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A divergent transcriptional landscape underpins the development and functional branching of MAIT cells

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Science Immunology  22 Nov 2019:
Vol. 4, Issue 41, eaay6039
DOI: 10.1126/sciimmunol.aay6039
  • Fig. 1 Transcriptomic analysis of mouse MAIT cell development.

    (A) Flow cytometric analysis of three stages of mouse thymic MAIT cells after MR1-5-OP-RU tetramer enrichment. MAIT cell stages are defined with CD24 and CD44; stage 1 (S1; CD24+CD44) in blue, stage 2 (S2; CD24CD44) in green, and stage 3 (S3; CD24CD44+) in red. (B) MD plot showing gene expression comparison of bulk cell–purified stage 3 versus stage 1 MAIT cells. Colored dots indicate genes that significantly up-regulated in stage 3 (red) and stage 1 (blue). Colored numbers represent the total number of DEGs. (C) Table lists the top 50 most DEGs within each subset. (B and C) Data are from three pooled biological replicates each generated from a pool of thymi from five mice. (D) Gene expression comparison of single cell–purified stage 2 versus stage 1 (left) and stage 3 versus stage 2 (right) MAIT cells. (E) Phenotypic analysis of thymic MAIT cell stages for expression of transcription factors LEF1 (encoded by gene Lef1), SATB1 (Satb1), TCF1 (Tcf7), and Bach2 (Bach2); SLAM molecules CD150 (Slamf1), Ly108 (Slamf6), and CD319 (Slamf7); costimulation receptors CD27 (Cd27) and CD28 (Cd28); glycoprotein CD138 (Sdc1); and chemokine receptors CCR2 (Ccr2), CCR6 (Ccr6), CCR7 (Ccr7), and CXCR6 (Cxcr6). Histograms depict stage 1 MAIT cells in blue, stage 2 MAIT cells in green, stage 3 MAIT cells in red, and CD4+CD8+ double-positive (DP) thymocytes in black. Colored numbers indicate mean fluorescence intensity (MFI) values of each marker for the respective cell population. (A and E) Data are representative of at least two independent experiments with a total of two separate samples (pools of three thymi).

  • Fig. 2 Bifurcation and transcriptomic comparison of mature mouse MAIT cells.

    (A) Flow cytometric analysis of mature mouse thymic MAIT cells after HSA complement–mediated depletion showing RORγt and T-bet expression in CD44+ stage 3 MAIT cells. (B) Panel of markers showing expression of CD138 (Sdc1), CD319 (Slamf7), CD43HG (Spn), CD122 (Il2rb), and CD127 (Il7r) relative to RORγt and T-bet in stage 3 MAIT cells. (C) Flow cytometry profile of stage 3 MAIT cells costained with CD138 and CD319. (D) Gene expression comparison of purified CD319+ versus CD138+ stage 3 MAIT cells. Colored dots indicate genes significantly up-regulated in CD319+ cells (red) and CD138+ cells (blue). Colored numbers represent the total number of DEGs. (E) Tables list genes enriched corresponding to CD319+ (red, top) and CD138+ (blue, bottom) MAIT cell subsets. (F) Flow cytometry analysis for expression of PLZF, CD44, ICOS, Ly6c, and NK1.1 relative to RORγt+ and T-bet+ mature MAIT cells. (G) LEF1, SATB1, CD28, and CD103 expression on CD138+- and CD319+-stained mature MAIT cells. Colored numbers indicate MFI of each marker for the respective CD138+ or CD319+ MAIT cells. (A to C, F, and G) Data are representative of at least two independent experiments.

  • Fig. 3 Lineage diversification within stage 3 of MAIT cell development.

