicity differences between a cancer neoantigen and its wild-type peptide counterpart. Next, we demonstrate that a supervised AI method for class I MHC(HLA-A2)-peptide complex classification significantly outperforms a sequence model on small datasets corrected for trivial sequence correlations. Furthermore, we show that both unsupervised and supervised approaches reveal determinants of immunogenicity based on time-dependent molecular fluctuations and anchor position dynamics outside the MHC binding groove. We discuss implications of these structural and dynamic immunogenicity correlates for the induction of T cell responses and therapeutic T cell receptor design.

Single-Cell Transcriptional Profiling Reveals Signatures of Helper, Effector, and Regulatory MAIT Cells during Homeostasis and Activation.

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

Genetic and environmental determinants of human TCR repertoire diversity.

T cell discrimination of self and non-self is the foundation of the adaptive immune response, and is orchestrated by the interaction between T cell receptors (TCRs) and their cognate ligands presented by major histocompatibility (MHC) molecules. However, the impact of host immunogenetic variation on the diversity of the TCR repertoire remains unclear. Here, we analyzed a cohort of 666 individuals with TCR repertoire sequencing. We show that TCR repertoire diversity is positively associated with polymorphism at the human leukocyte antigen class I (HLA-I) loci, and diminishes with age and cytomegalovirus (CMV) infection. Moreover, our analysis revealed that HLA-I polymorphism and age independently shape the repertoire in healthy individuals. Our data elucidate key determinants of human TCR repertoire diversity, and suggest a mechanism underlying the evolutionary fitness advantage of HLA-I heterozygosity.

Efficient and accurate mixed model association tool for single-cell eQTL analysis.

Understanding the genetic basis of gene expression can help us understand the molecular underpinnings of human traits and disease. Expression quantitative trait locus (eQTL) mapping can help in studying this relationship but have been shown to be very cell-type specific, motivating the use of single-cell RNA sequencing and single-cell eQTLs to obtain a more granular view of genetic regulation. Current methods for single-cell eQTL mapping either rely on the "pseudobulk" approach and traditional pipelines for bulk transcriptomics or do not scale well to large datasets. Here, we propose SAIGE-QTL, a robust and scalable tool that can directly map eQTLs using single-cell profiles without needing aggregation at the pseudobulk level. Additionally, SAIGE-QTL allows for testing the effects of less frequent/rare genetic variation through set-based tests, which is traditionally excluded from eQTL mapping studies. We evaluate the performance of SAIGE-QTL on both real and simulated data and demonstrate the improved power for eQTL mapping over existing pipelines.

The influence of HLA genetic variation on plasma protein expression.

Genetic variation in the human leukocyte antigen (HLA) loci is associated with risk of immune-mediated diseases, but the molecular effects of HLA polymorphism are unclear. Here we examined the effects of HLA genetic variation on the expression of 2940 plasma proteins across 45,330 Europeans in the UK Biobank, with replication analyses across multiple ancestry groups. We detected 504 proteins affected by HLA variants (HLA-pQTL), including widespread trans effects by autoimmune disease risk alleles. More than 80% of the HLA-pQTL fine-mapped to amino acid positions in the peptide binding groove. HLA-I and II affected proteins expressed in similar cell types but in different pathways of both adaptive and innate immunity. Finally, we investigated potential HLA-pQTL effects on disease by integrating HLA-pQTL with fine-mapped HLA-disease signals in the UK Biobank. Our data reveal the diverse effects of HLA genetic variation and aid the interpretation of associations between HLA alleles and immune-mediated diseases.

An immunogenetic basis for lung cancer risk.

Cancer risk is influenced by inherited mutations, DNA replication errors, and environmental factors. However, the influence of genetic variation in immunosurveillance on cancer risk is not well understood. Leveraging population-level data from the UK Biobank and FinnGen, we show that heterozygosity at the human leukocyte antigen (HLA)-II loci is associated with reduced lung cancer risk in smokers. Fine-mapping implicated amino acid heterozygosity in the HLA-II peptide binding groove in reduced lung cancer risk, and single-cell analyses showed that smoking drives enrichment of proinflammatory lung macrophages and HLA-II+ epithelial cells. In lung cancer, widespread loss of HLA-II heterozygosity (LOH) favored loss of alleles with larger neopeptide repertoires. Thus, our findings nominate genetic variation in immunosurveillance as a critical risk factor for lung cancer.

Neoantigen immunogenicity landscapes and evolution of tumor ecosystems during immunotherapy with nivolumab.

