(A) Expression of CXCR5, PD-1, and PSGL1 on CD44^hi CD4 T cells taken from spleens of B6 mice 8 days after immunization with NP-CGG in alum.
(B) Microscopy of spleens taken from B6 mice 8 days after immunization with NP-CGG in alum. Sections were stained with 4 antibodies. For simplicity, the top panel shows staining to detect IgD, PNA, and CD4; the bottom panel shows that for IgD, PSGL1, and CD4. Inset panels (1–3) show increased magnification of labeled areas. Arrows indicate PSGL1^hi (panel 1) or PSGL1^lo (panels 2 and 3) CD4 T cells.
(C) Expression of bcl6 and il21 mRNA levels in sorted CD44^hi CD4⁺ PSGL1^lo CXCR5^lo PD-1^lo or CD44^hi CD4⁺ PSGL1^lo CXCR5^hi PD-1^hi cell subsets. Results are representative of 2 independent experiments. Fold change is in comparison to sorted naïve CD4 T cells. p values: bcl6, p = 0.8765, il21, **p = 0.0016.

(A) Expression of PD-1 on naïve, CD62L^hi PSGL1^hi, CD62L^lo PSGL1^hi, and CD62L^lo PSGL1^lo cells from MRL/Fas^lpr mice. Grey shaded is isotype control, black line is PD-1.
(B & C) Expression of bcl6 and prdm1 (Blimp1) transcripts in sorted CD62L^hi PSGL1^hi, CD62L^lo PSGL1^hi, and CD62L^lo PSGL1^lo cells isolated from spleens of 12–16 week old MRL/Faslpr mice. Results are representative of 3 experiments. In (B), **p = 0.0029 (comparing CD62L^lo PSGL1^hi and CD62L^lo PSGL1^lo cells), **p = 0.0093 (CD62L^hi PSGL1^hi vs. CD62L^lo PSGL1^lo), and in (C), *p = 0.0427 (CD62L^lo PSGL1^hi vs. CD62L^lo PSGL1^lo), *p = 0.0391 (CD62L^hi PSGL1^hi vs. CD62L^lo PSGL1^lo).
(D) Protein expression of Bcl6 and Blimp1 in sorted CD62L^hi PSGL1^hi, CD62L^lo PSGL1^hi, and CD62L^lo PSGL1^lo cells isolated from spleens of 12–16 week old MRL/Faslpr mice. The human B cell line BJAB was used as a positive control for Bcl6 and purified B cells activated in vitro with LPS for 48 hours served as a positive control for Blimp1. Actin was used as a loading control. Results are representative of 2 experiments.
(E & F) Spleens from 7–8 week old Bcl6-intact or -deficient MRL/Fas^lpr mice were analyzed for the presence of PSGL1^lo cells. Results are representative of 3 independent experiments from 3 cohorts of mice, with 4–5 mice per group. ***p < 0.0001 (Bcl6^+/+ vs. Bcl6^−/−), **p = 0.0038 (Bcl6^+/− vs. Bcl6^−/−).
(G – I) Percentage and number of CD138⁺ plasma cells analyzed for intracellular expression of IgM and IgG isolated from spleens of 7–8 week-old Bcl6-intact or -deficient MRL/Fas^lpr mice. In (I), open bars represent Bcl6^+/+, grey bars represent Bcl6^+/− and black bars represent Bcl6^−/− MRL/Fas^lpr mice. Results are representative of 3 independent experiments from 3 cohorts of mice, with 4–5 mice per group. In (H), p = 0.1497 (Bcl6^+/+ vs. Bcl6^−/−), p = 0.0817 (Bcl6^+/− vs. Bcl6^−/−). In (I), IgM p = 0.1591 (Bcl6^+/+ vs. Bcl6^−/−), *p = 0.0491 (Bcl6^+/− vs. Bcl6^−/−); IgG, p = 0.1209 (Bcl6^+/+ vs. Bcl6^−/−), *p = 0.0285 (Bcl6^+/− vs. Bcl6^−/−).

(A) Development of T_FH cells in IL-21-intact or -deficient mice 7 days after immunization with NP-CGG in alum compared to unimmunized (naïve) mice, using PSGL1 (left panels) or CXCR5 and PD-1 (right panels) as markers.
(B) Percentages of PSGL1^lo T cells of CD44^hi CD4 cells 7 days after immunization with NP-CGG in alum or in unimmunized (naïve) mice. Bar graph shows data representative of that revealed in the left panels of (A), compiled from 2 independent experiments, using 5 mice per group. p = 0.0959.
(C) Percentages of CXCR5^hi PD-1^hi T cells of CD44^hi CD4 cells 7 days after immunization with NP-CGG in alum or in unimmunized (naïve) mice. Data represented in the right panels of (A) are compiled from 2 independent experiments using 5 mice per group. p= 0.1044
(D) Bcl6 expression in CD4 CD44^hi cells sorted into PSGL1^hi and PSGL1^lo subsets 7 days after immunization of IL-21-intact and –deficient mice with NP-CGG in alum. Expression is shown as fold over sorted naïve CD4 T cells. Data are representative of 2 independent experiments.
(E) Analysis of GC cell formation (GL-7⁺ PNA⁺) in IL-21-intact, -deficient or unimmunized (naïve) mice. TCRβ⁻ IgD⁻ cells were gated.
(F) Percentage of GC B cells (GL-7⁺ PNA⁺) among B220⁺ cells. Data shown are representative of that shown in (E), and of 2 independent experiments using 4 mice per group. **p = 0.0011.
(G) Levels of anti-NP antibodies in the serum of IL-21-intact or -deficient mice bled 10 days after immunization with NP-GCC in alum. IgM, p = 0.8110; total IgG, **p = 0.0043; IgG1, ***p < 0.0001. Data compiled from one experiment, 5 mice per group.

