CD44

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MR1-5-OP-RU CD24 CD24 CD44 MAIT cells 2.78 11.2 WT RORγt- GFP reporter 1 5 1 4 1 3 2.28 1 5 1 4 1 3 4.8 1.6 8.1 1 5 1 4 1 3 1 5 1 4 1 3 3.7 3.21 8.5 61.7 1 2 1 3 1 4 1 5 TCRβ 2 1 1 3 1 4 1 5 CD44 1 2 GFP 1 3 1 4 1 5 1 2 1 3 1 4 1 5 Supplementary Figure 1 ROR t expression on MAIT cells in ROR t-gfp reporter mice. Flow cytometric analysis of MAIT cells from MR1-5-OP-RU tetramer enriched RORγt-GFP reporter mouse thymi for CD24, CD44, and GFP expression. Data are representative of a total of 9 mice with 3-pooled mouse thymi from 2 independent experiments.

MR1-5-OP-RU CD24 CD4 MR1-5-OP-RU + cells 1 5 1 4 1 3 1 2 TCRβ 2.6 1 5 34.8 1 4 1 3 1 2 15.1 CD44 4.8 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 S1 S2 S3 1 5 29.1 1. 1 4 1 3 1 2 5.2 1.7 1 2 1 3 1 4 1 5 CD8 Supplementary Figure 2 Thymic MAIT cell subsets in mice with transgenic expression of the TRAV1-TRAJ33 TCR. Flow cytometric analysis of MR1-5-OP-RU tetramer reactive MAIT cells in TRAV1-TRAJ33 C -/- TCR transgenic mouse thymus for expression of CD4, CD8, CD24 and CD44. Data are representative of 3 independent experiments.

Supplementary Figure 3 NKT cells in Drosha-deficient mice and germ-free mice. (a) Flow cytometric analysis of CD1d-PBS44 tetramer + TCRβ + NKT cells from thymus, spleen, lymph nodes from Drosha fl/+ CD4-Cre heterozygous control mice and Drosha CD4-Cre mice. Absolute numbers and percentage NKT cells of TCRβ + cells in thymus, spleen and lymph nodes of Drosha fl/+ CD4-Cre heterozygous control mice and Drosha CD4-Cre mice. (b) Flow cytometric analysis of NKT cells from thymus, spleen, lymph nodes from control (SPF) mice and germ-free (GF) mice. Absolute numbers and percentage NKT cells of TCRβ + cells in thymus and spleen of SPF mice and GF mice. *P<.1 **P<.1 ***P<.1 NS = not significant (Mann-Whitney rank sum U test (a, b)). Data are representative of 3 independent experiments with a total of 8 mice per group (a; mean ± SEM) or of 2 independent experiments with a combined total of 11-15 mice per group (b; mean ± SEM).

Supplementary Figure 4 MAIT cells in IL-18-deficient mice and IL-18R -deficient mice. (a) Flow cytometric analysis of MAIT cells from MR1-5-OP-RU tetramer enriched thymus, spleen and lymph nodes from WT and IL-18- deficient mice for CD24 and CD44 expression. (b) Absolute numbers and percentage MAIT cells of TCRβ + cells in individual thymus, spleen and lymph nodes of WT and IL-18-deficient mice. (c) Flow cytometric analysis of MAIT cells from MR1-5-OP-RU tetramer enriched thymus, spleen and lymph nodes from WT and IL-18Rα-deficient mice for CD24 and CD44 expression. (d) Absolute numbers and percentage MAIT cells of TCRβ + cells in individual thymus, spleen and lymph nodes of WT and IL-18Rα-deficient mice. *P<.1 **P<.1 ***P<.1 NS = not significant (Mann-Whitney rank sum U test (b, d)). Data are representative of 3 independent experiments with a total of 12 mice per group (a, b; mean ± SEM) or 2 independent experiments with a total of 1 mice per group (c, d; mean ± SEM).

Supplementary Figure 5 MAIT cells in C57BL/6 CD1d-deficient mice. Flow cytometric analysis of MAIT cells from thymus, MR1-5-OP-RU enriched thymus and spleen from C57BL/6 WT and C57BL/6 CD1d-deficient mice for CD24, CD44 and CD4/CD8 co-receptor expression. Data are representative of 3 independent experiments with a total of 6 mice per group.

