Using T Cell Receptor Repertoires to Understand the Principles of Adaptive Immune Recognition.

Adaptive immune recognition is mediated by antigen receptors on B and T cells generated by somatic recombination during lineage development. The high level of diversity resulting from this process posed technical limitations that previously limited the comprehensive analysis of adaptive immune recognition. Advances over the last ten years have produced data and approaches allowing insights into how T cells develop, evolutionary signatures of recombination and selection, and the features of T cell receptors that mediate epitope-specific binding and T cell activation. The size and complexity of these data have necessitated the generation of novel computational and analytical approaches, which are transforming how T cell immunology is conducted. Here we review the development and application of novel biological, theoretical, and computational methods for understanding T cell recognition and discuss the potential for improved models of receptor:antigen interactions.

The Biology and Underlying Mechanisms of Cross-Presentation of Exogenous Antigens on MHC-I Molecules.

To monitor the health of cells, the immune system tasks antigen-presenting cells with gathering antigens from other cells and bringing them to CD8 T cells in the form of peptides bound to MHC-I molecules. Most cells would be unable to perform this function because they use their MHC-I molecules to exclusively present peptides derived from the cell's own proteins. However, the immune system evolved mechanisms for dendritic cells and some other phagocytes to sample and present antigens from the extracellular milieu on MHC-I through a process called cross-presentation. How this important task is accomplished, its role in health and disease, and its potential for exploitation are the subject of this review.

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.

T cell antigen receptor recognition of antigen-presenting molecules.

The Major Histocompatibility Complex (MHC) locus encodes classical MHC class I and MHC class II molecules and nonclassical MHC-I molecules. The architecture of these molecules is ideally suited to capture and present an array of peptide antigens (Ags). In addition, the CD1 family members and MR1 are MHC class I-like molecules that bind lipid-based Ags and vitamin B precursors, respectively. These Ag-bound molecules are subsequently recognized by T cell antigen receptors (TCRs) expressed on the surface of T lymphocytes. Structural and associated functional studies have been highly informative in providing insight into these interactions, which are crucial to immunity, and how they can lead to aberrant T cell reactivity. Investigators have determined over thirty unique TCR-peptide-MHC-I complex structures and twenty unique TCR-peptide-MHC-II complex structures. These investigations have shown a broad consensus in docking geometry and provided insight into MHC restriction. Structural studies on TCR-mediated recognition of lipid and metabolite Ags have been mostly confined to TCRs from innate-like natural killer T cells and mucosal-associated invariant T cells, respectively. These studies revealed clear differences between TCR-lipid-CD1, TCR-metabolite-MR1, and TCR-peptide-MHC recognition. Accordingly, TCRs show remarkable structural and biological versatility in engaging different classes of Ag that are presented by polymorphic and monomorphic Ag-presenting molecules of the immune system.

The immunostimulatory RNA RN7SL1 enables CAR-T cells to enhance autonomous and endogenous immune function.

Poor tumor infiltration, development of exhaustion, and antigen insufficiency are common mechanisms that limit chimeric antigen receptor (CAR)-T cell efficacy. Delivery of pattern recognition receptor agonists is one strategy to improve immune function; however, targeting these agonists to immune cells is challenging, and off-target signaling in cancer cells can be detrimental. Here, we engineer CAR-T cells to deliver RN7SL1, an endogenous RNA that activates RIG-I/MDA5 signaling. RN7SL1 promotes expansion and effector-memory differentiation of CAR-T cells. Moreover, RN7SL1 is deployed in extracellular vesicles and selectively transferred to immune cells. Unlike other RNA agonists, transferred RN7SL1 restricts myeloid-derived suppressor cell (MDSC) development, decreases TGFB in myeloid cells, and fosters dendritic cell (DC) subsets with costimulatory features. Consequently, endogenous effector-memory and tumor-specific T cells also expand, allowing rejection of solid tumors with CAR antigen loss. Supported by improved endogenous immunity, CAR-T cells can now co-deploy peptide antigens with RN7SL1 to enhance efficacy, even when heterogenous CAR antigen tumors lack adequate neoantigens.

Antibody Affinity Shapes the Choice between Memory and Germinal Center B Cell Fates.