(A) Intracellular staining of IL-21 in LCMV-specific CD4 T cells from spleens isolated 8 days after infection in IL-6-intact or -deficient mice. Cells were re-stimulated with LCMV I-A^b binding peptide gp61-80 for 5 hours in vitro.
(B) Percentages of IL-21⁺ cells of LCMV-specific CD4 T cells. Data are compiled from 2 independent experiments, using 9 mice per group.
(C, D, & E) IL-21 and IFNγ production by LCMV-specific CD44^hi CD4 T cells assessed using intracellular staining after re-stimulation with PMA and ionomycin. Data are compiled from 2 independent experiments, using 9 mice per group.
(F) Expression of bcl6 and il21 in T_FH cells (CD4⁺CD44^hiCXCR5^hiBTLA^hi) and non-T_FH (CD4⁺CD44^hiCXCR5^loBTLA^lo) cells sorted 8 days post-infection of IL-6-intact or -deficient mice.

(A) Expression of PSGL1 and CD62L on CD4⁺ CD44^hi B220^lo splenocytes in aged lupus-prone MRL/Fas^lpr mice.
(B) Selected genes are differentially expressed in CD4 PSGL1^lo T cells from lupus-prone MRL/Fas^lpr mice.
(C) Expression of PSGL1 and CD62L on transferred OT-II Thy1.1 CD4 T cells in unimmunized or immunized mice. Left panels are gated on CD4⁺ B220^lo cells.
(D) Expression of CXCR5 and PD-1 on gated OT-II Thy1.1 CD4 T cells 6 days after transfer to B6 mice and immunization. Bar graph represents the percentage of cells that are CXCR5^hi PD-1^hi of indicated group. Results are representative of 3 independent experiments using 4 mice per group. **p = 0.0075
(E) Expression of ICOS on OT-II Thy1.1 CD4 T cells 6 days after transfer to B6 mice and immunization. Naïve CD4 T cells are shown in grey shade, and PSGL1^lo T cells by the black line.
(F) Expression of il21, bcl6, prdm1, and rorc transcripts in sorted PSGL1^hi and PSGL1^lo OT-II Thy1.1 CD4 T cells 6 days after transfer to B6 mice and immunization with OVA in alum. Results are representative of 3 independent experiments. Fold change is in comparison to sorted naïve CD4 T cells; il21, **p = 0.0012, bcl6, *p = 0.0223, and rorc, *p = 0.0221.
(G) Expression of CXCR4 on naïve, PSGL1^lo T_FH, and B220⁺ cells 7 days after transfer to B6 mice and immunization. The PSGL1^lo cells are in the gated OT-II Thy1.1 CD4 T cell population. Results are representative of 2 experiments using 4 mice per experiment.

(A) Expansion of adoptively transferred OT-II CD4 T cells (left panels) and expression of CD62L and PSGL1 on transferred CD44^hi Bcl6^+/+ or Bcl6^−/− CD4 T cells (right panels) following immunization of recipient mice.
(B) Percentage of PSGL1^lo CD4 T cells among total transferred T cells, representative of the data shown in (A), compiled from 3 independent experiments using 4 mice per group. ***p < 0.0001.
(C) Mean fluorescence intensity of PSGL1 on transferred OT-II⁺ Bcl6^+/+ or Bcl6^−/− CD4 T cells following immunization of recipient mice with OVA in alum. Naïve CD4 T cells represented by shaded grey, OT-II CD44^hi Bcl6^+/+ CD4 T cells by the dashed line, and OT-II CD44^hi Bcl6^−/− CD4 T cells by the solid line.
(D) MFI of PSGL1 gated on OT-II CD44^hi PSGL1^hi CD4 T cells. Bar graph is representative of 3 independent experiments, using 4 mice for each group. *** p < 0.0001.
(E) Expansion of OT-II Thy1.1 CD4 T cells transduced with a GFP-expressing retrovirus 6 days after transfer to B6 mice and immunization with OVA in alum.
(F) Expression of CD62L and PSGL1 on OT-II Thy1.1 GFP⁺ CD44^hi CD4 T cells 6 days after transfer to B6 mice and immunization.
(G) Percentages of PSGL1^lo cells of total transferred OT-II GFP⁺ CD4 T cells, with results representative of the data in (E) and of 3 independent experiments using 3 mice for each group. **p = 0.0061.

