Biology subjects

McCluskey, J.

Publications and source records attributed to McCluskey, J..

5 recordsLinked to original sources

bioRxiv2026

Specific targeting of MR1-antigen complexes using nanobodies

T cell receptor mimic (TCRm) antibodies and nanobodies that specifically bind peptide-HLA complexes have great therapeutic potential, as they can target polymorphic HLA on tumour cells furnishing peptides derived from tumour-associated antigens. MR1 is an MHC class-I-like molecule that exhibits limited polymorphism that binds and presents conserved metabolites, such as 5-OP-RU, derived from microbial riboflavin biosynthesis. Whether antibodies targeting such MR1-5-OP-RU complexes can be generated remains unclear. Using yeast display technology and in vitro affinity maturation, a nanobody with high affinity and fine specificity toward MR1-5-OP-RU complex was generated. These nanobodies bind both mouse and human MR1-5-OP-RU and inhibited MAIT cell responses to 5-OP-RU in vitro and in vivo demonstrating therapeutic potential. Moreover, we provide a molecular basis underpinning the fine specificity of these nanobodies, solving the crystal structures of MR1 in complex with either 5-OP-RU or Ac-6-FP. Here, the nanobody co-bound MR1 and 5-OP-RU, akin to a TCRm antibody. Moreover, we engineer bispecific antibodies targeting both MR1-5-OP-RU and CD3, that drive broad T cell killing of bacterially-infected cells as well as tumour cells treated with 5-OP-RU, thereby providing proof-of-principle for targeting the MR1 molecule with with TCRm-based nanobodies. One Sentence SummaryWe report the development of a nanobody targeting MR1-5-OP-RU complex and demonstrate its utility to modulate MAIT cells responses, and as a bispecific engager.

immunology↗

bioRxiv2021

Dual TCR-alpha expression on MAIT cells as a potential confounder of TCR interpretation

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 T cell receptor (TCR) chain that uses TRAV1-2 joined to TRAJ33/20/12 and recognize metabolites from bacterial riboflavin synthesis bound to the antigen-presenting molecule, MR1. Recently, our attempts to identify alternative MR1-presented antigens led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered antigen specificity is likely to lead to altered affinity for the most potent known antigen, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), we performed bulk TCR and {beta} chain sequencing, and single cell-based paired TCR sequencing, on T cells that bound the MR1-5-OP-RU tetramer, but with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Whereas we initially interpreted these as diverse MR1-restricted TCRs, single cell TCR sequencing revealed that cells expressing atypical TCR chains also co-expressed an invariant MAIT TCR chain. Transfection of each non-TRAV1-2 TCR chain with the TCR{beta} chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCR chain expression in human T cells and competition for the endogenous {beta} chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and non-canonical 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 antigen specificity.

immunology↗

bioRxiv2018

Activation and in vivo evolution of the MAIT cell transcriptome in mice and humans reveals diverse functionality

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells conserved across mammalian species, including mice and humans. By sequencing RNA from sorted MR1-5-OP-RU tetramer+ cells derived from either human blood or murine lungs, we define the basic transcriptome of an activated MAIT cell in both species and demonstrate how this profile changes during resolution and reinfection phases of infection. We observe strong similarities between MAIT cells in humans and mice. Compared with previously published T cell transcriptomes, MAIT cells displayed most similarity to iNKT cells when activated, but to {gamma}{delta} T cells, after resolution of infection. In both species activation leads to strong expression of pro-inflammatory cytokines and chemokines, and also a strong tissue repair signature, recently described in murine commensal-specific H2-M3-restricted T cells. These data define the requirements for, and consequences of, MAIT cell activation, revealing a tissue repair phenotype expressed upon MAIT cell activation in both species.

immunology↗

bioRxiv2018

MAIT cells contribute to protection against lethal influenza infection in vivo

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulated and were activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post infection. Activation was modulated via cytokines independently of MR1. MAIT cell-deficient MR1-/- mice showed enhanced weight loss and mortality to severe (H1N1) influenza. This was ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2-/-{gamma}C-/- mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.

immunology↗

bioRxiv2017

Legionella protection and vaccination mediated by Mucosal Associated Invariant T (MAIT) cells

Mucosal associated invariant T (MAIT) cells recognize conserved microbial metabolites from riboflavin synthesis. Striking evolutionary conservation and pulmonary abundance implicate them in antibacterial host defense, yet their roles in protection against clinically significant pathogens are unknown. Murine Legionella infection induced MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells associated with immune protection detectable in fully immunocompetent host animals. MAIT cell protection was more evident in mice lacking CD4+ cells, whilst profoundly immunodeficient RAG2-/-{gamma}C-/- mice were substantially rescued from uniformly lethal Legionella infection by adoptively-transferred MAIT cells. This protection was dependent on MR1, IFN-{gamma} and GM-CSF, but not IL-17, TNF- or perforin. Protection was enhanced and observed earlier post-infection in mice that were Ag-primed to boost MAIT cells before infection. Our findings define a significant role for MAIT cells in protection against a major human pathogen and indicate a potential role for vaccination to enhance MAIT cell immunity.

immunology↗