    (A) Flow cytometry profile of mouse MAIT cell subsets present within the mouse thymus single cell–sorted for RNA-seq analysis. Graph depicts percentages of each stage 3 subpopulation across eight samples. (B) Principal components analysis on the respective sorted subsets in (A) (left), and Monocole DDRtree showing the clustering of respective MAIT cell subsets (right). (C) Heatmap showing the DEGs across MAIT cell subsets ordered by decreasing Cd4 (top) and Lef1 (bottom) expression, grouped across developmental subsets (n = 363 in total, n = 75 stage 1, n = 34 stage 2, n = 81 CD138 CD319 double-negative stage 3, n = 95 CD138+ stage 3, and n = 78 CD319 stage 3). (D) Flow cytometry analysis of mature CD44+ MAIT cells for CD138 and CD319 expression (left). Analysis of CD4+ subset (yellow) from mature CD44+ CD319 MAIT cells (middle). Histogram overlay analysis of LEF1 expression between CD319 and CD138CD319 CD4+ stage 3 MAIT cells (right). (E) Scatter plot graph shows percentage of CD4+ subset within stage 2 and CD319 stage 3 MAIT cells. Graph shows percentage of LEF1+ cells within CD4+ S2 and CD4+ S3 MAIT cell populations.

  • Fig. 4 Identification of SAP, SATB1, Zap70, CXCR6, and CCR7 as key factors that regulate MAIT cell development with gene-deleted mice.

    (A to E) Three panels depicting analysis of MAIT cells WT control mice and SAP KO (A), Satb1m1Anu/m1Anu (B), SKG (C), CXCR6 KO (D), and CCR7 KO (E) mice. Left panel of flow cytometry analysis shows detection of MAIT cells from whole and MR1-5-OP-RU tetramer–enriched thymus of respective mice, analysis of MR1-tetramer–enriched MAIT cells for CD24 and CD44 expression. Scatter graphs depict absolute numbers and percentage of MAIT cells of T cells in thymus and spleen (A to E) and lymph nodes (E) of the respective mouse strains. Bar graphs in (E) show the proportion of RORγt and T-bet subset of stage 3 MAIT cells in WT and CCR7 KO thymus, spleen, and lymph nodes. Data are representative of two independent experiments with a total of six to nine mice per group (A to E; mean ± SEM). *P < 0.1 **P < 0.01 ***P < 0.001, ****P < 0.0001 (Mann-Whitney U rank sum test; A to E).

  • Fig. 5 Transcriptomic analysis of human MAIT cell development.

    (A) Flow cytometric analysis of three stages of human Vα7.2+ thymic MAIT cells of CD3+ cells before and after TRAV1-2 enrichment. MAIT cell stages are defined with CD27 and CD161; stage 1 (S1, CD27CD161) in blue, stage 2 (S2, CD27+CD161) in green, and stage 3 (S3, CD27+/-CD161+) in red. (B) MD plot showing gene expression comparison of bulk cell–purified stage 3 versus stage 1 human MAIT cells. Colored dots indicate genes that significantly up-regulated in stage 3 (red) and stage 1 (blue). Colored numbers represent the total number of DEGs. (C) Table lists the top 50 most DEGs within each subset. (D) Gene expression comparison of purified stage 2 versus stage 1 human MAIT cells and stage 3 versus stage 2 human MAIT cells. (B to D) Data are representative from two separate sorts, with a total of eight donor samples. (E) Phenotypic analysis of human thymic MAIT cell stages for expression of transcription factors SATB1, LEF1, and TCF1; CD1 molecules CD1a, CD1b, and CD1d; HLA-A, HLA-B, HLA-C; and chemokine receptors CCR5 and CCR6. Histograms depict stage 1 MAIT cells in blue, stage 2 MAIT cells in green, stage 3 MAIT cells in red, CD4+CD8+ DP thymocytes in black. (F) Flow cytometry analysis of CD4+ (yellow) and CD4 (pink) CD161+ thymic MAIT cells for LEF1 expression. Scatter plots show percentage CD4+ cells within CD161+ thymic MAIT cells and percentage LEF1+ cells within this CD4+ population. (E and F) Data indicate mean ± SEM and are representative of at least three experiments using three to seven separate donor samples.