(A) Expansion of OT-II Thy1.1 CD4 T cells 6 days after adoptive transfer and immunization of recipient B6 mice with OVA in alum in the presence or absence of B cells (left panels). Expression of CD62L and PSGL1 (middle panels), and PD-1 and CXCR5 (right panels) on the expanded OT-II Thy1.1 CD44^hi CD4 T cells in B cell-intact or –deficient recipients.
(B) Percentages of T_FH cells of OT-II Thy1.1 CD44^hi CD4 T cells taken from B cell-intact and – deficient mice are shown, with **p = 0.0049 (PSGL1^lo T cells) and **p = 0.0093 (CXCR5^hi PD-1^hi T cells). Data shown are representative of that in (A), and of 4 independent experiments, using 4 mice per group.
(C) Percentages of T_FH cells of OT-II Thy1.1 CD44^hi CD4 T cells taken from HEL Ig transgenic (MD4), followed by immunization of recipient B6 mice, *p = 0.4702 (PSGL1^lo T cells) and *p = 0.0370 (CXCR5^hi PD-1^hi T cells). Data are representative of 2 independent experiments using 4 mice per group.
(D) Percentages of T_FH cells of OT-II Thy1.1 CD44^hi CD4 T cells 7 days after immunization of CD19-intact or -deficient recipients with OVA in alum. Bar graph shows data representative of 2 independent experiments using 4 mice per group. p = 0.7029 (PSGL1^lo cells), **p = 0.0036 (CXCR5^hi PD-1^hi cells).
(E) Percentages of CXCR5^hi PD-1^hi cells of OT-II Thy1.1 CD44^hi cells 3 days after transfer (7 days after immunization) from B cell-intact or B cell-deficient mice. Bar graph shows data representative of 3 independent experiments using 2–3 mice per group. *p = 0.0129 (WT→WT vs. KO→KO), p = 0.6576 (WT→WT vs. KO→ WT), *p = 0.0354 (WT→WT vs. WT→KO), *p = 0.0218 (KO→WT vs. KO→KO).
(F) Percentages of PSGL1^lo cells of OT-II Thy1.1 CD44^hi cells 3 days after transfer (7 days after immunization) to B cell-intact or B cell-deficient mice. Bar graph shows data representative of 3 independent experiments, using 2–3 mice per group. p = 0.4327 (WT→ WT vs. KO→KO), p = 0.8822 (WT→WT vs. KO→WT), p= 0.3921 (WT→WT vs. WT→KO), p = 0.3920 (KO→WT vs. KO→KO).
(G) Expression of bcl6 mRNA in naïve CD4 T cells, and OT-II Thy1.1 CD44^hi T cells sorted 7 days after transfer to B6 wild type (WT) or B cell-deficient (uMT) hosts, followed by immunization with OVA in alum. *p = 0.0112, transfer to naïve vs. WT recipients; *p= 0.0138, transfer to WT vs. uMT recipients; *p= 0.0163, transfer to naïve vs. uMT recipients. Data are representative of 3 independent experiments.

(A&B) Expansion of activated (CD44^hi CD62L⁻) CD8 (A) and CD4 (B) T cells from spleens of IL-6-intact and -deficient mice isolated 8 days after LCMV infection.
(C) Expression of BTLA and CXCR5 on CD44^hi CD4 T cells 8 days after LCMV challenge in IL-6-intact or -deficient mice.
(D) Percentages of T_FH cells among CD44^hi CD4 T cells 8 days after LCMV challenge in IL-6-intact or -deficient mice. Data are compiled from 3 independent experiments, using 9 mice per group. *p=0.0441.
(E) Intracellular staining for IFNγ production in CD4 T cells from spleens isolated 8 days after LCMV infection in IL-6-intact or -deficient mice. Cells were re-stimulated with LCMV I-A^b binding peptide gp61-80 for 5 hours in vitro.
(F) Expression of CXCR5 on LCMV-specific (CD44^hi IFNγ⁺) CD4 T cells. Gray shaded line represents naïve CD4 T cells; black line represents CD44^hi IFNγ ⁺ CD4 T cells from IL-6 intact mice; and red line represents CD44^hi IFNγ ⁺ CD4 T cells from IL-6-deficient mice.
(G) Percentages of T_FH cells of LCMV-specific (IFNγ⁺) CD4 T cells 8 days after LCMV infection in IL-6-intact or -deficient mice. Data are compiled from 2 independent experiments, using 9 mice per group.

(A) Analysis of GC formation (Fas⁺ GL7⁺) in spleens isolated 8 days after LCMV infection from IL-6-intact or -deficient mice. Cells are gated on B220⁺ B cells.
(B) GC B cells as a percentage of total B220⁺ B cells from infected IL-6-intact or -deficient mice 8 days after LCMV infection. Data are compiled from 3 independent experiments, using 9 mice per group.
(C & D) Titers of anti-LCMV IgG antibody in the serum of IL-6-intact or - deficient mice bled 8 and 15 days after LCMV infection. Day 8, **p < 0.001. Data are representative of 4 independent experiments.