Neoantigen immunoediting drives immune checkpoint blockade efficacy, yet the molecular features of neoantigens and how neoantigen immunogenicity shapes treatment response remain poorly understood. To address these questions, 80 patients with non-small cell lung cancer were enrolled in the biomarker cohort of CheckMate 153 (CA209-153), which collected radiographic guided biopsy samples before treatment and during treatment with nivolumab. Early loss of mutations and neoantigens during therapy are both associated with clinical benefit. We examined 1,453 candidate neoantigens, including many of which that had reduced cancer cell fraction after treatment with nivolumab, and identified 196 neopeptides that were recognized by T cells. Mapping these neoantigens to clonal dynamics, evolutionary trajectories and clinical response revealed a strong selection against immunogenic neoantigen-harboring clones. We identified position-specific amino acid and physiochemical features related to immunogenicity and developed an immunogenicity score. Nivolumab-induced microenvironmental evolution in non-small cell lung cancer shared some similarities with melanoma, yet critical differences were apparent. This study provides unprecedented molecular portraits of neoantigen landscapes underlying nivolumab's mechanism of action.

Translational genetics identifies a phosphorylation switch in CARD9 required for innate inflammatory responses.

Population genetics continues to identify genetic variants associated with diseases of the immune system and offers a unique opportunity to discover mechanisms of immune regulation. Multiple genetic variants linked to severe fungal infections and autoimmunity are associated with caspase recruitment domain-containing protein 9 (CARD9). We leverage the CARD9 R101C missense variant to uncover a biochemical mechanism of CARD9 activation essential for antifungal responses. We demonstrate that R101C disrupts a critical signaling switch whereby phosphorylation of S104 releases CARD9 from an autoinhibited state to promote inflammatory responses in myeloid cells. Furthermore, we show that CARD9 R101C exerts dynamic effects on the skin cellular contexture during fungal infection, corrupting inflammatory signaling and cell-cell communication circuits. Card9 R101C mice fail to control dermatophyte infection in the skin, resulting in high fungal burden, yet show minimal signs of inflammation. Together, we demonstrate how translational genetics reveals molecular and cellular mechanisms of innate immune regulation.

The centrosomal protein FGFR1OP controls myosin function in murine intestinal epithelial cells.

Recent advances in human genetics have shed light on the genetic factors contributing to inflammatory diseases, particularly Crohn's disease (CD), a prominent form of inflammatory bowel disease. Certain risk genes associated with CD directly influence cytokine biology and cell-specific communication networks. Current CD therapies primarily rely on anti-inflammatory drugs, which are inconsistently effective and lack strategies for promoting epithelial restoration and mucosal balance. To understand CD's underlying mechanisms, we investigated the link between CD and the FGFR1OP gene, which encodes a centrosome protein. FGFR1OP deletion in mouse intestinal epithelial cells disrupted crypt architecture, resulting in crypt loss, inflammation, and fatality. FGFR1OP insufficiency hindered epithelial resilience during colitis. FGFR1OP was crucial for preserving non-muscle myosin II activity, ensuring the integrity of the actomyosin cytoskeleton and crypt cell adhesion. This role of FGFR1OP suggests that its deficiency in genetically predisposed individuals may reduce epithelial renewal capacity, heightening susceptibility to inflammation and disease.

Rules of Engagement: The Lymphocyte Receptor Ecosystem in Renal Cell Carcinoma.

Immune receptor repertoires provide insight into the clonal distribution of tumor-infiltrating lymphocytes, yet the clinical implications of T-cell receptor (TCR) and B-cell receptor (BCR) repertoire diversity in cancer are unclear. In this issue of Cancer Research, Ferral-Fairbanks and colleagues reveal the interplay between repertoire diversity, tumor molecular features, and clinical outcome in renal cell carcinoma (RCC). The authors show that aggressive tumors harbor increasingly diverse TCR and BCR repertoires and that both repertoires are altered by common RCC driver mutations. Moreover, the authors demonstrate that high TCR diversity is associated with improved overall survival. This study highlights the contribution of lymphocyte receptor dynamics to the emerging complexity of RCC antitumor immune responses. See related article by Ferral-Fairbanks et al., p. 929.

Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity.

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-ß (TGF-ß) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

Improved prediction of immune checkpoint blockade efficacy across multiple cancer types.

Only a fraction of patients with cancer respond to immune checkpoint blockade (ICB) treatment, but current decision-making procedures have limited accuracy. In this study, we developed a machine learning model to predict ICB response by integrating genomic, molecular, demographic and clinical data from a comprehensively curated cohort (MSK-IMPACT) with 1,479 patients treated with ICB across 16 different cancer types. In a retrospective analysis, the model achieved high sensitivity and specificity in predicting clinical response to immunotherapy and predicted both overall survival and progression-free survival in the test data across different cancer types. Our model significantly outperformed predictions based on tumor mutational burden, which was recently approved by the U.S. Food and Drug Administration for this purpose1. Additionally, the model provides quantitative assessments of the model features that are most salient for the predictions. We anticipate that this approach will substantially improve clinical decision-making in immunotherapy and inform future interventions.