MAIT cell-MR1 reactivity is highly conserved across multiple divergent species

Abstract

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αβ T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.

Keywords: 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU); MHC-I related protein 1 (MR1); T cell biology; T cell receptor (TCR); antigen (Ag); comparative immunology; innate-like immunity; major histocompatibility complex (MHC); mucosal-associated invariant T (MAIT) cell.

Figure 1

Biochemical characterization of recombinant cattle and pig MR1–5-OP-RU and MR1–6-FP monomers.A and B, 15% SDS-PAGE under non-reducing conditions of 1.5 μg purified biotinylated cattle, pig, and human MR1 in complex with β2m and loaded with 5-OP-RU (5-OP) or 6-FP in comparison to a protein ladder (M) with molecular weights of proteins indicated as relevant. Accounting for loss of 4 H atoms and 2 H atoms due to the formation of two disulphide bonds in MR1 and one disulphide bond in β2m; the molecular weights of biotinylated MR1 and β2m are as follows: human: MR1: 32,258 Da, β2m: 11,860 Da; cattle MR1: 32403 Da, β2m: 11764 Da; pig MR1: 32526 Da, β2m: 11542 Da. C, 5-OP-RU- and 6-FP-loaded cattle and pig MR1 monomers in comparison to biotinylated human MR1–5-OP-RU (assessed previously alongside other species’ MR1 molecules, (64)) in ELISA with mAbs 26.5 and 8F2.F9 showing normalized, base-line corrected dose-response curves (n = 3, mean ± SD). EC₅₀ values, as summarized in the table, were determined based on non-linear curve fits shown in the charts. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil; 6-FP, 6-formylpterin; β2m, β2 microglobulin; MR1, MHC-I related protein 1.

Figure 2

Identification of MAIT cells in multiple species using species-specific MR1–5-OP-RU and MR1–6-FP tetramers.A, representative flow cytometry plots from staining of human PBMC, cattle PBMC, pig-tailed macaque PBMC, mouse lung-, spleen- and blood-derived lymphocytes, and pig PBMC with MR1–5-OP-RU and MR1–6-FP tetramers of the corresponding species. B, frequencies of species-matched MR1–5-OP-RU and MR1–6-FP tetramer⁺ MAIT cells from human CD3⁺ PBMC (n = 4), cattle CD3⁺ PBMC (n = 4), pig-tailed macaque CD8⁺ PBMC (n = 5), mouse lungs, spleen, and whole blood-derived αβTCR⁺ lymphocytes (n = 6), and pig CD3⁺ PBMC (n = 3). Mean ± SEM is depicted. Experiments were performed once in pig-tailed macaque and mouse and twice in all other species. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil; 6-FP, 6-formylpterin; MAIT, mucosal-associated invariant T; MR1, major histocompatibility complex-I related protein 1; PBMC, peripheral blood mononuclear cell; TCR, T cell receptor.

Figure 3

Flow cytometry plots of species-mismatched MR1–5-OP-RU and MR1–6-FP tetramer staining. Human, cattle, pig-tailed macaque, mouse, and pig MR1–5-OP-RU and MR1–6-FP tetramers were used to stain human PBMC, cattle PBMC, lymphocytes from MAIT cell boosted mouse spleen, pig-tailed macaque PBMC, sheep PBMC or pig PBMC. Representative final gating is shown for each species and each, MR1–5-OP-RU and MR1–6-FP tetramer. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil; 6-FP, 6-formylpterin; MAIT, mucosal-associated invariant T; MR1, MHC-I related protein 1; PBMC, peripheral blood mononuclear cell.

