Supplementary Figure S1. PTPN2 levels are not altered in proliferating CD8+ T cells. Lymph node (LN) CD8+ T cells from C57BL/6 mice were stained with
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1 Supplementary Figure S1. PTPN2 levels are not altered in proliferating CD8+ T cells. Lymph node (LN) CD8+ T cells from C57BL/6 mice were stained with CFSE and stimulated with plate-bound α-cd3ε (10µg/ml) and α-cd28 (5µg/ml). CD8 + T cells were sorted as indicated on days 1-4 according to their CFSE profiles and cell lysates resolved by SDS-PAGE and immunoblotted with antibodies to PTPN2 and tubulin. Results are shown representative of two independent experiments. 1
2 Supplementary Figure S2. PTPN2-deficiency enhances CD8+ T cell LIP. a) Naïve CD8 + LN T cells from control (CD ) mice and Lck-Cre;Ptpn2 fl/fl (CD ) mice were stained with CFSE and co-transferred into sub-lethally irradiated CD /CD hosts. At 3, 5 and 7 days post transfer spleen T cells were analysed by flow cytometry. b) Naive CD4 + LN T cells from Ptpn2 fl/fl (CD ) versus Lck- Cre; Ptpn2 fl/fl (CD ) mice were stained with CFSE and transferred into sublethally irradiated CD hosts. At 7 days post transfer splenic T cells were analysed by flow cytometry. Representative CFSE profiles and quantified results are shown. Quantified results are means ± SEM for the indicated number of mice and are representative of at least two independent experiments. 2
3 (Refer to figure legend on the next page) 3
4 Supplementary Figure S3. PTPN2-deficiency promotes naive CD8+ T cell LIP and the generation of antigen-experienced memory T cells. a) Naive CD8 + LN T cells from Ptpn2 fl/fl (CD ) versus Lck-Cre; Ptpn2 fl/fl (CD ) mice were stained with CFSE and transferred into sub-lethally irradiated CD hosts. At 8 days post transfer splenic T cells were analysed by flow cytometry. Representative CFSE profiles and CD44 versus CD49d, KLRG-1 or CD62L plots (numbers in outlined areas are the relative numbers in the gate) are shown. b) Splenocytes from 6 week old Ptpn2 fl/fl and Lck-Cre; Ptpn2 fl/fl mice were analysed by flow cytometry. Representative CD44 versus CD49d contour plots are shown. True Memory (TM) CD8 + T cells were classified as CD44 hi CD49d hi IL7Rα lo KLRG1 hi or CD44 hi CD49d hi CD62L lo and Homeostatic Memory (HM) CD8 + T cells were classified as CD44 hi CD49d lo CD62L hi, CD44 hi CD49d lo CD122L hi or CD44 hi CD49d lo Ly6C +. Quantified results in a-b are means ± SEM for the indicated number of mice and are representative of three independent experiments. Significance was determined using 2-tailed Mann-Whitney U test; *P<0.05, **P<
5 (Refer to figure legend on the next page) 5
6 Supplementary Figure S4. Cytokine signalling in PTPN2-deficient T cells. a) Naïve CD8 + and CD4 + LN T cells from Ptpn2 fl/fl and Lck-Cre;Ptpn2 fl/fl mice were stimulated with 0.01ng/ml IL-7 for the indicated times. b) CD4 + central-memory (CD8 + CD62L hi CD44 hi ; CM) LN T cells from Ptpn2 fl/fl and Lck-Cre;Ptpn2 fl/fl mice were stimulated with IL-7 or IL-15 for 15 min. In a-b cells were stained for pstat5 and MFI determined by flow cytometry. Quantified results are means ± SEM for the indicated number of mice and are representative of at least two independent experiments. Significance was determined using 2-tailed Mann-Whitney U test; *P<0.05. c) Naïve CD8 + and CD4 + LN T cells from Ptpn2 fl/fl and Lck-Cre;Ptpn2 fl/fl mice were analysed by flow cytometry. Representative histogram overlays of unstained controls (background staining) and those stained for the indicated cytokine receptor chains are shown. Results shown are representative of two independent experiments. 6
