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1 Supplementary Materials for Regulation of Zap70 Expression During Thymocyte Development Enables Temporal Separation of CD4 and CD8 Repertoire Selection at Different Signaling Thresholds Manoj Saini, Charles Sinclair, Daniel Marshall, Mauro Tolaini, Shimon Sakaguchi, Benedict Seddon* *To whom correspondence should be addressed. Published 23 March 2010, Sci. Signal. 3, ra23 (2010) DOI: /scisignal The PDF file includes: Fig. S1. Rapid induction of Zap70 expression after the administration of dox to TetZap70 mice. Fig. S2. Abundance of co-receptors on DP thymocytes after induced production of Zap70 protein. Fig. S3. Rapid induction of ThPOK production in CD4 + SP cells from dox-fed TetZap70 mice. Fig. S4. Cell populations of peripheral lymph nodes in dox-fed TetZap70 mice. Fig. S5. Intrathymic development of Foxp3 + T regs from TetZap70 thymocytes. Fig. S6. NK1.1 + NK T cells fail to develop in TetZap70 mice fed a dox-containing diet. Fig. S7. TCR and CD5 define developmentally distinct populations of DP thymocytes. Fig. S8. The abundance of Zap70, Lck, SLP-76, LAT, and ERK in various thymocyte subsets from wild-type and TetZap70 mice. Fig. S9. INDO dye loading, Ca 2+ flux responses to ionomycin, and generation of perk in response to phorbol ester by wild-type and TetZap70 thymocytes. Fig. S10. The increased abundance of Zap70 protein in thymocytes from SKG mice. References

2 Fig. S1. Rapid induction of Zap70 expression after the administration of dox to TetZap70 mice. TetZap70 mice were injected i.p. with 1 mg dox in PBS. At 8, 10, 12, and 16 hours post-injection, groups of mice were taken and their thymi were characterized by flow cytometry. Histograms show Zap70 (column 1) and hucd2 (column 2) for TetZAP70 thymocytes (solid lines) and Zap70 -/- thymocytes (gray fill). Density plots show hucd2 against Zap70 in total thymocytes (column 3), TCR against CD5 for hucd2 - DP thymocytes (column 4), and TCR against CD5 for hucd2 + DP thymocytes (column 6). Histograms (column 6) show CD5 on hucd2 + DP thymocytes from TetZap70 mice (solid line), DP thymocytes from Zap70 -/- mice (gray fill), and DP thymocytes from WT mice (dashed line). Data are representative of two independent experiments.

3 Fig. S2. Abundance of co-receptors on DP thymocytes after induced production of Zap70 protein. In the same experiment as described in Fig. 2, the abundance of the coreceptors CD4 and CD8 on developing DP2 and DP3 subsets was examined. Density plots are of CD4 against CD8 on total live hucd2 + thymocytes (row 1) and CD5 against TCR on hucd2 + DP cells (row 2). Columns for d0, WT and I-A b-/- samples have no hucd2 gate applied. The gates used to define DP cells and the DP1 to DP3 subsets are shown together with their relative frequencies. Contour plot overlays show CD4 against CD8 on DP1 (gray) compared with DP2 (red) cells (row 3) and on DP1 (gray) compared with DP3 (blue) cells (row 4) for the corresponding time points. Data are representative of four independent experiments.

4 Fig. S3. Rapid induction of ThPOK production in CD4 + SP cells from dox-fed TetZap70 mice. Bar chart shows the relative abundance of ThPOK mrna, as determined by real-time RT-PCR, in total Rag1 -/- thymus, purified DP1 thymocytes from WT mice, CD4 + SP cells purified from the thymi of TetZap70 mice fed dox-containing food for 2 or 3 days, CD4 + CD8 lo thymocytes from WT or MHC class II deficient mice, and CD4 + SP and CD8 + SP cells purified from the thymi of WT mice. Purified populations were isolated by cell sorting. Data are representative of two independent experiments.

