Microbiology & Immunology - Research Publications
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ItemBiparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2(CELL PRESS, 2022-11-18)The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.
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ItemCD8 coreceptor engagement of MR1 enhances antigen responsiveness by human MAIT and other MR1-reactive T cells(Rockefeller University Press, 2022)Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αβ coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αβ enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αβ act as functional coreceptors for MAIT and other MR1-reactive T cells.
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ItemNo Preview AvailableCD36 family members are TCR-independent ligands for CD1 antigen-presenting molecules.(American Association for the Advancement of Science, 2021-06-25)CD1c presents lipid-based antigens to CD1c-restricted T cells, which are thought to be a major component of the human T cell pool. However, the study of CD1c-restricted T cells is hampered by the presence of an abundantly expressed, non-T cell receptor (TCR) ligand for CD1c on blood cells, confounding analysis of TCR-mediated CD1c tetramer staining. Here, we identified the CD36 family (CD36, SR-B1, and LIMP-2) as ligands for CD1c, CD1b, and CD1d proteins and showed that CD36 is the receptor responsible for non-TCR-mediated CD1c tetramer staining of blood cells. Moreover, CD36 blockade clarified tetramer-based identification of CD1c-restricted T cells and improved identification of CD1b- and CD1d-restricted T cells. We used this technique to characterize CD1c-restricted T cells ex vivo and showed diverse phenotypic features, TCR repertoire, and antigen-specific subsets. Accordingly, this work will enable further studies into the biology of CD1 and human CD1-restricted T cells.
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ItemDifferential location of NKT and MAIT cells within lymphoid tissue(NATURE PORTFOLIO, 2022-03-08)Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αβ T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.
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ItemImmune recognition of phosphoantigen-butyrophilin molecular complexes by γδ T cells(WILEY, 2020-11)Gamma-delta (γδ) T cells are an important component of the immune system. They are often enriched in non-lymphoid tissues and exhibit diverse functional attributes including rapid activation, cytokine production, proliferation, and acquisition of cytotoxicity following both TCR-dependent and TCR-independent stimulation, but poor capacity for immunological memory. They can detect a broad range of antigens, although typically not peptide-MHC complexes in contrast to alpha-beta (αβ) T cells. In humans, a prominent population of γδ T cells, defined as Vγ9Vδ2+ cells, reacts to small phosphorylated non-peptide "phosphoantigens" (pAgs). The molecular mechanism underpinning this recognition is poorly defined, but is known to involve butyrophilin family members and appears to involve indirect pAg recognition via alterations to butyrophilin molecular complexes. In this review, we discuss recent advances in our understanding of pAg recognition by γδ T cells including the role of butyrophilins and in particular, a newly described role for butyrophilin 2A1.
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ItemButyrophilin 2A1 is essential for phosphoantigen reactivity by gamma delta T cells(American Association for the Advancement of Science, 2020-02-07)Gamma delta (γδ) T cells are essential to protective immunity. In humans, most γδ T cells express Vγ9Vδ2+ T cell receptors (TCRs) that respond to phosphoantigens (pAgs) produced by cellular pathogens and overexpressed by cancers. However, the molecular targets recognized by these γδTCRs are unknown. Here, we identify butyrophilin 2A1 (BTN2A1) as a key ligand that binds to the Vγ9+ TCR γ chain. BTN2A1 associates with another butyrophilin, BTN3A1, and these act together to initiate responses to pAg. Furthermore, binding of a second ligand, possibly BTN3A1, to a separate TCR domain incorporating Vδ2 is also required. This distinctive mode of Ag-dependent T cell activation advances our understanding of diseases involving pAg recognition and creates opportunities for the development of γδ T cell-based immunotherapies.
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Itemα-Glucuronosyl and α-glucosyl diacylglycerides, natural killer T cell-activating lipids from bacteria and fungi(ROYAL SOC CHEMISTRY, 2020-02-28)Natural killer T cells express T cell receptors (TCRs) that recognize glycolipid antigens in association with the antigen-presenting molecule CD1d. Here, we report the concise chemical synthesis of a range of saturated and unsaturated α-glucosyl and α-glucuronosyl diacylglycerides of bacterial and fungal origins from allyl α-glucoside with Jacobsen kinetic resolution as a key step. These glycolipids are recognized by a classical type I NKT TCR that uses an invariant Vα14-Jα18 TCR α-chain, but also by an atypical NKT TCR that uses a different TCR α-chain (Vα10-Jα50). In both cases, recognition is sensitive to the lipid fine structure, and includes recognition of glycosyl diacylglycerides bearing branched (R- and S-tuberculostearic acid) and unsaturated (oleic and vaccenic) acids. The TCR footprints on CD1d loaded with a mycobacterial α-glucuronosyl diacylglyceride were assessed using mutant CD1d molecules and, while similar to that for α-GalCer recognition by a type I NKT TCR, were more sensitive to mutations when α-glucuronosyl diacylglyceride was the antigen. In summary, we provide an efficient approach for synthesis of a broad class of bacterial and fungal α-glycosyl diacylglyceride antigens and demonstrate that they can be recognised by TCRs derived from type I and atypical NKT cells.