7 Supplementary Figure S5. STAT5 levels are not reduced in Lck-Cre;Ptpn2 fl/+ heterozygous T cells. Cell lysates from naive CD4 + and CD8 + lymph node (LN) T cells from four Ptpn2 fl/+ and four Lck-Cre;Ptpn2 fl/+ mice were resolved by SDS- PAGE and immunoblotted with antibodies to STAT5, PTPN2 and tubulin. STAT5 levels were quantified by densitometric analysis and normalised for tubulin; each lane corresponds to T cells from a different mouse. Quantified results are means ± SEM for the indicated number of mice and are representative of at least two independent experiments; arbitrary units (a.u.) are shown. 7
8 Supplementary Figure S6. PTPN2 deficiency skews the TCR repertoire in CD8+ T cells that have undergone LIP. Naive CD8 + LN T cells isolated from wild type (WT; CD ) mice and Lck-Cre;Ptpn2 fl/fl (CD ) mice were pooled and transferred into sub-lethally irradiated CD /CD hosts. At 8 days post transfer, splenic and LN CD8+ T cells were stained for the indicated TCRVβ-chains and NK1.1 and analysed by flow cytometry. a) Representative FACS profiles of CD8+ T cells stained for NK1.1 and TCRVβ8.1/8.2 are shown. b) TCRVβ-chain frequencies in spleen and lymph nodes after transfer are shown. In b quantified results are means ± SEM for the indicated number of mice and are representative of at least three independent experiments. Significance was determined using 2-tailed Mann- Whitney U test; *P<0.05, **P<0.01, *** p<
9 Supplementary Figure S7. PTPN2 deficiency increases the proportion of T cells undergoing fast-paced LIP. Naïve CD8 + LN T cells from control (CD ) mice and Lck-Cre;Ptpn2 fl/fl (CD ) mice were stained with CFSE and co-transferred into sub-lethally irradiated CD /CD hosts. At 8 days post-transfer spleen and liver T cells were analysed by flow cytometry. Quantified results are means ± SEM for the indicated number of mice and are representative of two independent experiments. Both fast and slow proliferating and total relative T cell numbers were determined. 9
10 Supplementary Figure S8. PTPN2 deficiency exacerbates CD8 + T cell responses to self- and gut-antigens in irradiated mice. a) Genomic DNA from faeces of antibiotic treated CD mice was extracted using the DNA stool isolation kit. Bacterial DNA was amplified by PCR using a specific primer pair for bacterial 16sDNA resolved on an agarose gel and visualised by ethidium bromide staining. Primers for epithelial gut DNA were used as positive loading control. b) Naive CD8 + lymph node (LN) T cells from Ptpn2 fl/fl (CD ) versus Lck-Cre;Ptpn2 fl/fl (CD ) were stained with CFSE and transferred into antibiotic treated, sub-lethally irradiated CD hosts. At 8 days post transfer splenic and liver T cells were analysed by flow cytometry. Quantified results are means ± SEM for the indicated number of mice and are representative of at least three independent experiments; significance was determined using one-way ANOVA with bonferroni correction; *P<0.05, **P<0.01, *** p<
11 Supplementary Figure S9. Lck-Cre;Ptpn2 fl/+ CD8+ T cells promote signs of autoimmunity in irradiated mice. Naive CD8 + and CD4 + LN T cells isolated from Ptpn2 fl/+ (CD ) and Lck-Cre;Ptpn2 fl/+ (CD ) mice were co-transferred into sub-lethally irradiated CD hosts. At 12 weeks post transfer blood lymphocytes were analysed by flow cytometry. Relative numbers of total, naïve (CD62L hi CD44 lo ), central memory (CD62L hi CD44 hi ; CM) and effector/memory (CD62L lo CD44 hi ; EM) CD4 + T cells were determined. Representative CD44 versus CD62L plots before and after adoptive transfer are shown. At 20 weeks post transfer splenic, liver and lung lymphocytes were analysed by flow cytometry. Absolute numbers of total, naïve, CM and EM CD4 + T cells were determined. Results shown are means ± SEM for the indicated number of mice; significance was determined using a two-tailed Mann- Whitney U Test; *p < 0.05 ** p <
12 Supplementary Figure S10. Full scans of the immunoblots shown in the main figures 12
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