Nature Immunology: doi: /ni Supplementary Figure 1. Id2 and Id3 define polyclonal T H 1 and T FH cell subsets.
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1 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 (SLAM lo CXCR5 + or CXCR5 + PD-1 ) or GC T FH (CXCR5 + PD-1 + ) differentiation for the indicated antigen-experienced (CD49d + CD11a + ) CD4 + T cell populations was analyzed by flow cytometry and quantified. *p<0.01, **p<0.001 and ***p< (two-tailed unpaired Student's t test). Data are representative of three experiments (a,b), each with n = 3 mice per group (mean ± s.e.m.).
2 Supplementary Figure 2 Knockdown of Id2 results in increased T FH differentiation. SMARTA CD4 + T cells transduced with the indicated shrnamir-rv were transferred into B6 SMARTA CD4 + T cells were transduced with the indicated shrnamir-rv. (a) RNA was isolated from shrnamir-rv + CD4 + T cells and Id2 expression was determined by qrt- PCR. shrnamir-rv + CD4 + T cells were transferred into B6 mice and analyzed 6 (b-e) or 3 (f-i) days after LCMV infection. (b,f) Quantitation of shrna + SMARTA CD4 + T cells. (c-e) GC T FH (CXCR5 + PSGL-1 ) cell development was analyzed by flow cytometry (c) and quantified as a fraction of SMARTA CD4 + T cells (d) or total splenocytes (e). (g-i) T FH (CXCR5 + CD25 ) and T H 1 (CD25 + CXCR5 ) differentiation was analyzed by flow cytometry (g) and quantified as a fraction of SMARTA CD4 + T cells (h) or total splenocytes (i). (j,k) OT-II CD4 + T cells transduced with the indicated shrnamir-rv were transferred into B6 mice and analyzed 11 days after footpad immunization with NP-OVA in alum. (j) T FH (CXCR5 + PD-1 + ) and (k) GC T FH (CXCR5 + Bcl6 + ) differentiation was analyzed by flow cytometry and quantified as a fraction of OT-II CD4 + T cells or total lymph node cells. *p<0.05, **p<0.01, ***p<0.001, ****p< (twotailed unpaired Student's t test). Data are pooled from two (a) experiments or representative of two (j,k), four (b-e) or five (f-i) independent experiments with n=6-14 mice per group (mean ± s.e.m.).
3 Supplementary Figure 3 Expression of Id proteins orchestrates CD4 + T cell differentiation. (a-c) Id2 +/+ CD4-Cre + (Id2 +/+ ) or Id2 fl/fl CD4-Cre + (Id2 / ) SMARTA CD4 + T cells were transferred into B6 mice and analyzed 4 (a) and 7 (a-c) days after LCMV infection. (a) Flow cytometric analysis of CXCR5 and SLAM expression quantified as gmfi on days 4 and 7. (b) Flow cytometric analysis of granzyme B and TCF1 expression (left panels), quantification as a frequency of SMARTA CD4 + T cells
4 (right panels). (c) Flow cytometric analysis of IFN- and T-bet expression in total SMARTA CD4 + T cells. gmfi of T-bet expression and total number of IFN-y + SMARTA CD4 + T cells is shown. (d) Id2 +/+ CD4-Cre + and Id2 fl/fl CD4-Cre + mice were analyzed 7 days after LCMV infection, SLAM hi CXCR5, SLAM mid CXCR5 mid and SLAM lo CXCR5 + cells were analyzed by flow cytometry (left panels) and quantified as a frequency of antigen-experienced (CD49d + CD11a + ) CD4 + T cells (middle panel) or as total splenic numbers (right panel). (e) SMARTA CD4 + T cells transduced with the indicated shrnamir-rv were transferred into B6 mice and analyzed 3 days after LCMV infection. Quantitation of Bcl6 expression in SMARTA T H 1 (CXCR5 PD-1 ) and T FH (CXCR5 + PD-1 + ) cells is shown. (f) Id2 +/+ CD4-Cre + or Id2 fl/fl CD4-Cre + SMARTA CD4 + T cells were transferred into Bcl6 fl/fl CD4-Cre + mice and analyzed 8 days after LCMV infection. SLAM hi CXCR5, SLAM mid CXCR5 mid and SLAM lo CXCR5 + cells were analyzed by flow cytometry (left panels) and quantified as a frequency of SMARTA CD4 + T cells (middle panel) or as total numbers (right panel) *p<0.05, **p<0.01, ***p<0.001, ****p< (two-tailed unpaired Student's t test). Data are representative of one (e) or three (a-d,f) independent experiments with n=3-10 mice per group (mean ± s.e.m.).
