B6.SJL (Ly5.2) mice were obtained from Taconic Farms. Rag1-deficient mice were

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1 Supplementary Methods Mice. B6.SJL (Ly5.2) mice were obtained from Taconic Farms. Rag1-deficient mice were purchased from The Jackson Laboratory. Real-time PCR Total cellular RNA was extracted from the indicated sorted cells supplemented with 10 μg glycogen using Trizol reagent (Invitrogen). RNA samples were treated with RNasefree DNase I (Invitrogen) before reverse-transcription to eliminate contaminating genomic DNA. Total RNA was reverse transcribed using Superscript II reversetranscriptase and oligo(dt) primer (Invitrogen). Real-time PCR was performed on a 7300 Real Time PCR system (Applied Biosystems). Analyses were performed using primers (Invitrogen), an internal fluorescent Taqman probe (Biosearch Technologies) specific to ThPOK and HPRT, and Universal PCR Master Mix (Applied Biosystems). The primer and Taqman probe sequences were as follows. ThPOK primers: 5 - AGAAGCCCTTTGCCTGTGA-3 and 5 -TGTGGATCTTCAGCTTGT CATTC-3 ; ThPOK probe: 5 -FAM-TCTGCGGCGTCCGCTTCAC-3 ; HPRT primers: 5 - TGAAGAGCTACTGTAATGATCAGTCAAC-3 and 5 -AGCAAGCTTGCAACC TTAACCA-3 ; HPRT probe: 5 -FAM-TGCTTTCCCTGGTTAAGCAGTACAGCC C- 3. Generation of mixed bone marrow chimeric mice Bone marrow cells from femur and tibia were incubated with anti-cd3-biotin, anti- 1

2 CD4-biotin, and anti-cd8-biotin antibodies (ebioscience) followed by anti-biotin MACS beads (Milteneyi). After T cell depletion, 3 x 10 6 bone marrow cells from each of the indicated donors were injected intravenously into sublethally irradiated Rag1 deficient hosts (600 rad). Mice were infected 8-12 weeks after reconstitution. 2

3 Supplementary Reference S1. Beere, H. M., and D. R. Green. Stress management- heat shock protein-70 and the regulation of apoptosis Trends Cell Biol 11: 6-10 S2. Panaretou, B., Siligardi, G., Meyer, P., Maloney, A., Sullivan, J. K., Singh, S., Millson, S. H., Clarke, P. A., Naaby-Hansen, S., Stein, R., Cramer, R., Mollapour, M., Workman, P., Piper, P. W., Pearl, L. H., Prodromou, C Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1. Mol Cell 10: S3. Ohtsu, M., Sakai, N., Fujita, H., Kashiwagi, M., Gasa, S., Shimizu, S., Eguchi, Y., Tsujimoto, Y., Sakiyama, Y., Kobayashi, K., Kuzumaki, N Inhibition of apoptosis by the actin-regulatory protein gelsolin. Embo J 16: S4. de Souza-Pinto, N. C., Maynard, S., Hashiguchi, K., Hu, J., Muftuoglu, M., Bohr, V The Recombination Protein Rad52 Cooperates with the Excision Repair Protein Ogg1 for the Repair of Oxidative Lesions in Mammalian Cells. Mol Cell Biol. S5. Potts, P. R., Porteus, M. H., Yu, H Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks. Embo J 25:

4 CD4+ day46 +LCMV day 20 day 8 CD44 low CD8+ N.D Normalized ThPOK Supplementary Figure 1. ThPOK mrna levels increase in polyclonal Ag-specific CD8 T cells after LCMV infection. B6 mice were infected with LCMV. D b /GP33 tetramer-positive CD8 T cells in infected mice at the indicated time-points, and CD44 low CD8 T cells or CD4 T cells in naïve mice were purified using FACS sorting. ThPOK mrna expression levels were assessed by real-time quantitative PCR (normalized to HPRT). The ThPOK mrna level in CD4 T cells was defined as 100. N.D., none detected.

5 ThPOK/GFP Supplementary Figure 2. Downregulation of ThPOK expression levels in activated CD4 + T cells. hpok expression levels in naïve CD4 T cells (black line) and activated CD44 hi CD4 T cells (blue ine) in ThPOK wt/gfp mice infected with LCMV 8 days post-infection were analyzed by flow cytometry. Numbers in the histograms are mean fluorescent intensity of gfp in naïve CD4 + T cells (black) and ctivated CD44 hi CD4 + T cells (blue). Results are representative of three independent experiments.

