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, 211 (8), 1601-10

MR1-restricted MAIT Cells Display Ligand Discrimination and Pathogen Selectivity Through Distinct T Cell Receptor Usage

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MR1-restricted MAIT Cells Display Ligand Discrimination and Pathogen Selectivity Through Distinct T Cell Receptor Usage

Marielle C Gold et al. J Exp Med.

Abstract

Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRβ chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.

Figures

Figure 1.
Figure 1.
Pathogen-reactive MAIT TCRα chains are not confined to TRAV1-2/TRAJ33 rearrangements. Human CD8+ MAIT cells from four individuals were stimulated overnight using A549 cells infected with M. smegmatis, S. typhimurium, or C. albicans. Live, pathogen-reactive MAIT cells were then isolated by flow cytometry based on coexpression of TRAV1-2 and TNF. Unbiased molecular analysis of expressed TRA gene products was performed as described in Materials and methods. Aggregate TRAJ gene profiles from all four donors in response to each pathogen (n = 3) are shown at the top; data from individual donors are shown underneath as indicated. Each sequence was counted once regardless of frequency. Total sequence counts are indicated in each case.
Figure 2.
Figure 2.
Pathogen-reactive MAIT TCRβ chains are diverse. Details as described in the legend to Fig. 1, except that TRBV gene usage was analyzed. Aggregate TRBV gene profiles from all four donors in response to each pathogen (n = 3) are shown at the top; data from individual donors are shown underneath as indicated. Each sequence was counted once regardless of frequency. Total sequence counts are indicated in each case.
Figure 3.
Figure 3.
MAIT TRAV1-2+ CDR3α sequences display length and sequence similarity. (A) Representation of unique TRAV1-2+ CDR3α sequences from each donor in response to each pathogen as indicated. Each bar represents a distinct sequence, the relative frequency of which is depicted on the y axis. TRAJ gene usage is shown on the x axis labeled for dominant sequences. (B) Distribution of CDR3α amino acid lengths from TRAV1-2+ MAIT cells responsive to each pathogen as indicated in the key. (C) Visual representation of amino acid enrichments at each position across the CDR3α core compiled from pathogen-reactive MAIT cell TRAV1-2+ sequences. Analysis was confined to CDR3α sequences with a length of 11 amino acids, encompassing 39 sequences for M. smegmatis, 61 for S. typhimurium, and 30 for C. albicans. Graphics were generated using Seq2Logo.
Figure 4.
Figure 4.
MAIT CDR3β usage is unique across individuals and pathogens. (A) Representation of unique TRBV20-1+ CDR3β sequences from each donor in response to each pathogen as indicated. Each bar represents a distinct sequence, the relative frequency of which is depicted on the y axis. TRBJ gene usage is shown on the x axis labeled for dominant sequences. (B) Distribution of CDR3β amino acid lengths from all TRBV gene–defined MAIT cells responsive to each pathogen as indicated in the key. (C) Visual representation of amino acid enrichments at each position across the CDR3β core compiled from pathogen-reactive MAIT cell TRBV+ sequences. Analysis was confined to CDR3β sequences with a length of 13 or 14 amino acids, encompassing 34 sequences for M. smegmatis, 75 for S. typhimurium, and 39 for C. albicans. Graphics were generated using Seq2Logo.
Figure 5.
Figure 5.
MR1-restricted MAIT cell clones display ligand discrimination for the vitamin B metabolite RL-6,7-diMe. (A) A549 cells (20,000 per well) were pulsed with 5 µg/ml RL-6,7-diMe overnight and tested for their ability to stimulate MR1-restricted clones (10,000 per well) in IFN-γ ELISPOT assays. Clones D426 B1 and D481 A9 express TRAJ33; clones D481 F12 and D481 C7 express TRAJ20. TRBV usage and CDR3α and CDR3β sequences are indicated. Error bars represent SEM of duplicate determinations. (B) A549 cells (25,000 per well) were infected with S. typhimurium, C. albicans, or M. smegmatis at an MOI of 50 (hi), 25 (med), or 12.5 (lo) and then incubated overnight with the indicated MR1-restricted MAIT cell clones (10,000 per well) in duplicate wells. IFN-γ production was quantified by ELISPOT. Similar results were obtained from a minimum of three independent experiments.

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