Genetics of Natural Killer Cells in Human Health, Disease, and Survival.

Natural killer (NK) cells have vital functions in human immunity and reproduction. In the innate and adaptive immune responses to infection, particularly by viruses, NK cells respond by secreting inflammatory cytokines and killing infected cells. In reproduction, NK cells are critical for genesis of the placenta, the organ that controls the supply of oxygen and nutrients to the growing fetus. Controlling NK cell functions are interactions of HLA class I with inhibitory NK cell receptors. First evolved was the conserved interaction of HLA-E with CD94:NKG2A; later established were diverse interactions of HLA-A, -B, and -C with killer cell immunoglobulin-like receptors. Characterizing the latter interactions is rapid evolution, which distinguishes human populations and all species of higher primate. Driving this evolution are the different and competing selections imposed by pathogens on NK cell-mediated immunity and by the constraints of human reproduction on NK cell-mediated placentation. Promoting rapid evolution is independent segregation of polymorphic receptors and ligands throughout human populations.

Antigen Presentation by Extracellular Vesicles from Professional Antigen-Presenting Cells.

The initiation and maintenance of adaptive immunity require multifaceted modes of communication between different types of immune cells, including direct intercellular contact, secreted soluble signaling molecules, and extracellular vesicles (EVs). EVs can be formed as microvesicles directly pinched off from the plasma membrane or as exosomes secreted by multivesicular endosomes. Membrane receptors guide EVs to specific target cells, allowing directional transfer of specific and complex signaling cues. EVs are released by most, if not all, immune cells. Depending on the type and status of their originating cell, EVs may facilitate the initiation, expansion, maintenance, or silencing of adaptive immune responses. This review focusses on EVs from professional antigen-presenting cells, their demonstrated and speculated roles, and their potential for cancer immunotherapy.

Regulation of the Cell Biology of Antigen Cross-Presentation.

Antigen cross-presentation is an adaptation of the cellular process of loading MHC-I molecules with endogenous peptides during their biosynthesis within the endoplasmic reticulum. Cross-presented peptides derive from internalized proteins, microbial pathogens, and transformed or dying cells. The physical separation of internalized cargo from the endoplasmic reticulum, where the machinery for assembling peptide-MHC-I complexes resides, poses a challenge. To solve this problem, deliberate rewiring of organelle communication within cells is necessary to prepare for cross-presentation, and different endocytic receptors and vesicular traffic patterns customize the emergent cross-presentation compartment to the nature of the peptide source. Three distinct pathways of vesicular traffic converge to form the ideal cross-presentation compartment, each regulated differently to supply a unique component that enables cross-presentation of a diverse repertoire of peptides. Delivery of centerpiece MHC-I molecules is the critical step regulated by microbe-sensitive Toll-like receptors. Defining the subcellular sources of MHC-I and identifying sites of peptide loading during cross-presentation remain key challenges.

IRGQ-mediated autophagy in MHC class I quality control promotes tumor immune evasion.

The autophagy-lysosome system directs the degradation of a wide variety of cargo and is also involved in tumor progression. Here, we show that the immunity-related GTPase family Q protein (IRGQ), an uncharacterized protein to date, acts in the quality control of major histocompatibility complex class I (MHC class I) molecules. IRGQ directs misfolded MHC class I toward lysosomal degradation through its binding mode to GABARAPL2 and LC3B. In the absence of IRGQ, free MHC class I heavy chains do not only accumulate in the cell but are also transported to the cell surface, thereby promoting an immune response. Mice and human patients suffering from hepatocellular carcinoma show improved survival rates with reduced IRGQ levels due to increased reactivity of CD8+ T cells toward IRGQ knockout tumor cells. Thus, we reveal IRGQ as a regulator of MHC class I quality control, mediating tumor immune evasion.

Variations in MHC Class II Antigen Processing and Presentation in Health and Disease.

MHC class II (MHC-II) molecules are critical in the control of many immune responses. They are also involved in most autoimmune diseases and other pathologies. Here, we describe the biology of MHC-II and MHC-II variations that affect immune responses. We discuss the classic cell biology of MHC-II and various perturbations. Proteolysis is a major process in the biology of MHC-II, and we describe the various components forming and controlling this endosomal proteolytic machinery. This process ultimately determines the MHC-II-presented peptidome, including cryptic peptides, modified peptides, and other peptides that are relevant in autoimmune responses. MHC-II also variable in expression, glycosylation, and turnover. We illustrate that MHC-II is variable not only in amino acids (polymorphic) but also in its biology, with consequences for both health and disease.

Gasdermin D licenses MHCII induction to maintain food tolerance in small intestine.

The intestinal epithelial cells (IECs) constitute the primary barrier between host cells and numerous foreign antigens; it is unclear how IECs induce the protective immunity against pathogens while maintaining the immune tolerance to food. Here, we found IECs accumulate a less recognized 13-kD N-terminal fragment of GSDMD that is cleaved by caspase-3/7 in response to dietary antigens. Unlike the 30-kD GSDMD cleavage fragment that executes pyroptosis, the IEC-accumulated GSDMD cleavage fragment translocates to the nucleus and induces the transcription of CIITA and MHCII molecules, which in turn induces the Tr1 cells in upper small intestine. Mice treated with a caspase-3/7 inhibitor, mice with GSDMD mutation resistant to caspase-3/7 cleavage, mice with MHCII deficiency in IECs, and mice with Tr1 deficiency all displayed a disrupted food tolerance phenotype. Our study supports that differential cleavage of GSDMD can be understood as a regulatory hub controlling immunity versus tolerance in the small intestine.

