Nature Immunology: doi: /ni Supplementary Figure 1. Id2 and Id3 define polyclonal T H 1 and T FH cell subsets.

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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<0.0001 (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.).

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<0.0001 (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.).

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

(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<0.0001 (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.).

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<0.0001 (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.).

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<0.0001 (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.).

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.

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