Immunological memory is required for protection against repeated infections and is the basis of all effective vaccines. Antibodies produced by memory B cells play an essential role in many of these responses. We have combined lineage tracing with antibody cloning from single B cells to examine the role of affinity in B cell selection into germinal centers (GCs) and the memory B cell compartment in mice immunized with an HIV-1 antigen. We find that contemporaneously developing memory and GC B cells differ in their affinity for antigen throughout the immune response. Whereas GC cells and their precursors are enriched in antigen binding, memory B cells are not. Thus, the polyclonal memory B cell compartment is composed of B cells that were activated during the immune response but whose antigen binding affinity failed to support further clonal expansion in the GC.

Kinetic proofreading through the multi-step activation of the ZAP70 kinase underlies early T cell ligand discrimination.

T cells recognize a few high-affinity antigens among a vast array of lower affinity antigens. According to the kinetic proofreading model, antigen discrimination properties could be explained by the gradual amplification of small differences in binding affinities as the signal is transduced downstream of the T cell receptor. Which early molecular events are affected by ligand affinity, and how, has not been fully resolved. Here, we used time-resolved high-throughput proteomic analyses to identify and quantify the phosphorylation events and protein-protein interactions encoding T cell ligand discrimination in antigen-experienced T cells. Although low-affinity ligands induced phosphorylation of the Cd3 chains of the T cell receptor and the interaction of Cd3 with the Zap70 kinase as strongly as high-affinity ligands, they failed to activate Zap70 to the same extent. As a result, formation of the signalosome of the Lat adaptor was severely impaired with low- compared with high-affinity ligands, whereas formation of the signalosome of the Cd6 receptor was affected only partially. Overall, this study provides a comprehensive map of molecular events associated with T cell ligand discrimination.

VLR-based adaptive immunity.

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy.

Immune checkpoint therapy (ICT) shows encouraging results in a subset of patients with metastatic castration-resistant prostate cancer (mCRPC) but still elicits a sub-optimal response among those with bone metastases. Analysis of patients' bone marrow samples revealed increased Th17 instead of Th1 subsets after ICT. To further evaluate the different tumor microenvironments, we injected mice with prostate tumor cells either subcutaneously or intraosseously. ICT in the subcutaneous CRPC model significantly increases intra-tumoral Th1 subsets and improves survival. However, ICT fails to elicit an anti-tumor response in the bone CRPC model despite an increase in the intra-tumoral CD4 T cells, which are polarized to Th17 rather than Th1 lineage. Mechanistically, tumors in the bone promote osteoclast-mediated bone resorption that releases TGF-ß, which restrains Th1 lineage development. Blocking TGF-ß along with ICT increases Th1 subsets and promotes clonal expansion of CD8 T cells and subsequent regression of bone CRPC and improves survival.

Inflammasomes and adaptive immune responses.

A fundamental concept in immunology is that the innate immune system initiates or instructs downstream adaptive immune responses. Inflammasomes are central players in innate immunity to pathogens, but how inflammasomes shape adaptive immunity is complex and relatively poorly understood. Here we highlight recent work on the interplay between inflammasomes and adaptive immunity. We address how inflammasome-dependent release of cytokines and antigen activates, shapes or even inhibits adaptive immune responses. We consider how distinct tissue or cellular contexts may alter the effects of inflammasome activation on adaptive immunity and how this contributes to beneficial or detrimental outcomes in infectious diseases, cancer and autoimmunity. We aspire to provide a framework for thinking about inflammasomes and their connection to the adaptive immune response.

High-throughput and high-dimensional single-cell analysis of antigen-specific CD8+ T cells.

Although critical to T cell function, antigen specificity is often omitted in high-throughput multiomics-based T cell profiling due to technical challenges. We describe a high-dimensional, tetramer-associated T cell antigen receptor (TCR) sequencing (TetTCR-SeqHD) method to simultaneously profile cognate antigen specificities, TCR sequences, targeted gene expression and surface-protein expression from tens of thousands of single cells. Using human polyclonal CD8+ T cells with known antigen specificity and TCR sequences, we demonstrate over 98% precision for detecting the correct antigen specificity. We also evaluate gene expression and phenotypic differences among antigen-specific CD8+ T cells and characterize phenotype signatures of influenza- and Epstein-Barr virus-specific CD8+ T cells that are unique to their pathogen targets. Moreover, with the high-throughput capacity of profiling hundreds of antigens simultaneously, we apply TetTCR-SeqHD to identify antigens that preferentially enrich cognate CD8+ T cells in patients with type 1 diabetes compared to healthy controls and discover a TCR that cross-reacts with diabetes-related and microbiome antigens. TetTCR-SeqHD is a powerful approach for profiling T cell responses in humans and mice.