5 Fig. S4. Cell populations of peripheral lymph nodes in dox-fed TetZap70 mice. Adult TetZap70 mice were fed dox-containing food, and groups of mice (n = 3) taken at different times after feeding had their lymph nodes analyzed for the presence of CD4, CD8, and TCR proteins by flow cytometry. Graphs on the left show the mean percentage of CD4 + TCR hi (A) and CD8 + TCR hi (B) cells among lymph node cells (LNC) after the indicated days of feeding with dox-containing food. Plots on the right in each panel show the percentages of CD4 + TCR hi or CD8 + TCR hi cells in the lymph nodes of TetZap70 mice (Tet) continuously fed dox-containing food and from C57Bl6/J (WT) mice as a control. Data are pooled from two independent experiments.

6 Fig. S5. Intrathymic development of Foxp3 + T regs from TetZap70 thymocytes. Bone marrow chimeras (n = 20 mice) were generated by reconstituting irradiated Rag1 -/- mice with bone marrow from TetZap70 mice. Six weeks after reconstitution, mice were fed dox-containing food and groups of mice (n = 3 to 4 mice) were taken at different times and their thymocytes were analysed for the presence of CD4, CD8, hucd2, TCR, CD25, and FoxP3. (A) Dot plots show CD4 against CD8α (top row) on total cells from WT mice or on thymocytes from TetZap70 mice and CD25 against FoxP3 (bottom row) on TCR hi CD4 + SP cells at the indicated days after dox feeding. Numbers indicate the percentage of cells in the corresponding gate. (B) Graphs show frequency of hucd2 + TetZap70 thymocytes that were CD4 + SP (filled circles), CD8 + SP (empty diamonds), or TCR hi FoxP3 + CD4 + SP cells (filled squares) against the time on dox-containing food and compared with the frequencies of the corresponding populations in WT thymus. Data are representative of three independent experiments.

7 Fig. S6. NK1.1 + NK T cells fail to develop in TetZap70 mice fed a dox-containing diet. Thymocytes from WT mice and from TetZap70 mice continuously fed with dox-containing food were analyzed by flow cytometry for the presence of TCR, CD4, CD8, CD5, Zap70, and NK1.1. (A) Dot plots show TCR against NK1.1 on total thymocytes and CD5 against NK1.1 on CD4 + SP cells. Numbers indicate the frequency of cells in the corresponding gate. (B) Histograms show Zap70 in DP1 thymocytes (black line), CD4 + SP cells (red line), and NK1.1 + TCR int thymocytes (broken blue line) from WT mice. Gray fill shows background staining of DP thymocytes from Zap70 -/- mice. Data are representative of three independent experiments.

8 Fig. S7. TCR and CD5 define developmentally distinct populations of DP thymocytes. To investigate lineage relationships between DP and SP subsets of thymocytes, we examined DP populations in MHC class I and MHC class II restricted TCR transgenic mice, which are capable of producing only CD8 + or CD4 + lineage cells, respectively. DP thymocytes from class II restricted OTII and DO11.10 TCR transgenic strains had enlarged DP2 populations, as defined by intermediate amounts of TCR, but no TCR hi DP3 subset (A), which suggested that DP2 thymocytes directly gave rise to CD4 + SP cells. In contrast, DPs from class I restricted F5 and OTI TCR transgenic mice, which develop only CD8 + T cells, had pronounced DP2 and DP3 populations (A), consistent with serial transition through both DP stages. We also examined WT DP populations for the expression of genes associated with CD4 + or CD8 + lineages by real-time RT-PCR. Gata3 has been implicated in the development of CD4 + T cells (1), and was specifically increased in expression in the DP2 population. However, ThPOK transcripts were not detectable at this stage, consistent with other studies (2, 3). Runx3 and cathepsin W (CtsW) (4) are associated with the development of the CD8 + lineage and were specifically increased in expression in DP3, but not DP2 or DP1, subsets (B). (A) Upper row shows plots of CD4 against CD8 for live gated thymocytes from WT, class II restricted TCR transgenic OTII and D mice, and class I restricted TCR transgenic F5 and OTI mice. Lower rows show CD5 against TCR on DP, CD4 + SP, and CD8 + SP thymocytes from the corresponding mice with gates to indicate the abundance of TCR and CD5 on DP2 and DP3 populations relative to that of SP cells. (B) DP1 to DP3 subsets, CD4 + and CD8 + SP thymocytes were sorted from WT mice and relative amounts of mrna for the indicated genes indicated were determined by real-time RT-PCR analysis. *, P < (n = 3 mice),, P < 0.02 (n = 3 mice). Data are representative of four or more independent experiments.