  • Fig. 6 Transcriptomic analysis of extrathymic MAIT cell development.

    (A) Flow cytometry profile with CD27 and CD161 expression of Vα7.2+ MAIT cells of CD3+ cells from young (thymus donor) and adult peripheral blood sample. (B) MD plot showing gene expression comparison of bulk cell–purified stage 3 MAIT cells from young peripheral blood versus thymus sample and associated list of the most DEGs. Data were derived from five young blood and three thymus donor samples. (C) Flow cytometry analysis of CCR7 and LEF1 expression on stage 3 thymus MAIT cells, stage 3 young blood MAIT cells, and young blood conventional T cells. Data are representative of at least three experiments using three separate donor samples. (D) Classification of known DEGs between MAIT and conventional T cells and known core transcriptional signature of CD161-associated up-regulated genes into intrathymic (between thymic stage 3 and stage 1) and extrathymic (between young blood and thymus) regulation events.

  • Fig. 7 A new model of MAIT cell development.

    Dashed lines depict likely pathways based on transcriptome analysis. Phenotypic signature of each stage is shown in blue text in boxes. Factors that regulate each differentiation step are depicted above the arrow for each relevant step: Green text represents newly defined factors that support the relevant step, and black text represents previously defined factors from this study. This model is based on mouse MAIT cell development.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/41/eaay6039/DC1

    Fig. S1. Full heatmap data for Fig. 3C.

    Fig. S2. GO and KEGG pathway enrichment analysis in MAIT cell development.

    Fig. S3. Trajectory analysis on sorted single cells.

    Fig. S4. MD profiles of DEGs of Lef1+ stage 3 MAIT cells compared with other subsets.

    Fig. S5. Conventional TCRβ+, NKT, and γδT cell compartments in SAP, Satb1m1Anu/m1Anu, SKG, Cxcr6 KO, and Ccr7 KO mice.

    Fig. S6. Abnormal Foxp3+ Treg phenotype of mice homozygous for the Satb1m1Anu allele.

    Fig. S7. MAIT cell development appears to be normal in Bach2-deficient mice.

    Fig. S8. Full heatmap data for Fig. 5.

    Fig. S9. DEGs between bulk splenic and thymic mature MAIT cells.

    Fig. S10. Flow cytometry gating strategies and representative sorting purities.

    Table S1. Full list of DEGs from bulk (100 cells) purified thymic stage 3 versus stage 1 mouse MAIT cells.

    Table S2. Full list of top DEGs between stage 3 and stage 1 human MAIT cells.

    Table S3. List of antibodies used.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Full heatmap data for Fig. 3C.
    • Fig. S2. GO and KEGG pathway enrichment analysis in MAIT cell development.
    • Fig. S3. Trajectory analysis on sorted single cells.
    • Fig. S4. MD profiles of DEGs of Lef1+ stage 3 MAIT cells compared with other subsets.
    • Fig. S5. Conventional TCRβ+, NKT, and γδT cell compartments in SAP, Satb1m1Anu/m1Anu, SKG, Cxcr6 KO, and Ccr7 KO mice.
    • Fig. S6. Abnormal Foxp3+ Treg phenotype of mice homozygous for the Satb1m1Anu allele.
    • Fig. S7. MAIT cell development appears to be normal in Bach2-deficient mice.
    • Fig. S8. Full heatmap data for Fig. 5.
    • Fig. S9. DEGs between bulk splenic and thymic mature MAIT cells.
    • Fig. S10. Flow cytometry gating strategies and representative sorting purities.
    • Table S1. Full list of DEGs from bulk (100 cells) purified thymic stage 3 versus stage 1 mouse MAIT cells.
    • Table S2. Full list of top DEGs between stage 3 and stage 1 human MAIT cells.
    • Table S3. List of antibodies used.

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