How α-Helical Motifs Form Functionally Diverse Lipid-Binding Compartments.

Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.

Antigen receptor signaling and cell death resistance controls intestinal humoral response zonation.

Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.

Accurate structure prediction of biomolecular interactions with AlphaFold 3.

The introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2-6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein-ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein-nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody-antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

The ß1-adrenergic receptor links sympathetic nerves to T cell exhaustion.

CD8+ T cells are essential components of the immune response against viral infections and tumours, and are capable of eliminating infected and cancerous cells. However, when the antigen cannot be cleared, T cells enter a state known as exhaustion1. Although it is clear that chronic antigen contributes to CD8+ T cell exhaustion, less is known about how stress responses in tissues regulate T cell function. Here we show a new link between the stress-associated catecholamines and the progression of T cell exhaustion through the ß1-adrenergic receptor ADRB1. We identify that exhausted CD8+ T cells increase ADRB1 expression and that exposure of ADRB1+ T cells to catecholamines suppresses their cytokine production and proliferation. Exhausted CD8+ T cells cluster around sympathetic nerves in an ADRB1-dependent manner. Ablation of ß1-adrenergic signalling limits the progression of T cells towards the exhausted state in chronic infection and improves effector functions when combined with immune checkpoint blockade (ICB) in melanoma. In a pancreatic cancer model resistant to ICB, ß-blockers and ICB synergize to boost CD8+ T cell responses and induce the development of tissue-resident memory-like T cells. Malignant disease is associated with increased catecholamine levels in patients2,3, and our results establish a connection between the sympathetic stress response, tissue innervation and T cell exhaustion. Here, we uncover a new mechanism by which blocking ß-adrenergic signalling in CD8+ T cells rejuvenates anti-tumour functions.

The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability.

The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I-peptide complex formation.

CD8α(+) dendritic cells (DCs) are specialized at cross-presenting extracellular antigens on major histocompatibility complex (MHC) class I molecules to initiate cytotoxic T lymphocyte (CTL) responses; however, details of the mechanisms that regulate cross-presentation remain unknown. We found lower expression of the lectin family member Siglec-G in CD8α(+) DCs, and Siglec-G deficient (Siglecg(-/-)) mice generated more antigen-specific CTLs to inhibit intracellular bacterial infection and tumor growth. MHC class I-peptide complexes were more abundant on Siglecg(-/-) CD8α(+) DCs than on Siglecg(+/+) CD8α(+) DCs. Mechanistically, phagosome-expressed Siglec-G recruited the phosphatase SHP-1, which dephosphorylated the NADPH oxidase component p47(phox) and inhibited the activation of NOX2 on phagosomes. This resulted in excessive hydrolysis of exogenous antigens, which led to diminished formation of MHC class I-peptide complexes for cross-presentation. Therefore, Siglec-G inhibited DC cross-presentation by impairing such complex formation, and our results add insight into the regulation of cross-presentation in adaptive immunity.

Antigen-presenting innate lymphoid cells orchestrate neuroinflammation.

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases1-6, but the pathways that control these responses remain unclear. Here we define a population of inflammatory group 3 innate lymphoid cells (ILC3s) that infiltrate the CNS in a mouse model of multiple sclerosis. These ILC3s are derived from the circulation, localize in proximity to infiltrating T cells in the CNS, function as antigen-presenting cells that restimulate myelin-specific T cells, and are increased in individuals with multiple sclerosis. Notably, antigen presentation by inflammatory ILC3s is required to promote T cell responses in the CNS and the development of multiple-sclerosis-like disease in mouse models. By contrast, conventional and tissue-resident ILC3s in the periphery do not appear to contribute to disease induction, but instead limit autoimmune T cell responses and prevent multiple-sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data define a population of inflammatory ILC3s that is essential for directly promoting T-cell-dependent neuroinflammation in the CNS and reveal the potential of harnessing peripheral tissue-resident ILC3s for the prevention of autoimmune disease.