9 Fig. S8. The abundance of Zap70, Lck, SLP-76, LAT, and ERK in various thymocyte subsets from wild-type and TetZap70 mice. (A) Total cell lysates were prepared from sorted populations of DP1, DP2, DP3, CD4 + SP, and CD8 + SP cells from the thymi of WT mice. Lysates (2 x 10 5 T cells/lane) were resolved by SDS-PAGE and analyzed by Western blotting for the abundance of Zap70 protein. Blots were also analyzed for GAPDH as a loading control. Numbers indicate the ratios of the intensities of the Zap70 bands to those of GAPDH as determined by densitometric analysis with NIH Image software. Data are representative of two independent experiments. (B) Histograms show flow cytometric analysis of the abundance of Lck, LAT, SLP-76, and ERK2 in DP1, DP2, and DP3 thymocytes from WT (black line) and from TetZap70 (red line) mice fed dox (3 mg per g of body weight) for 6 days. Gray fills indicating negative control stains are from splenic B cells incubated with antibodies against Lck, LAT, and SLP-76 or from incubation of thymocytes with an isotypematched control antibody in the case of ERK2. Data are representative of two or more independent experiments.

10 Fig. S9. INDO dye loading, Ca 2+ flux responses to ionomycin, and generation of perk in response to phorbol ester by wild-type and TetZap70 thymocytes. Thymocytes were surface-labelled with mab against CD4, CD8, TCR, CD5, and hucd2 and were labelled with Indo dye to analyze Ca 2+ flux as described in the legend for Fig. 7. (A) Histograms show the log of fluorescence of the Indo label in DP1, DP2, and DP3 thymocytes from WT (black line) and TetZap70 mice (red broken line) prior to stimulation. Graphs show the Ca 2+ flux responses of DP1, DP2, and DP3 thymocytes from WT (black line) or TetZap70 (red broken line) mice following stimulation with ionomycin. (B) Histograms show the abundance of perk in DP1, DP2, and DP3 thymocyte subsets from WT (black line) and TetZap70 mice (red broken line) in response to 2-min stimulation with PDBu (1 μg/ml) and as compared with WT thymic subsets pretreated with UO126 for 30 min prior to stimulation with PDBu. Data are representative of two independent experiments.

11 Fig. S10. The increased abundance of Zap70 protein in thymocytes from SKG mice. Thymocytes from BALB/c and SKG mice were analyzed by flow cytometry for the presence of CD4, CD8, TCR, and CD5 on the cell surface and for intracellular Zap70. Density plots show CD4 against CD8 on total live cells and CD5 against TCR on DP thymocytes for the indicated mice. The upper histograms show the abundance of Zap70 in DP1 (thin broken line), DP2 (red line), DP3 (blue line), and CD4 + SP (thick broken line) thymocytes as compared with incubation of total thymocytes with the isotype control antibody (gray fill). Lower histograms show isotype control background staining in DP1 (grey fill), DP2 (red line), DP3 (blue line), and CD4 + SP (thick broken line) thymocytes. Data are representative of three independent experiments.

12 References 1. S. Y. Pai, M. L. Truitt, C. N. Ting, J. M. Leiden, L. H. Glimcher, I. C. Ho, Critical roles for transcription factor GATA-3 in thymocyte development. Immunity 19, (2003). 2. T. Egawa, D. R. Littman, ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat. Immunol. 9, (2008). 3. S. Muroi, Y. Naoe, C. Miyamoto, K. Akiyama, T. Ikawa, K. Masuda, H. Kawamoto, I. Taniuchi, Cascading suppression of transcriptional silencers by ThPOK seals helper T cell fate. Nat. Immunol. 9, (2008). 4. A. Bhandoola, B. Kithiganahalli, L. Granger, A. Singer, Programming for cytotoxic effector function occurs concomitantly with CD4 extinction during CD8(+) T cell differentiation in the thymus. Internat. Immunol. 12, (2000).

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