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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.

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Supplementary Figure S2. PTPN2-deficiency enhances CD8+ T cell LIP.    a) Naïve

CD8+ LN T cells from control (CD45.1+) mice and Lck-Cre;Ptpn2fl/fl (CD45.2+) mice

were stained with CFSE and co-transferred into sub-lethally irradiated

CD45.1+/CD45.2+ hosts. At 3, 5 and 7 days post transfer spleen T cells were analysed

by flow cytometry. b) Naive CD4+ LN T cells from Ptpn2fl/fl (CD45.2+) versus Lck-

Cre; Ptpn2fl/fl (CD45.2+) mice were stained with CFSE and transferred into sub-

lethally irradiated CD45.1+ 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.

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(Refer to figure legend on the next page)

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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 Ptpn2fl/fl (CD45.2+) versus Lck-Cre; Ptpn2fl/fl (CD45.2+) mice were stained with

CFSE and transferred into sub-lethally irradiated CD45.1+ 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

Ptpn2fl/fl and Lck-Cre; Ptpn2fl/fl mice were analysed by flow cytometry. Representative

CD44 versus CD49d contour plots are shown. ‘True Memory’ (TM) CD8+ T cells

were classified as CD44hiCD49dhiIL7RαloKLRG1hi or CD44hiCD49dhiCD62Llo and

‘Homeostatic Memory’ (HM) CD8+ T cells were classified as

CD44hiCD49dloCD62Lhi, CD44hiCD49dloCD122Lhi or CD44hiCD49dloLy6C+.

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<0.01.

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(Refer to figure legend on the next page)

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Supplementary Figure S4. Cytokine signalling in PTPN2-deficient T cells. a) Naïve

CD8+ and CD4+ LN T cells from Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl mice were stimulated

with 0.01ng/ml IL-7 for the indicated times. b) CD4+ central-memory

(CD8+CD62LhiCD44hi; CM) LN T cells from Ptpn2fl/fl and Lck-Cre;Ptpn2fl/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 Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl

mice were analysed by flow cytometry. Representative histogram overlays of un-

stained controls (background staining) and those stained for the indicated cytokine

receptor chains are shown. Results shown are representative of two independent

experiments.

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Supplementary Figure S5. STAT5 levels are not reduced in Lck-Cre;Ptpn2fl/+

heterozygous T cells. Cell lysates from naive CD4+ and CD8+ lymph node (LN) T

cells from four Ptpn2fl/+ and four Lck-Cre;Ptpn2fl/+ 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.

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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; CD45.1+) mice and Lck-Cre;Ptpn2fl/fl (CD45.2+) mice were pooled and

transferred into sub-lethally irradiated CD45.1+/CD45.2+ 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<0.001.

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Supplementary Figure S7. PTPN2 deficiency increases the proportion of T cells

undergoing fast-paced LIP. Naïve CD8+ LN T cells from control (CD45.1+) mice

and Lck-Cre;Ptpn2fl/fl (CD45.2+) mice were stained with CFSE and co-transferred into

sub-lethally irradiated CD45.1+/CD45.2+ 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.

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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 CD45.1+ 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 Ptpn2fl/fl (CD45.2+) versus Lck-Cre;Ptpn2fl/fl (CD45.2+) were stained with CFSE

and transferred into antibiotic treated, sub-lethally irradiated CD45.1+ 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<0.001.

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Supplementary Figure S9. Lck-Cre;Ptpn2fl/+ CD8+ T cells promote signs of

autoimmunity in irradiated mice. Naive CD8+ and CD4+ LN T cells isolated from

Ptpn2fl/+ (CD45.2+) and Lck-Cre;Ptpn2fl/+ (CD45.2+) mice were co-transferred into

sub-lethally irradiated CD45.1+ hosts. At 12 weeks post transfer blood lymphocytes

were analysed by flow cytometry. Relative numbers of total, naïve (CD62LhiCD44lo),

central memory (CD62LhiCD44hi; CM) and effector/memory (CD62LloCD44hi; 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 < 0.01.

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                                                            Supplementary Figure S10. Full scans of the immunoblots shown in the main figures