Human blood MAIT cell subsets defined using MR1 tetramers

Abstract

Mucosal-associated invariant T (MAIT) cells represent up to 10% of circulating human T cells. They are usually defined using combinations of non-lineage-specific (surrogate) markers such as anti-TRAV1-2, CD161, IL-18Rα and CD26. The development of MR1-Ag tetramers now permits the specific identification of MAIT cells based on T-cell receptor specificity. Here, we compare these approaches for identifying MAIT cells and show that surrogate markers are not always accurate in identifying these cells, particularly the CD4⁺ fraction. Moreover, while all MAIT cell subsets produced comparable levels of IFNγ, TNF and IL-17A, the CD4⁺ population produced more IL-2 than the other subsets. In a human ontogeny study, we show that the frequencies of most MR1 tetramer⁺ MAIT cells, with the exception of CD4⁺ MAIT cells, increased from birth to about 25 years of age and declined thereafter. We also demonstrate a positive association between the frequency of MAIT cells and other unconventional T cells including Natural Killer T (NKT) cells and Vδ2⁺ γδ T cells. Accordingly, this study demonstrates that MAIT cells are phenotypically and functionally diverse, that surrogate markers may not reliably identify all of these cells, and that their numbers are regulated in an age-dependent manner and correlate with NKT and Vδ2⁺ γδ T cells.

Keywords: Human immunology; MAIT; MR1; T cell; unconventional T cell.

Figure 1

Enumeration of unconventional T‐cell subsets. (a) Flow cytometric plots showing example gating of (i) MAIT cells and atypical MR1 tetramer⁺ TRAV1‐2⁻ T cells, (ii) Type I NKT cells and (iii) γδ T cells, gated on total CD3⁺ T cells, and (iv) Vδ1 versus Vδ2 expression on γδ T cells. (b) Box and whisker plots showing the percentage of innate‐like T‐cell subsets of total CD3⁺ T cells. MAITs n = 47; atypical MR1 tet⁺ n = 12; Type I NKT cells n = 27; γδ T cells n = 33; derived from eight experiments. (c) Scatter plots showing donor‐matched percentages of (i) MAIT and NKT cells, or (ii) MAIT and Vδ2⁺ T cells as a proportion of total CD3⁺ cells (Spearman correlations (i) r = 0.53, P = 0.005, n = 27; (ii) r = 0.45, P = 0.018, n = 27).

Figure 2

Co‐receptor distribution on MAIT cells. Flow cytometric plots showing example gating of CD4 and CD8α expression (a) and CD8α and CD8β expression (b i) on non‐MAIT αβ T cells (upper panels) and MAIT cells (lower panels). Box and whisker plots showing CD8β (b ii) and CD8α (b iii) expression on CD8αβ⁺, cells on MAIT cells and non‐MAIT αβ T cells (n = 4, from two experiments). CD8β gate set based on fluorescence minus one (FMO) control. (c) Box and whisker plots showing: (i) the percentage of MAIT cells expressing each co‐receptor; (ii) the percentage of CD4⁺CD8⁺ DP MAIT cells that are CD8αα⁺ or CD8αβ⁺, and; (iii) the percentage of total T cells expressing each co‐receptor that are MAIT cells (total CD8⁺, DN, CD4⁺ and DP n = 33; CD8αα⁺ and CD8αβ⁺ n = 10; from eight experiments).

Figure 3

Selective decrease in MAIT cell subsets with age. (a) Box plot showing MAIT cell frequency of total CD3⁺ T cells. (b–f) Scatter plots showing donor age versus the proportion of total CD3⁺ T cells that are: (b) MAIT cells (c) CD8α ⁺ MAIT cells (d) DN MAIT cells (e) DP MAIT cells or (f) CD4⁺ MAIT cells. Red data points = ages 0–25; blue data points = ages 26–70. Statistical tests are Spearman correlations. Linear regression lines and 95% confidence intervals are also depicted. Samples from young donors (ages 0–14) had been partially analyzed in a previous study,16 but without full CD4/CD8 subset analysis as depicted here.

