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
Supplementary Figure 1. Characterization of basophils after reconstitution of SCID mice
Supplementary figure legends Supplementary Figure 1. Characterization of after reconstitution of SCID mice with CD4 + CD62L + T cells. (A-C) SCID mice (n = 6 / group) were reconstituted with 2 x 1 6 CD4
T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice
SUPPLEMENTAL METHODS T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice Florian Wiede 1, Benjamin J. Shields 1, Sock Hui Chew 1, Konstantinos Kyparissoudis 2,
% of live splenocytes. STAT5 deletion. (open shapes) % ROSA + % floxed
Supp. Figure 1. a 14 1 1 8 6 spleen cells (x1 6 ) 16 % of live splenocytes 5 4 3 1 % of live splenocytes 8 6 4 b 1 1 c % of CD11c + splenocytes (closed shapes) 8 6 4 8 6 4 % ROSA + (open shapes) % floxed
and follicular helper T cells is Egr2-dependent. (a) Diagrammatic representation of the
Supplementary Figure 1. LAG3 + Treg-mediated regulation of germinal center B cells and follicular helper T cells is Egr2-dependent. (a) Diagrammatic representation of the experimental protocol for the
Supplementary Figure Legends. group) and analyzed for Siglec-G expression utilizing a monoclonal antibody to Siglec-G (clone SH2.1).
Supplementary Figure Legends Supplemental Figure : Naïve T cells express Siglec-G. Splenocytes were isolated from WT B or Siglec-G -/- animals that have not been transplanted (n= per group) and analyzed
MATERIALS AND METHODS. Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All
MATERIALS AND METHODS Antibodies (Abs), flow cytometry analysis and cell lines Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All other antibodies used
Supplementary. presence of the. (c) mrna expression. Error. in naive or
Figure 1. (a) Naive CD4 + T cells were activated in the presence of the indicated cytokines for 3 days. Enpp2 mrna expression was measured by qrt-pcrhr, infected with (b, c) Naive CD4 + T cells were activated
Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured
Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured under Th0, Th1, Th2, Th17, and Treg conditions. mrna
The autoimmune disease-associated PTPN22 variant promotes calpain-mediated Lyp/Pep
SUPPLEMENTARY INFORMATION The autoimmune disease-associated PTPN22 variant promotes calpain-mediated Lyp/Pep degradation associated with lymphocyte and dendritic cell hyperresponsiveness Jinyi Zhang, Naima
CD44
MR1-5-OP-RU CD24 CD24 CD44 MAIT cells 2.78 11.2 WT RORγt- GFP reporter 1 5 1 4 1 3 2.28 1 5 1 4 1 3 4.8 1.6 8.1 1 5 1 4 1 3 1 5 1 4 1 3 3.7 3.21 8.5 61.7 1 2 1 3 1 4 1 5 TCRβ 2 1 1 3 1 4 1 5 CD44 1 2 GFP
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic
Supplementary Figure 1: Expression of NFAT proteins in Nfat2-deleted B cells (a+b) Protein expression of NFAT2 (a) and NFAT1 (b) in isolated splenic B cells from WT Nfat2 +/+, TCL1 Nfat2 +/+ and TCL1 Nfat2
Supplementary Figure 1. Efficiency of Mll4 deletion and its effect on T cell populations in the periphery. Nature Immunology: doi: /ni.
Supplementary Figure 1 Efficiency of Mll4 deletion and its effect on T cell populations in the periphery. Expression of Mll4 floxed alleles (16-19) in naive CD4 + T cells isolated from lymph nodes and
Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice
Research article Related Commentary, page 4618 T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice Florian Wiede, 1 Benjamin J. Shields, 1 Sock Hui Chew, 1 Konstantinos
Nature Immunology: doi: /ni Supplementary Figure 1. Cellularity of leukocytes and their progenitors in naive wild-type and Spp1 / mice.
Supplementary Figure 1 Cellularity of leukocytes and their progenitors in naive wild-type and Spp1 / mice. (a, b) Gating strategies for differentiated cells including PMN (CD11b + Ly6G hi and CD11b + Ly6G
Nature Immunology: doi: /ni Supplementary Figure 1. Id2 and Id3 define polyclonal T H 1 and T FH cell subsets.