Figure 4

Frequency and staining characteristics of MR1–5-OP-RU and MR1–6-FP tetramer⁺populations identified with species-matched and species-mismatched tetramer reagents. Bar charts and heatmap summaries, displaying the following: A, frequencies of species-matched (red histograms) and species-mismatched (black histograms) MR1–5-OP-RU (RU, black dots) and MR1–6-FP (FP, white dots) tetramer⁺ cells in human CD3⁺ PBMC (n = 8), cattle CD3⁺ PBMC (n = 8), pig-tailed macaque CD8⁺ PBMC (n = 5), MAIT cell boosted mouse spleen (n = 7)–derived αβTCR⁺ lymphocytes, and sheep CD8⁺ PBMC (n = 6). Histograms depict mean frequency ± SEM with each individual datapoint shown. B, species-matched (red histograms) and species-mismatched (black histograms) MR1–5-OP-RU tetramer (RU) geometric mean fluorescence intensity (gMFI) fold change over the MR1–5-OP-RU tetramer^- population (background) and (C) SD of the MR1–5-OP-RU tetramer⁺ (RU) gMFI as described in (A). Differences between the frequencies obtained from species-matched and species-mismatched MR1–5-OP-RU staining were evaluated using a one-way ANOVA with Giesser-Greenhouse correction or, where there are missing values, a mixed effect model with repeated measures, followed by Dunnett’s multiple comparison test, comparing staining with species-matched MR1–5-OP-RU tetramer and all other species-mismatched MR1–5-OP-RU tetramers, with individual variance computed for each comparison. Only statistically significant differences (p value < 0.05) are indicated. Data are combined from either one (pig-tailed macaque), two (human, cattle, and sheep), or three (mouse) separate experiments. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil; 6-FP, 6-formylpterin; PBMC, peripheral blood mononuclear cell; MAIT, mucosal-associated invariant T; MR1, MHC-I related protein 1; TCR, T cell receptor.

Figure 5

Detailed characterization of MAIT cells in pigs and sheep.A, (I) representative flow cytometry plots of cells stained with pig MR1–5-OP-RU (pMR1–5-OP-RU) or MR1–6-FP (pMR1–6-FP) tetramers conjugated to PE or BV421 among CD3⁺ γδTCR^- pig PBMC, BAL, lungs spleen, and liver (and for comparison among CD3⁺ cattle PBMC), (II) the proportion of cells double positive for pig MR1–5-OP-RU (RU) or MR1–6-FP (FP) tetramers conjugated to PE or BV421 among CD3⁺ γδTCR^- T cells in pig PBMC (n = 5) (and for comparison among CD3⁺ MHC-II^- cattle PBMC (n = 1)), (III) the proportion of cells double positive for pig MR1–5-OP-RU (RU) or MR1–6-FP (FP) tetramers conjugated to PE or BV421 among CD3⁺ MHC-II^- pig PBMC (n = 18) (and for comparison among CD3⁺ human PBMC (n = 1)), and (IV) the proportion of cells double positive for pig MR1–5-OP-RU (RU) or MR1–6-FP (FP) tetramers conjugated to PE or BV421 among CD3⁺ γδTCR⁻ T cells in pig (n = 2 or 3) BAL, lungs, spleen, and liver lymphocytes. B, representative flow cytometry plots (I) and quantification of the frequency (II) of the IFNγ and TNF intracellular cytokine response in cattle CD8⁺ PBMC (n = 1) and CD3⁺ γδTCR^- pig (n = 3) PBMC, BAL, lungs, liver, and spleen lymphocytes, following stimulation with 1 μM 5-OP-RU for 6 h. Mean ± SEM are shown. C, representative images (I) and quantification (II) of secreted IFNγ assessed by ELISpot following coincubation of human (n = 3), pig (n = 3), cattle (n = 4), and sheep (n = 3) PBMC with medium, 1 μM Ac-6-FP, 1 μM 5-OP-RU, or 4 μg/ml ConA for 18 h. Mean SCF/10⁶ cells ± SEM are shown. Ac-6-FP, acetyl-6-FP; 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil; 6-FP, 6-formylpterin; MAIT, mucosal-associated invariant T; MHC, major histocompatibility complex; MR1, MHC-I related protein 1; TCR, T cell receptor; PBMC, peripheral blood mononuclear cell.

Figure 6

Frequency and TCRα usage of putative MAIT cells in cattle, sheep and pig PBMC.A, frequencies of TRAV1⁺ (I) and putative MAIT TCR CDR3α sequence⁺ (II) of total TRA bulk sequenced cattle, sheep, and pig PBMC (n = 2). B, frequency of TRAJ33, TRAJ12, or TRAJ20 gene segment usage among TRAV1⁺TRA sequences from cattle, sheep, and pig PBMC. C, relative frequency and sequence of putative MAIT TCR CDR3α-loops in each animal. D, sequence logo (Seq2Logo) of the amino acid weighting of the CDR3α-loops from putative MAIT TCRs in each species (note that the Y95, highlighted in green, is conserved between all species). MAIT, mucosal-associated invariant T; PBMC, peripheral blood mononuclear cell; TCR, T cell receptor.