Figure 4

Comparison of MR1‐Ag tetramers and surrogate phenotyping techniques. (a) (i) Representative flow cytometric pseudo‐color plots showing example gating of MAIT cells using anti‐TRAV1‐2 mAb versus CD161 in one cocktail, or IL‐18Rα or CD26 in another cocktail on total T cells, followed by MR1‐5‐OP‐RU tetramer staining on TRAV1‐2⁺ CD161/IL‐18Rα/CD26 high, intermediate or negative cells. (ii) Box and whisker plots showing percentage TRAV1‐2⁺, CD161 (green; n = 24), IL‐18Rα (red; n = 12) and CD26 (blue; n = 12) High (HI), Intermediate (INT) and Low (LOW) cells that are MR1‐5‐OP‐RU tetramer^+, derived from three experiments. (b) (i) Representative flow cytometric pseudo‐color plots showing example gating of MAIT cells using anti‐TRAV1‐2 mAb versus CD161 for T cell co‐receptor subsets (upper panel) and MR1‐5‐OP‐RU tetramer staining on TRAV1‐2⁺ CD161^HI cells for each co‐receptor (lower panel). (ii) Box and whisker plots showing percentage TRAV1‐2⁺, CD161^HI cells that are MR1‐5‐OP‐RU tetramer⁺ for each co‐receptor (CD4⁺, DN and CD8⁺ n = 24; DP, CD8αα⁺ and CD8αβ⁺ n = 12, from three experiments). (c) (i) Representative flow cytometric pseudo‐color plots showing example gating of MAIT cells using MR1‐5‐OP‐RU tetramer versus CD161, IL‐18Rα or CD26 on total T cells (left panels) or MR1‐5‐OP‐RU tetramer⁺ TRAV1‐2⁺ T cells (right panel). (ii) Box and whisker plots showing percentage TRAV1‐2⁺ MR1‐5‐OP‐RU tetramer⁺ cells that are HI (blue), INT (Red) or NEG (green) for CD161 (n = 24), IL‐18Rα (n = 12) and CD26 (n = 12), derived from three experiments.

Figure 5

Surface phenotyping of MAIT cells. (a) Representative histogram overlays of surface marker expression on MAIT cells (blue histograms), non‐MAIT CD3⁺ T cells (red histograms) and CD3⁻, CD19⁻ lymphocytes (green histograms) (b) Box and whisker plots showing the percentage of MAIT cell subsets expressing CCR6, CXCR6, CD27, CD56, NKG2D and NKG2A (n = 12 from two separate experiments). MAIT cell subsets were gated as per Figure 2a after initially gating on CD3⁺TRAV1‐2⁺MR1‐5‐OP‐RU tetramer⁺, viable lymphocytes. The statistical test was a Wilcoxon matched‐pairs signed‐rank test. (c) Box and whisker plot showing the percentage of total CD56⁺ T cells that are MAIT cells (n = 28, from three experiments).

Figure 6

Transcription factor and cytokine profile of MAIT cell subsets. (a) Upper panel: Representative flow cytometric histograms showing transcription factor staining on MAIT cells (blue histograms) overlaid above total non‐MAIT αβ T cells (red histograms). Lower panel: Box and whisker plots showing the MFI of transcription factor staining on MAIT cells (blue boxes) and total non‐MAIT T cells (red boxes) for PLZF, RORγt, T‐bet and GATA‐3. (n = 12 from three experiments). (b) Box and whisker plots showing the MFI of transcription factor staining between MAIT cell subsets for PLZF, T‐bet and RORγt. (n = 10 from two separate experiments). (c) Box and whisker plots showing the percentage of IFNγ⁺ or TNF⁺ MAIT cells subsets after 7 h stimulation of donor PBMCs with PMA and ionomycin. (n = 11, from three experiments). (d) Box plots showing supernatant cytokine levels after FACS‐sorted MAIT cell subsets were stimulated for 24 h with PMA and ionomycin. (n = 7 for CD4⁺, DN, CD8αα⁺ and CD8αβ⁺ MAIT cells, and n = 4 for DP MAIT cells from two separate experiments). Statistical analyses in b‐d were performed using Friedman tests with Dunn's multiple comparison post hoc tests.

Figure 7

TRAJ gene usage by TRAV1‐2⁺ MAIT cells. (a) Box and whisker plots showing percent total productive TRAV1‐2⁺ reads recombined with TRAJ12, TRAJ20, TRAJ33 or other TRAJ genes in amplified cDNA from MR1‐5‐OP‐RU tetramer⁺ TRAV1‐2⁺ T cells subjected to deep sequencing. Data are from 4 human donors (n = 43 969, 45 454, 46 126 and 46 553 functional TRAV1‐2⁺ reads for donors 1–4, respectively). (b) Pie charts showing distribution of TRAJ genes by noncanonical TRAV1‐2⁺ TCR‐α chain transcripts from four individual donors. (c) Bar graphs showing CDR3‐α junction amino acid length distribution for TRAJ12⁺, TRAJ20⁺, TRAJ33⁺ and Other TRAV1‐2⁺ TCR‐α chain transcripts pooled from four individual donors (derived from n = 9513, 19 522, 141 853 and 3006 transcripts, respectively). (d) Sequence logos depicting the amino acid distribution at the CDR3‐α junction in TRAJ12⁺, TRAJ20⁺ or TRAJ33⁺ TCR‐α chain transcripts that are 10 amino acids in junctional length (derived from n = 9451, 18 910 and 141 274 transcripts, respectively).