Supplementary Figure 1 Id2 and Id3 define polyclonal T H 1 and T FH cell subsets. Id2 YFP/+ (a) or Id3 GFP/+ (b) mice were analyzed 7 days after LCMV infection. T H 1 (SLAM + CXCR5 or CXCR5 PD-1 ), T FH
SUPPLEMENT Supplementary Figure 1: (A) (B)
SUPPLEMENT Supplementary Figure 1: CD4 + naïve effector T cells (CD4 effector) were labeled with CFSE, stimulated with α-cd2/cd3/cd28 coated beads (at 2 beads/cell) and cultured alone or cocultured with
Supplemental Materials for. Effects of sphingosine-1-phosphate receptor 1 phosphorylation in response to. FTY720 during neuroinflammation
Supplemental Materials for Effects of sphingosine-1-phosphate receptor 1 phosphorylation in response to FTY7 during neuroinflammation This file includes: Supplemental Table 1. EAE clinical parameters of
Supporting Information Table of Contents
Supporting Information Table of Contents Supporting Information Figure 1 Page 2 Supporting Information Figure 2 Page 4 Supporting Information Figure 3 Page 5 Supporting Information Figure 4 Page 6 Supporting
SUPPLEMENTARY INFORMATION
doi:1.138/nature1554 a TNF-α + in CD4 + cells [%] 1 GF SPF 6 b IL-1 + in CD4 + cells [%] 5 4 3 2 1 Supplementary Figure 1. Effect of microbiota on cytokine profiles of T cells in GALT. Frequencies of TNF-α
Supplementary Information
Supplementary Information Supplementary Figure 1! a! b! Nfatc1!! Nfatc1"! P1! P2! pa1! pa2! ex1! ex2! exons 3-9! ex1! ex11!!" #" Nfatc1A!!" Nfatc1B! #"!" Nfatc1C! #" DN1! DN2! DN1!!A! #A!!B! #B!!C! #C!!A!
Supplementary Figures
Supplementary Figures Supplementary Fig. 1. Surface thiol groups and reduction of activated T cells. (a) Activated CD8 + T-cells have high expression levels of free thiol groups on cell surface proteins.
Blocking antibodies and peptides. Rat anti-mouse PD-1 (29F.1A12, rat IgG2a, k), PD-
Supplementary Methods Blocking antibodies and peptides. Rat anti-mouse PD-1 (29F.1A12, rat IgG2a, k), PD- L1 (10F.9G2, rat IgG2b, k), and PD-L2 (3.2, mouse IgG1) have been described (24). Anti-CTLA-4 (clone
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Nature Immunology: doi: /ni Supplementary Figure 1. Gene expression profile of CD4 + T cells and CTL responses in Bcl6-deficient mice.
Supplementary Figure 1 Gene expression profile of CD4 + T cells and CTL responses in Bcl6-deficient mice. (a) Gene expression profile in the resting CD4 + T cells were analyzed by an Affymetrix microarray
SUPPLEMENTARY INFORMATION. Supp. Fig. 1. Autoimmunity. Tolerance APC APC. T cell. T cell. doi: /nature06253 ICOS ICOS TCR CD28 TCR CD28
Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1 Supp. Fig. 2 CD4 + CD44 low CD4
NK cell flow cytometric assay In vivo DC viability and migration assay
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Supplementary Figure 1
Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition
Supplementary Figure 1. ETBF activate Stat3 in B6 and Min mice colons
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Supplementary Figure 1. Example of gating strategy
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Supplemental Materials
Supplemental Materials Programmed death one homolog maintains the pool size of regulatory T cells by promoting their differentiation and stability Qi Wang 1, Jianwei He 1, Dallas B. Flies 2, Liqun Luo
Cell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice
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Spleen. mlns. E Spleen 4.1. mlns. Spleen. mlns. Mock 17. Mock CD8 HIV-1 CD38 HLA-DR. Ki67. Spleen. Spleen. mlns. Cheng et al. Fig.