5 Supplementary Figure 4 Expression of Id3 limits unregulated differentiation of T FH cells and GC T FH cells. (a) Id3 +/+ CD4-Cre + (Id3 +/+ ) or Id3 fl/fl CD4-Cre + (Id3 / ) SMARTA CD4 + T cells were transferred into B6 mice and analyzed 7 days after LCMV infection. (a) Flow cytometric analysis of CXCR5 + Bcl6 hi (top), SLAM hi CXCR5 (T H 1) and SLAM lo CXCR5 + (T FH ) (middle); or PD-1 CXCR5 (T H 1), PD-1 CXCR5 + (T FH ) and PD-1 + CXCR5 + (GC T FH ) (bottom) populations and quantification as a frequency of SMARTA CD4 + T cells (right panels). (b) Id3 +/+ CD4-Cre + and Id3 fl/fl CD4-Cre + mice were analyzed 7 days after LCMV and PD-1 CXCR5 (T H 1), PD-1 CXCR5 + (T FH ) and PD-1 + CXCR5 + (GC T FH ) expression was analyzed by flow cytometry (left) and quantified as a frequency of antigen-specific (gp66-77) CD4 + T cells (right). (c-d) NIP CD4 + T cells transduced with the indicated RV were transferred into B6 mice and analyzed 6 days (c) or 3 days (d) after LCMV infection. (c) T FH (CXCR5 + SLAM lo ) differentiation was analyzed by flow cytometry and quantified as a frequency of NIP CD4 + T cells. (d) Early T FH (CXCR5 + Bcl6 + ) differentiation was analyzed by flow cytometry and quantified as a frequency of NIP CD4 + T cells. *p<0.05, **p<0.01, ***p<0.001, ****p< (two-tailed unpaired Student's t test). Data are pooled from two (c,d) experiments or representative of two (a,b) independent experiments with n=8-10 mice per group (mean ± s.e.m.).
6 Supplementary Figure 5 E proteins drive CXCR5 expression and inhibit the formation of T H 1 cells. (a) Id2 +/+ CD4-Cre + or Id2 fl/fl CD4-Cre + SMARTA CD4 + T cells were transduced with the indicated shrnamir-rv and expanded in vitro for 4 days. Graph indicates relative mrna expression of Tcf3 by dsred + SMARTA CD4 + T cells. (b) Gene expression of E proteins and related genes of interest in T H 1 and T FH SMARTA at day 3 after acute LCMV infection measured by RNA-Seq. Cd8a and Cd19 are included as negative controls. Data are from ref. 32 (GSE67336). SMARTA (c) or NIP (d-k) CD4 + T cells transduced with the indicated RV were transferred into B6 mice and analyzed 3 days (c-f) or 6 days (g-k) after LCMV infection. (c) CXCR5 expression by SMARTA T H 1 (CXCR5 Bcl6 ) and T FH (CXCR5 + Bcl6 + ) cells. (d,g) Quantitation of RV + NIP CD4 + T cells. (e) CXCR5 expression by NIP T H 1 (CXCR5 Bcl6 ) and T FH (CXCR5 + Bcl6 + ) cells. (f) Quantitation of Tbet expression by NIP T H 1 (CD25 + CXCR5 ) and T FH (CXCR5 + CD25 ) cells. (h-j) T FH (CXCR5 + SLAM lo ) and T H 1 (SLAM + CXCR5 ) differentiation was analyzed by flow cytometry (h) and quantified as a fraction of NIP CD4 + T cells (i) or total splenocytes (j). (k) CXCR5 expression by NIP T H 1 (SLAM + CXCR5 ) and T FH (CXCR5 + SLAM lo ) cells.. **p<0.01, ***p< (two-tailed unpaired Student's t test). Data are pooled from two (i,j) experiments or are representative of two (a,c) or three (d-h,k) independent experiments with n=8-10 mice per group (mean ± s.e.m.).
7 Supplementary Figure 6 Bcl6 inhibits Id2 expression. (a) Bcl6 fl/fl CD4-Cre + SMARTA CD4 + T cells transduced with the indicated vectors were transferred into B6 mice. (b) WT, Bcl6 fl/wt CD4- Cre + (Bcl6 +/ ), Bcl6 fl/fl CD4-Cre + (Bcl6 / ) SMARTA CD4 + T cells were transferred into B6 mice. Gates used to sort IL-2R + and IL-2R SMARTA CD4 + T cells 3 days after LCMV infection are indicated. (c) Sequences of the primers used in the study. (d) Model for the role of Id and E proteins in orchestrating CD4 + T cell differentiation. Data are representative of 2 independent experiments.
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
Nature Immunology: doi: /ni Supplementary Figure 1. Transcriptional program of the TE and MP CD8 + T cell subsets.
