Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.Career Development Award: (#IK2 CX000538); U.S. Department of Veterans Affairs Clinical Sciences Research and Development Program (MJH); U.S.Department of Veterans Affairs Biomedical Laboratory Research and Development Program (DML) Merit Award: (#I01 BX000533); American Lung Association: (RT-350058)

Multiple receptors converge on H2-Q10 to regulate NK and gamma delta T-cell development

Class Ib major histocompatibility complex (MHC) is an extended family of molecules, which demonstrate tissue-specific expression and presentation of monomorphic antigens. These characteristics tend to imbue class Ib MHC with unique functions. H2-Q10 is potentially one such molecule that is overexpressed in the liver but its immunological function is not known. We have previously shown that H2-Q10 is a ligand for the natural killer cell receptor Ly49C and now, using H2-Q10-deficient mice, we demonstrate that H2-Q10 can also stabilize the expression of Qa-1b. In the absence of H2-Q10, the development and maturation of conventional hepatic natural killer cells is disrupted. We also provide evidence that H2-Q10 is a new high affinity ligand for CD8αα and controls the development of liver-resident CD8αα γδT cells. These data demonstrate that H2-Q10 has multiple roles in the development of immune subsets and identify an overlap of recognition within the class Ib MHC that is likely to be relevant to the regulation of immunity

Study of MAIT cell activation in viral infections in vivo

MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation

Correlations in daily deviations from the median of f0F2, M(3000)F2 and VF

The statistical analysis of the hourly daily deviations from the corresponding monthly-median values of the factor M(3000)F2 and of the critical frequency of the F2-layer, f0F2, reveals that the correlation between these parameters is poor. A similar analysis between the hourly daily deviations from the corresponding monthly-median of the height h’F and the f0F2 leads to similar results. These results indicate that the factor M(3000)F2, the height h’F and the critical frequency f0F2 may have a different daily variability

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.Supported by the National Health and Medical Research Council of Australia (1083942, 1013667 and 1016629; CDF 1035858 to A.E.; ECF 1054431 to D.G.P.; Senior Principal Research Fellowships 1020770 and 1027369 to D.I.G. and D.P.F.; Australia Fellowship AF50 to J.R.; CDF2 Fellowship 1047025 to M.C.; CDF2 Fellowship 1023294 to K.K.; and CDF1 Fellowship 1106004 to L.K.M.), the Australian Research Council (CE140100011 and LE110100106; Future Fellowship FT140100278 to A.P.U.), the Leukaemia Foundation of Australia (Postgraduate Scholarship for N.A.G.), the National Heart Foundation of Australia (Future Leader Fellowship for C.A.N.-P.), the Hudson Institute (Star Recruitment Fellowship for M.F.N.) and the Ritchie Centre (Victor Yu Fellowship for M.F.N.)