Supplementary Figure 1 Cytokine receptors on developing thymocytes that can potentially signal Runx3d expression.

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1 Supplementary Figure 1 Cytokine receptors on developing thymocytes that can potentially signal Runx3d expression.

2 (a) Characterization of c-independent SP8 cells. Stainings for maturation markers (top) and CD8 lineage markers (bottom) are shown for B6 (black) and c cko (red) SP8 (CD8SP TCR hi ) cells, compared to DP (CD4 + CD8 + ) cells (shaded curves). (b) List of cytokines, cytokine receptor chains, and signaling molecules examined in this study. (c) Identification of intermediate stage (CD4 + CD8 lo CD69 + ) thymocytes that have been signaled by MHC class I-dependent ligands to undergo positive selection and to differentiate into SP8 thymocytes. MHC class I-signaled thymocytes were obtained from MHCII KO CD1d KO mice because MHCI-signaled thymocytes in these mice do not differentiate into CD4 + NKT cells. (d) Surface expression of non- c cytokine receptor chains on intermediate stage thymocytes from MHCII KO CD1d KO mice. Cytokine receptor stainings are shown in red, control stainings are in gray. One of two experiments is shown. (e) Schematic of the DP stimulation assay. Pre-selection DP thymocytes (CD69 - CD4 + CD8 + ) were electronically sorted and stimulated with PMA+Ionomycin for 16h; washed and rested in medium for 10h; and then cultured with cytokines for 16-20h. Cells were then harvested and their gene expression analyzed by quantitative PCR. (f) List of cytokines that we tested in the B6 DP stimulation assay and their upregulation of mrnas encoding Runx3d, Runx1, Runx2, Cbfb and Bcl2 (, no gene up-regulation; +, minimal gene up-regulation; ++, intermediate gene up-regulation; +++, high gene up-regulation).

3 Supplementary Figure 2 Effect of cytokines on the generation of SP8 cells in fetal thymic organ culture. (a) Thymocyte profiles of E16.5 B6 thymic lobes before and after 5 days FTOC in medium (top and middle rows). Profile of TCR hi thymocytes on d5 of FTOC (bottom row) and numbers in boxes within

4 the profiles indicate frequency of cells within that box. (b) SP8 cell generation in c cko FTOCs by non- c cytokines. Bar graph of SP8 cell frequencies from c cko FTOCs cultured with the indicated cytokines, with SP8 cell frequencies in each cytokine group compared to medium alone (horizontal red dashed line). As an additional comparison, SP8 cell frequencies from B6 FTOCs are also shown. Data are from 4-17 individual lobes combined from 2-6 experiments. (c) IL-13 fails to induce SP8 cell generation in FTOCs. Comparison of SP8 cell frequencies (left) and numbers (right) in c cko FTOCs cultured for 5 days with or without IL-13. SP8 cells from B6 FTOC are shown for comparison (black bar). (d) Exogenous addition of any lineage-specifying cytokine increases SP8 cell generation in B6 FTOCs. Bar graph of SP8 cell frequencies (left) and numbers (right) in B6 FTOCs cultured for 5 days with the indicated cytokines compared to medium alone cultures. Mean and s.e.m. are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P <

5 Supplementary Figure 3 Lymph node SP8 cells in CytoQuad mice. Numbers of CD8 TCR + LN T cells in the indicated mice. Mean and s.e.m. are shown.

6 Supplementary Figure 4 c cytokines and non- c cytokines signal overlapping CD8 + T cell populations. Onion diagram for schematic representation of cytokine redundancy. We think that, in normal mice, all SP8 cell generation is signaled by the c cytokine IL-7. In the absence of IL-7 signaling, fewer SP8 cells are generated and these are signaled by the c cytokine IL-15. In the absence of c signaling, still fewer SP8 cells are generated and these are signaled by four non- c cytokines (IL-6, IFN-, TSLP, and TGF- ). It is only in the absence of in vivo signaling by the other three non- c cytokines (IL-6, IFN-, and TSLP) that the contribution of TGF- signaling to SP8 cell generation is appreciated. Most importantly, elimination of in vivo signaling by all six of these cytokines eliminates SP8 cell generation.

7 Supplementary Figure 5 Analysis of STAT- and SMAD-binding sites in the Runx3d locus. (a) Vista conservation analysis of murine and human Runx3d genomic regions. Peaks represent degree of conservation. Colored circles indicate potential binding sites for STATs (red) and SMADs (blue). (b) Comparison of surface CD103 expression on SP8 thymocytes from the indicated mice.

8 Supplementary Figure 6 Lineage-specifying transcription factors and the generation of SP8 cells. (a) The Runx3d YFP/YFP DP stimulation assay (schematized in supplementary Fig. 1e) was performed with pre-selection DP thymocytes from Runx3d-deficient (Runx3d YFP/YFP ) mice. After PMA+ionomycin stimulation, the Runx3d YFP/YFP DP thymocytes were stimulated with various cytokines and assessed for expression of Runx1 mrna. (b) Generation of MHC-II specific SP8 cells in ThPOK-deficient mice requires cytokine signaling. Thymocyte profiles from the indicated mice. Numbers in boxes in the profiles indicate CD8SP cell frequencies. Mean and s.e.m. are shown.

9 Supplementary Figure 7 Signaling events in the thymus that generate SP8 thymocytes: all generation of SP8 cells requires signaling by lineage-specifying cytokines. TCR signaling of DP thymocytes selectively terminates Cd8 gene expression, causing surface CD8

10 protein expression to decline. (a) In wildtype mice, MHC-I-specific TCR signaling eventually ceases because of the loss of surface CD8 protein expression, which allows cells to then be signaled by lineage-specifying cytokines. Signaling of positively selected thymocytes by lineage-specifying cytokines induces high Runx3d expression which both inhibits Runx1 and mediates co-receptor reversal (i.e. Cd4 gene silencing and Cd8 gene re-expression), resulting in differentiation into SP8 cells. (b) In the absence of intra-thymic signaling by lineage-specifying cytokines, Runx3d expression is not induced and Runx1 is unable to generate SP8 cells. (c) In Runx3d-deficient mice, intra-thymic signaling by lineage-specifying cytokines augments the ability of Runx1 to mediate coreceptor reversal and promote SP8 cell generation. (d) In ThPOK-deficient mice, CD4-lineage specification cannot occur. Because MHC-II-specific TCR signaling in the thymus must eventually cease, perhaps because of limiting ligand expression, cessation of TCR signaling allows the cells to then be signaled by lineage-specifying cytokines that induce high Runx3d expression which then inhibits Runx1 and mediates co-receptor reversal with the result that MHC-II-specific thymocytes differentiate into SP8 cells.