A membrane-associated MHC-I inhibitory axis for cancer immune evasion.

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.

Lymph node colonization induces tumor-immune tolerance to promote distant metastasis.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

T-Scan: A Genome-wide Method for the Systematic Discovery of T Cell Epitopes.

T cell recognition of specific antigens mediates protection from pathogens and controls neoplasias, but can also cause autoimmunity. Our knowledge of T cell antigens and their implications for human health is limited by the technical limitations of T cell profiling technologies. Here, we present T-Scan, a high-throughput platform for identification of antigens productively recognized by T cells. T-Scan uses lentiviral delivery of antigen libraries into cells for endogenous processing and presentation on major histocompatibility complex (MHC) molecules. Target cells functionally recognized by T cells are isolated using a reporter for granzyme B activity, and the antigens mediating recognition are identified by next-generation sequencing. We show T-Scan correctly identifies cognate antigens of T cell receptors (TCRs) from viral and human genome-wide libraries. We apply T-Scan to discover new viral antigens, perform high-resolution mapping of TCR specificity, and characterize the reactivity of a tumor-derived TCR. T-Scan is a powerful approach for studying T cell responses.

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

Evolutionary Pressure against MHC Class II Binding Cancer Mutations.

The anti-cancer immune response against mutated peptides of potential immunological relevance (neoantigens) is primarily attributed to MHC-I-restricted cytotoxic CD8+ T cell responses. MHC-II-restricted CD4+ T cells also drive anti-tumor responses, but their relation to neoantigen selection and tumor evolution has not been systematically studied. Modeling the potential of an individual's MHC-II genotype to present 1,018 driver mutations in 5,942 tumors, we demonstrate that the MHC-II genotype constrains the mutational landscape during tumorigenesis in a manner complementary to MHC-I. Mutations poorly bound to MHC-II are positively selected during tumorigenesis, even more than mutations poorly bound to MHC-I. This emphasizes the importance of CD4+ T cells in anti-tumor immunity. In addition, we observed less inter-patient variation in mutation presentation for MHC-II than for MHC-I. These differences were reflected by age at diagnosis, which was correlated with presentation by MHC-I only. Collectively, our results emphasize the central role of MHC-II presentation in tumor evolution.

Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair.

Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.

LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade.

Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation.

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

Machine learning predictions of MHC-II specificities reveal alternative binding mode of class II epitopes.

CD4+ T cells orchestrate the adaptive immune response against pathogens and cancer by recognizing epitopes presented on class II major histocompatibility complex (MHC-II) molecules. The high polymorphism of MHC-II genes represents an important hurdle toward accurate prediction and identification of CD4+ T cell epitopes. Here we collected and curated a dataset of 627,013 unique MHC-II ligands identified by mass spectrometry. This enabled us to precisely determine the binding motifs of 88 MHC-II alleles across humans, mice, cattle, and chickens. Analysis of these binding specificities combined with X-ray crystallography refined our understanding of the molecular determinants of MHC-II motifs and revealed a widespread reverse-binding mode in HLA-DP ligands. We then developed a machine-learning framework to accurately predict binding specificities and ligands of any MHC-II allele. This tool improves and expands predictions of CD4+ T cell epitopes and enables us to discover viral and bacterial epitopes following the aforementioned reverse-binding mode.

CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma.

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with αCTLA-4, but not αPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ T cells but not CD8+ T cells. αCTLA-4 treatment increased frequencies of intratumoral IFNγ-producing CD4+ T cells, and IFNγ blockade negated the therapeutic impact of αCTLA-4. The anti-tumor activity of CD4+ T cells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ T cells interacted directly with microglia, promoting IFNγ-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary for tumor suppression. Thus, αCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ T cell-microglia circuit wherein IFNγ triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ T cell response.

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

HLA-II immunopeptidome profiling and deep learning reveal features of antigenicity to inform antigen discovery.

CD4+ T cell responses are exquisitely antigen specific and directed toward peptide epitopes displayed by human leukocyte antigen class II (HLA-II) on antigen-presenting cells. Underrepresentation of diverse alleles in ligand databases and an incomplete understanding of factors affecting antigen presentation in vivo have limited progress in defining principles of peptide immunogenicity. Here, we employed monoallelic immunopeptidomics to identify 358,024 HLA-II binders, with a particular focus on HLA-DQ and HLA-DP. We uncovered peptide-binding patterns across a spectrum of binding affinities and enrichment of structural antigen features. These aspects underpinned the development of context-aware predictor of T cell antigens (CAPTAn), a deep learning model that predicts peptide antigens based on their affinity to HLA-II and full sequence of their source proteins. CAPTAn was instrumental in discovering prevalent T cell epitopes from bacteria in the human microbiome and a pan-variant epitope from SARS-CoV-2. Together CAPTAn and associated datasets present a resource for antigen discovery and the unraveling genetic associations of HLA alleles with immunopathologies.