Supplementary Figure 6 Schematic of the three development stages of MAIT cells. Mouse and human MAIT cell development in the thymus can be defined by three separate stages. Mouse thymic MAIT cells can be defined by a three-stage sequential pathway from CD24 + CD44 - (stage 1), via CD24 - CD44 - (stage 2), to CD24 - CD44 + (stage 3), while in humans thymic MAIT cells can be defined by three distinct stages from CD161 - CD27 - (stage 1), via CD161 - CD27 + (stage 2), to CD161 + CD27 +/lo (stage 3). These stages are regulated by several factors including MR1, PLZF, Drosha and commensal bacteria.

Supplementary Table 1. TCR sequences of mouse MAIT cell subsets MR1-5-OP-RU tetramer + stage 1 CD24 + CD44 - and stage 3 CD24 - CD44 + MAIT cells were sorted as single cells and their TCRα and β chains were determined using single cell TCR sequencing. X defines undetermined amino acid. Cell TRAV TRAJ CDR3 alpha TRBV TRBJ CDR3 beta Stage 1 CD24+ CD44- MR1-5- OP- RU+ TCRβ+ 1 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13 TRBJ1-3 CASGDASXSGNTLYF 2 TRAV1 TRAJ33 CAVTDSNYQLIW TRBV13-3 TRBJ2-1 CASSEQGGYAEQFF 3 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13-2 TRBJ2-5 CASGGWGFQDTQYF 4 TRAV1 TRAJ33 CAVRXXNYXLIW TRBV13-3 N.D. 5 TRAV1 TRAJ33 CAVRDRDYQLIW TRBV19 TRBJ1-6 CASTPGDNSPLYF 6 i) TRAV1 ii) TRAV9 i) TRAJ33 ii) TRAJ29 CAPMDSNYQLXW TRBV13-2 ND 7 i) TRAV1 ii) TRAV7 TRAJ33 CAVRDSNYQLIW TRBV13-2 TRBJ2-5 CASGDGGGWDTQYF 8 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13-3 TRBJ1-6 CASSDAGVNSPLYF 9 TRAV1 TRAJ33 CAVSNSNYQLIW TRBV13-2 TRBJ2-1 CASGDGDSYAEQFF 1 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13-2 TRBJ2-3 CASGDGDSAETLYF 11 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13-2 TRBJ2-3 CASGGDGDSAETLYF 12 TRAV1 TRAJ33 CAVMDSNYQLIW TRBV13-2 TRBJ2-1 CASGDGDSYAEQFF 13 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13-3 TRBJ2-7 CASSAGTSSYEQYF Stage 3 CD24- CD44+ MR1-5- OP- RU+ TCRβ+ 14 TRAV1 TRAJ33 CAVRDSNYQLIW TRBV13-3 TRBJ2-2 CASSDKGDTGQLYF 15 TRAV1 TRAJ33 CAVLDSNYQLIW TRBV13-3 TRBJ1-6 CASSDAGVNSPLYF 16 TRAV1 TRAJ33 CAVMDSNYQLIW TRBV19 TRBJ2-7 CASSPGLESSYEQYF 17 TRAV1 TRAJ33 CAVKDSNYXLIW TRBV13-3 TRBJ2-2 CASTRTENTGQLYF

Supplementary Table 2. List of flow cytometry antibodies used Antibody Manufacturer Clone Mouse TCRβ BD Pharmingen H57-597 CD4 Biolegend RM4-5 CD8α BD Pharmingen 53-6.7 CD8β ebioscience H35-17.2 CD16/32 produced in-house 2.4G2 CD24 BD Pharmingen M1/69 CD44 BD Pharmingen, ebioscience IM7 CD62L ebioscience MEL-14 CD69 ebioscience H1.2F3 CD13 Biolegend 2E7 CD127 (IL-7R) ebioscience A7R34 CD218 (IL-18Rα) Biolegend BG NK1.1 BD Pharmingen PK136 B22 BD Pharmingen RA3-6B2 IFN-γ BD Pharmingen XMG1.2 IL-17A BD Pharmingen TC11-18H1 PLZF ebioscience Mags.21F7 T-bet ebioscience ebio4b1 RORγt ebioscience B2D Human CD3ε BD Biosciences UCHT1 CD4 Biolegend OKT4 CD8α Biolegend SK1 CD8β ebioscience SIDI8BEE CD14 Biolegend M5E2 CD19 Biolegend HIB19 CD27 Biolegend O323 CD161 Biolegend HP-3G1 CD218 (IL-18Rα) Biolegend H44 TRAV1-2 Biolegend 3C1 IFN-γ BD Pharmingen B27 TNF BD Pharmingen MAb11

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