Supplementary Figure 1 Transcriptional program of the TE and MP CD8 + T cell subsets. (a) Comparison of gene expression of TE and MP CD8 + T cell subsets by microarray. Genes that are 1.5-fold upregulated
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SUPPLEMENTARY INFORMATION doi:1.138/nature1458 Grosso et al. Barbosa et al. 74 72 45 33 47 7 51 Rappsilber et al. Supplementary Figure 1 a, Venn-Diagram of identified splice factors in the work of Barbossa
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immunology.sciencemag.org/cgi/content/full/2/16/eaan6049/dc1 Supplementary Materials for Enzymatic synthesis of core 2 O-glycans governs the tissue-trafficking potential of memory CD8 + T cells Jossef
Supplementary Figures
Supplementary Figures Supplementary Figure 1 DOT1L regulates the expression of epithelial and mesenchymal markers. (a) The expression levels and cellular localizations of EMT markers were confirmed by
Nature Genetics: doi: /ng Supplementary Figure 1
Supplementary Figure 1 MSI2 interactors are associated with the riboproteome and are functionally relevant. (a) Coomassie blue staining of FLAG-MSI2 immunoprecipitated complexes. (b) GO analysis of MSI2-interacting
Supplementary Figure 1
Supplementary Figure 1 YAP negatively regulates IFN- signaling. (a) Immunoblot analysis of Yap knockdown efficiency with sh-yap (#1 to #4 independent constructs) in Raw264.7 cells. (b) IFN- -Luc and PRDs
Supplemental Figures Supplemental Figure 1:
Supplemental Figures Supplemental Figure 1: Representative FACS data showing Concurrent Brain cell type Acquisition using either Percoll PLUS (top row) or myelin removal beads (bottom two rows). Debris
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SUPPLEMENTARY INFORMATION Articles https://doi.org/1.138/s41591-18-135-2 In the format provided by the authors and unedited. Sequestration of T cells in bone marrow in the setting of glioblastoma and other
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Supplementary Figure 1. BMS enhances human T cell activation in vitro in a
Supplementary Figure 1. BMS98662 enhances human T cell activation in vitro in a concentration-dependent manner. Jurkat T cells were activated with anti-cd3 and anti-cd28 antibody in the presence of titrated
Peer review correspondence
OX40 controls effector CD4 + T-cell expansion, not follicular T helper cell generation in acute Listeria infection Clare L. Marriott, Emma C. Mackley, Cristina Ferreira, Marc Veldhoen, Hideo Yagita and
Supplemental Table S1
Supplemental Table S. Tumorigenicity and metastatic potential of 44SQ cell subpopulations a Tumorigenicity b Average tumor volume (mm ) c Lung metastasis d CD high /4 8. 8/ CD low /4 6./ a Mice were injected
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Supplementary Data Table of Contents: - Supplementary Methods - Supplementary Figures S1(A-B) - Supplementary Figures S2 (A-B) - Supplementary Figures S3 - Supplementary Figures S4(A-B) - Supplementary
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doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
Supplementary Figure 1
Supplementary Figure 1 Expression of apoptosis-related genes in tumor T reg cells. (a) Identification of FOXP3 T reg cells by FACS. CD45 + cells were gated as enriched lymphoid cell populations with low-granularity.
Supplementary Figure S1. Flow cytometric analysis of the expression of Thy1 in NH cells. Flow cytometric analysis of the expression of T1/ST2 and
Supplementary Figure S1. Flow cytometric analysis of the expression of Thy1 in NH cells. Flow cytometric analysis of the expression of T1/ST2 and Thy1 in NH cells derived from the lungs of naïve mice.
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Supplemental Figure Legends Supplemental Figure 1. Western blot analysis indicated that was detected in the fractions of plasma membrane and cytosol but not in nuclear fraction isolated from Pkd1 null
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Cell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice
Supplementary Methods: Cell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice and gently meshed in DMEM containing 10% FBS to prepare for single cell suspensions. CD4 + CD25
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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 -/- mice were stained for expression of CD4 and CD8.
Bezzi et al., Supplementary Figure 1 *** Nature Medicine: doi: /nm Pten pc-/- ;Zbtb7a pc-/- Pten pc-/- ;Pml pc-/- Pten pc-/- ;Trp53 pc-/-
Gr-1 Gr-1 Gr-1 Bezzi et al., Supplementary Figure 1 a Gr1-CD11b 3 months Spleen T cells 3 months Spleen B cells 3 months Spleen Macrophages 3 months Spleen 15 4 8 6 c CD11b+/Gr1+ cells [%] 1 5 b T cells
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