Microbiology & Immunology - Research Publications
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ItemTranscriptional profiling of human Vδ1 T cells reveals a pathogen-driven adaptive differentiation program.(Elsevier BV, 2022-05-24)γδ T cells are generally considered innate-like lymphocytes, however, an "adaptive-like" γδ compartment has now emerged. To understand transcriptional regulation of adaptive γδ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult Vδ1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ αβ T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive Vδ1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive Vδ1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant Vγ9+Vδ2+ T cells exhibit a distinct "innate-effector" transcriptional program established by early childhood. In summary, adaptive-like γδ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells.
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ItemCollision-Induced Affinity Selection Mass Spectrometry for Identification of Ligands.(American Chemical Society (ACS), 2022-10-19)Hyphenated mass spectrometry has been used to identify ligands binding to proteins. It involves mixing protein and compounds, separation of protein-ligand complexes from unbound compounds, dissociation of the protein-ligand complex, separation to remove protein, and injection of the supernatant into a mass spectrometer to observe the ligand. Here we report collision-induced affinity selection mass spectrometry (CIAS-MS), which allows separation and dissociation inside the instrument. The quadrupole was used to select the ligand-protein complex and allow unbound molecules to be exhausted to vacuum. Collision-induced dissociation (CID) dissociated the protein-ligand complex, and the ion guide and resonance frequency were used to selectively detect the ligand. A known SARS-CoV-2 Nsp9 ligand, oridonin, was successfully detected when it was mixed with Nsp9. We provide proof-of-concept data that the CIAS-MS method can be used to identify binding ligands for any purified protein.
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ItemNo Preview AvailableDual TCR-α Expression on Mucosal-Associated Invariant T Cells as a Potential Confounder of TCR Interpretation(AMER ASSOC IMMUNOLOGISTS, 2022-03-15)Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRβ-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRβ-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous β-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.
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ItemNo Preview AvailableVaricella Zoster Virus Impairs Expression of the Nonclassical Major Histocompatibility Complex Class I-Related Gene Protein (MR1)(OXFORD UNIV PRESS INC, 2023-02-01)The antigen presentation molecule MR1 (major histocompatibility complex, class I-related) presents ligands derived from the riboflavin (vitamin B) synthesis pathway, which is not present in mammalian species or viruses, to mucosal-associated invariant T (MAIT) cells. In this study, we demonstrate that varicella zoster virus (VZV) profoundly suppresses MR1 expression. We show that VZV targets the intracellular reservoir of immature MR1 for degradation, while preexisting, ligand-bound cell surface MR1 is protected from such targeting, thereby highlighting an intricate temporal relationship between infection and ligand availability. We also identify VZV open reading frame (ORF) 66 as functioning to suppress MR1 expression when this viral protein is expressed during transient transfection, but this is not apparent during infection with a VZV mutant virus lacking ORF66 expression. This indicates that VZV is likely to encode multiple viral genes that target MR1. Overall, we identify an immunomodulatory function of VZV whereby infection suppresses the MR1 biosynthesis pathway.
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ItemNo Preview AvailableSARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans(CELL PRESS, 2022-02-17)SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.
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ItemSARS-CoV-2-specific T cell memory with common TCRαβ motifs is established in unvaccinated children who seroconvert after infection(CELL PRESS, 2022-07-12)As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαβ repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαβ motifs in unvaccinated seroconverted children after their first virus encounter.
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ItemIL-23 costimulates antigen-specific MAIT cell activation and enables vaccination against bacterial infection(AMER ASSOC ADVANCEMENT SCIENCE, 2019-11-01)Mucosal-associated invariant T (MAIT) cells are activated in a TCR-dependent manner by antigens derived from the riboflavin synthesis pathway, including 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), bound to MHC-related protein-1 (MR1). However, MAIT cell activation in vivo has not been studied in detail. Here, we have found and characterized additional molecular signals required for optimal activation and expansion of MAIT cells after pulmonary Legionella or Salmonella infection in mice. We show that either bone marrow–derived APCs or non–bone marrow–derived cells can activate MAIT cells in vivo, depending on the pathogen. Optimal MAIT cell activation in vivo requires signaling through the inducible T cell costimulator (ICOS), which is highly expressed on MAIT cells. Subsequent expansion and maintenance of MAIT-17/1-type responses are dependent on IL-23. Vaccination with IL-23 plus 5-OP-RU augments MAIT cell–mediated control of pulmonary Legionella infection. These findings reveal cellular and molecular targets for manipulating MAIT cell function under physiological conditions.
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ItemNo Preview AvailableStructural bases of T cell antigen receptor recognition in celiac disease.(Elsevier BV, 2022-06)Celiac disease (CeD) is a human leukocyte antigen (HLA)-linked autoimmune-like disorder that is triggered by the ingestion of gluten or related storage proteins. The majority of CeD patients are HLA-DQ2.5+, with the remainder being either HLA-DQ8+ or HLA-DQ2.2+. Structural studies have shown how deamidation of gluten epitopes engenders binding to HLA-DQ2.5/8, which then triggers an aberrant CD4+ T cell response. HLA tetramer studies, combined with structural investigations, have demonstrated that repeated patterns of TCR usage underpins the immune response to some HLADQ2.5/8 restricted gluten epitopes, with distinct TCR motifs representing common landing pads atop the HLA-gluten complexes. Structural studies have provided insight into TCR specificity and cross-reactivity towards gluten epitopes, as well as cross-reactivity to bacterial homologues of gluten epitopes, suggesting that environmental factors may directly play a role in CeD pathogenesis. Collectively, structural immunology-based studies in the CeD axis may lead to new therapeutics/diagnostics to treat CeD, and also serve as an exemplar for other T cell mediated autoimmune diseases.
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ItemNo Preview AvailableHost immunomodulatory lipids created by symbionts from dietary amino acids(NATURE PORTFOLIO, 2021-12-09)Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.
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ItemNo Preview AvailableA high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation(AMER ASSOC ADVANCEMENT SCIENCE, 2021-08)Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4+ T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.