The molecular and cellular basis of antigen recognition by unconventional T cells

Abstract

Unconventional T cells are evolutionarily conserved populations of T cells, many of which recognise non-peptide antigens in the context of monomorphic antigen presentation molecules related to the major histocompatibility complex (MHC). These MHC related molecules, known as CD1 and MR1 present lipid and small metabolite antigens respectively.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that are highly abundant in humans and recognise the potent riboflavin biosynthesis derived metabolite 5-OP-RU presented by MR1. The majority of MAIT cells in the peripheral blood of humans express the CD8 co-receptor, a molecule also expressed by other T cells that contributes to antigen recognition and T cell activation. In contrast to other T cells, a role for CD8 on MAIT cells has not been formally tested. The first chapter of results in this thesis examines whether CD8 is able to bind MR1, and if CD8 is important for the activation of MAIT cells and other MR1-reactive T cells. The data revealed that CD8 binds directly to MR1, in a manner concordant with MHC class I, and that this interaction is important for the functional response of MAIT cells. Specifically, both isoforms of CD8 (CD8aa and CD8ab) bound to MR1 tetramers and the CD8-MR1 interaction could be abrogated by mutating MR1 in the putative CD8 binding site. The effects of the CD8-MR1 interaction were examined on primary MAIT cells, where MR1 tetramers bound more strongly to CD8+ compared CD8- MAIT cell subsets. Importantly, mutating MR1 tetramers reduced CD8+ MAIT cell engagement to CD8- MAIT cell levels. To determine the effect of the CD8-MR1 interaction on function, primary MAIT cells were activated in the presence of wild type or mutant MR1. In line with the importance of CD8 on MR1 recognition, the cytokine secretion by CD8+ MAIT cells was decreased in the absence of CD8 binding to MR1. Furthermore, the data here establishes that CD8 is vital for the recognition of MR1 presenting less potent, lower affinity antigens such as folate derivatives. Similarly, low affinity MR1-antigen recognition and cytokine secretion by other MR1-reactive T cells was completely abrogated in the absence of CD8 binding. Thus, CD8 is an important co-receptor for the function of MAIT cells and may expand the diversity of ligands recognised by MAIT and other MR1-reactive T cells.

CD1b-restricted T cells are a subset of unconventional T cells in humans that recognise lipid antigens derived from endogenous and microbial sources that are presented by CD1b. Comparatively little is known about the biology of CD1b-restricted T cells, particularly autoreactive CD1b-restricted T cells that recognise endogenous lipid antigens such as phospholipids. The second chapter of results examines autoreactive TCR recognition of CD1b, including the breadth of permissive endogenous lipid antigens that are bound by mammalian CD1b. Autoreactive CD1b-restricted T cells were identified from healthy blood donors using CD1b tetramers presenting heterogeneous mammalian lipids and the autoreactive T cells expressed distinct T cell receptors (TCRs) which were cloned to generate autoreactive T cell lines. Different CD1b restricted T cell lines exhibited altered staining reactivity with CD1b tetramers loaded with different mammalian phospholipid antigens and strikingly, some autoreactive T cell lines recognised CD1b in an antigen independent manner. An activation assay using the autoreactive T cell lines cocultured with a series of mutant CD1b expressing cell lines revealed several CD1b binding ‘hotspots’ along the a1 and a2 helices that were critical for TCR-mediated activation. Using soluble proteins, autoreactive TCR permissive ligands were isolated from mammalian CD1b by separating ternary TCR-CD1b-lipid complexes from binary CD1b-lipid complexes that did not bind to the TCR using size-exclusion chromatography. Mass spectra analysis of the fractionated proteins revealed an abundance of phospholipids, particularly phosphatidylcholine as permissive CD1b lipid antigens. These data suggest autoreactive TCR antigen-specificity is more diverse than previously described and that these TCRs may adopt novel docking modes onto CD1b to recognise distinct endogenous antigens.

In the final chapter of results, the phenotype and functional characteristics of M. tuberculosis-reactive CD1b-restricted T cells was investigated. Glucose monomycolate (GMM) is a cell wall lipid expressed by pathogenic Mycobacteria, including M. tuberculosis, that is presented by CD1b to T cells. GMM-reactive T cells were first isolated directly ex vivo from the peripheral blood of latent M. tuberculosis infected donors using CD1b tetramers. Herein, an optimised CD1b tetramer enrichment was developed to isolate very small frequencies of GMM-reactive T cells from healthy donor blood directly ex vivo. In contrast to M. tuberculosis infected patients, GMM-reactive T cells from healthy donors expressed a diverse TCR repertoire. GMM-reactive T cells were exclusively CD4+CD8- and most cells expressed the memory marker CD45RO. Similar to other unconventional T cells, GMM-reactive T cells from healthy donors expressed the transcription factor promyelocytic leukaemia zinc finger (PLZF). In vitro stimulation revealed GMM-reactive T cells secrete both TNF and IFNg, similar to MAIT cells. To characterise these cells further, single-cell transcriptomic analysis was performed on GMM-reactive T cells isolated using CD1b-GMM tetramers. GMM-reactive T cell expressed unique transcriptomic signatures that were accurately distinguishable from the transcriptomes of natural killer T (NKT) cells and CD4+ T cells, indicating that these cells may be functionally distinct from other unconventional and conventional T cells populations.

In summary, these data describe a role for the CD8 co-receptor on MAIT and other MR1-reactive T cells and expand on the limited knowledge of CD1b-restricted T cells in healthy blood in regard to TCR repertoire, antigen-specificity and phenotype.