Activation and In Vivo Evolution of the MAIT Cell Transcriptome in Mice and Humans Reveals Tissue Repair Functionality

Abstract

Mucosal-associated invariant T (MAIT) cells are MR1-restricted innate-like T cells conserved across mammalian species, including mice and humans. By sequencing RNA from sorted MR1-5-OP-RU tetramer⁺ cells derived from either human blood or murine lungs, we define the basic transcriptome of an activated MAIT cell in both species and demonstrate how this profile changes during the resolution of infection and during reinfection. We observe strong similarities between MAIT cells in humans and mice. In both species, activation leads to strong expression of pro-inflammatory cytokines and chemokines as well as a strong tissue repair signature, recently described in murine commensal-specific H2-M3-restricted T cells. Transcriptomes of MAIT cells and H2-M3-specific CD8+ T cells displayed the most similarities to invariant natural killer T (iNKT) cells when activated, but to γδ T cells after the resolution of infection. These data define the requirements for and consequences of MAIT cell activation, revealing a tissue repair phenotype expressed upon MAIT cell activation in both species.

Keywords: MHC-related protein 1; T cell; activation; human; lung; mouse; mucosal-associated invariant T cell; riboflavin; tissue repair; transcriptome.

Graphical abstract

Figure 1

Costimulatory Requirements for MAIT Cell Activation In Vivo (A) Representative flow-cytometry plots showing MAIT cell percentage among TCRβ⁺ lymphocytes in the lungs of C57BL/6 mice with or without prior stimulation with intranasal CpG and 5-OP-RU. (B and C) Relative (B) and absolute (C) numbers of MR1-5-OP-RU tetramer⁺ MAIT cells in the lungs of C57BL/6 mice 7 days after intranasal exposure to specific TLR agonists either alone or in combination with 76 pmol 6-FP, or with 76 pmol 5-OP-RU. Control mice received nothing (n = 4, naive) or CpG with 5-OP-RU (n = 3). Experiments used n = 5 (5-OP-RU treated), n = 3 (6-FP treated), or n = 2 (TLR agonist alone) mice per group. The experiment was subsequently repeated with similar results. Statistical tests: unpaired t tests, comparing TLR + 5-OP-RU with naive control (n = 4), on untransformed (B) or log-transformed (C) data with Bonferroni corrections ^∗p < 0.05, ^∗∗∗p < 0.001.

Figure 2

Reactome Pathway Analysis of Activated MAIT Cells Pathway analysis of human and murine activated MAIT cell transcriptomes. (A and B) Human peripheral blood 5-OP-RU-stimulated MR1-5-OP-RU-tetramer⁺ MAIT cells compared with (A) naive CD8⁺CD45RA⁺ cells or (B) unstimulated MAIT cells. (C and D) Murine pulmonary MR1-5-OP-RU-tetramer⁺ MAIT cells day 7 post infection with Legionella were compared with (C) naive CD8⁺CD44⁻CD62L⁺ T cells from uninfected mice or (D) MR1-tetramer⁺ MAIT cells from mice 12 weeks post infection with Legionella. Plots show the extent to which named pathways from the curated Reactome database are upregulated. Color intensity represents statistical significance of the upregulation, dot size represents the number of genes upregulated in the pathway, x axis represents the proportion of all differentially expressed genes included in the pathway (“gene ratio”). n = 3 biological replicates per group performed once. Pathways were selected using a significance threshold of a log fold change >2 and p < 0.01.

Figure 3

Comparison of Murine MAIT Cell Transcriptomes with Other Cells in Immunological Genome Project Dataset and with Commensal-Induced H2-M3-Restricted T Cells Hierarchical clustering was used to compare transcriptomes of murine pulmonary MAIT cells or naive CD8⁺CD44⁻CD62L⁺ cells in this study with 88 other cell types deposited in the Immunological Genome Project (ImmGen) database and three cell types selected from Linehan et al., (2018) (gray lozenges). Figure shows a dendrogram (left), ImmGen identifiers (middle), and the full name of each cell type (right). Further details on cell types are presented in Table S2. ImmGen samples are identified in white lettering. Samples from the current study are identified in black lettering, with extended lozenges. Each cell type is represented by 2 to 3 replicates, identified by a numerical suffix, of which each replicate (“batch”) represents pooled tissue from three animals. Cell types are color coded: invariant natural killer T cells (iNKT, orange), natural killer (NK) cells (brown), γδ T cells (light green), innate lymphoid cells (ILC, dark green), conventional CD8 T cells (blue), and MAIT cells (purple). CD, clonal designation; NCR, NK cell receptor; Teff, effector T cell; Tmem, memory T cell.

Figure 4

Gene Set Enrichment Analysis for Tissue Repair Gene Signature in Human and Murine MAIT Cells (A–D) Gene set enrichment analysis (GSEA) was used to determine potential enrichment of a tissue repair signature (Linehan et al., 2018) in gene expression profiles from human (A and B) and murine (C and D) MAIT cells. (A) GSEA summary plots for 5-OP-RU-stimulated human peripheral blood MAIT cells compared with unstimulated MAIT cells. The gene set is highly enriched: enrichment score (ES) = 0.62; normalized enrichment score (NES) = 1.38; nominal p value < 0.01; familywise error rate (FWER) p value < 0.01. (B) Heatmap of expression of leading-edge subset genes within the gene set (red, highest expression; blue, lowest). (C) GSEA summary plots for murine pulmonary MAIT cells 7 days post i.n. L. longbeachae infection (“Acute infection”), compared with MAIT cells 12 weeks post infection (“Resolved infection”). The gene set is highly enriched: enrichment score (ES) = 0.85; normalized enrichment score (NES) = 1.23; nominal p value < 0.01; familywise error rate (FWER) p value < 0.01. (D) Heatmap of expression of leading-edge subset genes within the gene set (red, highest expression; blue, lowest). n = 3 biological replicates per group performed once.