6 TCRβ+ CD8+CD4-T Vα2 CD4 92 CD8 CD8+CD4-CD44 low T CD8 CD44 CD8+CD4-CD44 hi T ThPOK/GFP Supplementary Figure 3. ThPOK expression in naïve T cells of ThPOK gfp/gfp mice. ThPOK expression levels in CD44 hi CD4 - and CD44 low CD4 - T cells of naïve ThPOK gfp/gfp mice were analyzed by flow cytometry. Numbers in the dot plots are the percentages. Results are representative of three independent experiments.

7 A CD28 CD27 KLRG1 CD127 CD62L CCR7 B CD28 CD27 KLRG1 CD127 CD62L CCR7 C CD28 CD27 KLRG1 CD127 CD62L CCR7 Supplementary Figure 4. Surface phenotypes of control and ThPOK-mutant naïve, effector, and memory T cells. A, The expression levels of representative surface molecules on naïve CD44 low ThPOK hd/hd and ThPOK wt/wt T cells in the spleen. B, The expression pattern of epresentative surface markers on effector ThPOK hd/hd and ThPOK wt/wt T cells in the spleen days after LCMV infection. C, The surface phenotype of memory ThPOK hd/hd and ThPOK wt/wt 14 T cells in the spleen. The expression levels were analyzed more than 90 days post-infection. esults are representative of two independent experiments.

8 A number per spleen (x 10-3 ) 10 1 p= ThPOK wt/wt ThPOK hd/hd B 100 p= p=0.004 % thy p= days post infection Supplementary Figure 5. The kinetics of expansion of ThPOK hd/hd and ThPOK wt/wt T cells fter primary infection. A, Four days after LCMV infection, the percentages of CD8 T cells were nalyzed in total spleen cells. The percentages of T cells (Thy1.1) among CD8 T cells in the pleen were determined after enrichment of CD8 T cells. The absolute numbers of T cells are hown. B, The percentages of ThPOK hd/hd (open circles) and ThPOK wt/wt (closed circles) T cells in blood are shown on 5, 6, and 7 days post-infection.

9 A 60 p=0.02 % reduction B6 vs B6 ThPOK wt/hd vs B6 B C number per spleen (x 10-6 ) ThPOK wt/wt p= ThPOK wt/hd number per spleen (x 10-6 ) ThPOK wt/wt p= ThPOK wt/gfp Supplementary Figure 6. Polyclonal ThPOK wt/hd Ag-specific CD8 T cells, ThPOK wt/hd T cells, nd ThPOK wt/gfp T cells show a mild impairment in accumulation. A, Sublethally irradiated Rag1 -deficient mice were reconstituted with a 1:1 mixture of T cell-depleted bone marrow cells from either Ly5.1 B6 and Ly5.2 B6 mice, or from Ly5.1 B6 and Ly5.2 ThPOK wt/hd mice. Ten to twelve weeks later, he distribution of CD8 T cells in peripheral blood of the chimeras was checked using congenic markers. he distribution among GP33-specific CD8 T cells in peripheral blood was analyzed 8 days after LCMV nfection. The percentages of reduction signify a relative reduction of Ly5.2 + BM-derived cells among P33-specific CD8 T cells 8 days after infection. B, The absolute numbers of transferred ThPOK wt/hd T cells and ThPOK wt/wt T cells in the spleen 8 days after LCMV infection. C, The absolute numbers of transferred ThPOK wt/gfp T cells and ThPOK wt/w t T cells in the spleen 8 days ost-infection.

10 gene name fold change known function reference Heat shock protein 1B DNA repair/anti apoptotic Heat shock protein 1A DNA repair/anti apoptotic Integrin alpha cell adhesion Integrin alpha cell adhesion AHA response to stimulus CD160 antigen receptor activity/negative signaling Alcam cell adhesion gelsolin barbed-end actin filament capping/anti apoptotic Rad DNA repair S4 Smc DNA repair S5 S1 S1 S2 ref. 21 S3 Supplementary Table 1. Gene expression in ThPOK hd/hd T cells versus ThPOK wt/wt T cells during the memory phase. Genes encoding products involved in survival or immune responses are listed among genes whose expression levels differ more than 1.5 fold between the two groups.