C D E F Mock 17 Mock 4.1 CD38 57 CD8 23.7 HLA-DR Ki67 G H I Cheng et al. Fig.S1 Supplementary Figure 1. persistent infection leads to human T cell depletion and hyper-immune activation. Humanized mice
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Supplementary Figure 1. mrna expression of chitinase and chitinase-like protein in splenic immune cells. Each splenic immune cell population was
Supplementary Figure 1. mrna expression of chitinase and chitinase-like protein in splenic immune cells. Each splenic immune cell population was sorted by FACS. Surface markers for sorting were CD11c +
Supplementary Information:
Supplementary Information: Follicular regulatory T cells with Bcl6 expression suppress germinal center reactions by Yeonseok Chung, Shinya Tanaka, Fuliang Chu, Roza Nurieva, Gustavo J. Martinez, Seema
Supplemental Table I.
Supplemental Table I Male / Mean ± SEM n Mean ± SEM n Body weight, g 29.2±0.4 17 29.7±0.5 17 Total cholesterol, mg/dl 534.0±30.8 17 561.6±26.1 17 HDL-cholesterol, mg/dl 9.6±0.8 17 10.1±0.7 17 Triglycerides,
Nature Medicine doi: /nm.3957
Supplementary Fig. 1. p38 alternative activation, IL-21 expression, and T helper cell transcription factors in PDAC tissue. (a) Tissue microarrays of pancreatic tissue from 192 patients with pancreatic
pro-b large pre-b small pre-b CCCP (µm) Rag1 -/- ;33.C9HCki
a TMRM FI (Median) b TMRM FI (Median) c 20 15 10 5 0 8 6 4 2 0 pro-b large pre-b small pre-b 0 10 20 30 40 50 60 70 80 90 100 TMRM (nm) pro-b large pre-b small pre-b 0 1 2 4 8 16 32 64 128 256 CCCP (mm)
Supplemental Figure 1. Activated splenocytes upregulate Serpina3g and Serpina3f expression.
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Supplementary Fig. 1 No relative growth advantage of Foxp3 negative cells.
Supplementary Fig. 1 Supplementary Figure S1: No relative growth advantage of Foxp3 negative cells. itreg were induced from WT (A) or FIR (B) CD4 + T cells. FIR itregs were then removed from the TCR signal
SUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES Supplementary Figure 1: Chemokine receptor expression profiles of CCR6 + and CCR6 - CD4 + IL-17A +/ex and Treg cells. Quantitative PCR analysis of chemokine receptor transcript abundance
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Supplementary Figure 1. Normal T lymphocyte populations in Dapk -/- mice. (a) Normal thymic development in Dapk -/- mice. Thymocytes from WT and Dapk
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Supplemental Figure Legends
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Supplementary Figure 1
d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
Supplementary table I. Real-time primers used in the study. The fold change was obtained by
Supplementary table I. Real-time primers used in the study. The fold change was obtained by normalizing the gene expression number to those of HPRT, then comparing the samples to untreated or naive mice.
Tbk1-TKO! DN cells (%)! 15! 10!
a! T Cells! TKO! B Cells! TKO! b! CD4! 8.9 85.2 3.4 2.88 CD8! Tbk1-TKO! 1.1 84.8 2.51 2.54 c! DN cells (%)! 4 3 2 1 DP cells (%)! 9 8 7 6 CD4 + SP cells (%)! 5 4 3 2 1 5 TKO! TKO! TKO! TKO! 15 1 5 CD8
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IFNγ Supplemental Figure 1. CD T cell number (x1 6 ) 18 15 1 9 6 3 CD CD T cells CD6L C CD5 CD T cells CD6L D CD8 T cell number (x1 6 ) 1 8 6 E CD CD8 T cells CD6L F Log(1)CFU/g Feces 1 8 6 p
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Tim-3 Supplementary Figure 1 Tc0 49.5 0.6 Tc1 63.5 0.84 Un 49.8 0.16 35.5 0.16 10 4 61.2 5.53 10 3 64.5 5.66 10 2 10 1 10 0 31 2.22 10 0 10 1 10 2 10 3 10 4 IL-10 28.2 1.69 IL-27 Supplementary Figure 1.
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Pearson r = P (one-tailed) = n = 9
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