Volume 48, Issue 4, Pages 702-715.e4 (April 2018) T Follicular Helper Cell-Germinal Center B Cell Interaction Strength Regulates Entry into Plasma Cell or Recycling Germinal Center Cell Fate  Wataru Ise, Kentaro Fujii, Katsuyuki Shiroguchi, Ayako Ito, Kohei Kometani, Kiyoshi Takeda, Eiryo Kawakami, Kazuo Yamashita, Kazuhiro Suzuki, Takaharu Okada, Tomohiro Kurosaki  Immunity  Volume 48, Issue 4, Pages 702-715.e4 (April 2018) DOI: 10.1016/j.immuni.2018.03.027 Copyright © 2018 Terms and Conditions

Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions

Figure 1 Bcl6loCD69hi LZ GC B Cells Express IRF4 (A) Expression of Bcl6-YFP, IRF4, and CD69 in NP-specific LZ (NP+CXCR4lo CD86hi) GC B cells from draining lymph nodes of Bcl6-YFP reporter mice 12 days after s.c. (subcutaneous) immunization with NP-CGG in CFA. (B) Expression of Bcl6-YFP in naive B cells (black line, B220+CD38hi), NP-specific plasmablasts (orange line, CD44hi CD138hi NP+), NP-specific GC LZ Fraction 1 (red line, Bcl6lo CD69hi), Fraction 2 (blue line, Bcl6hi CD69hi), or Fraction 3 (green line, Bcl6hi CD69lo). Expression of Bcl6-YFP was analyzed before (for naive B cells) or 12 days after s.c. immunization of Bcl6-YFP reporter mice with NP-CGG in CFA (for plasmablasts or GC B cells). (C) The number of NP-specific GC B cells or GC-derived plasmablasts and the frequency of Bcl6lo CD69hi (Fraction 1) or Bcl6hi CD69hi (Fraction 2) cells in NP-specific LZ GC B cells from popliteal draining lymph nodes of Bcl6-YFP reporter mice at 7, 14, and 21 days after immunization with NP-CGG in CFA. The number of NP-specific GC B cells (278 ± 109) or NP-specific plasmablasts (18.2 ± 6.4) at day 14 after immunization with CGG in CFA is shown in open circles. ∗p < 0.05; n.s., non-significant; unpaired Student’s t test. Bars indicate mean ± SD. Data are from at least three (A and B, n = 3) or two (C, n = 5) independent experiments. Please also see Figure S1. Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions

Figure 2 Bcl6lo CD69hi LZ GC B Cells Are Plasma Cell Precursors (A) The clonal similarity between plasmablasts (left graph) or DZ cells (right graph) and LZ Fraction 1–3 cells. NP+IgG1+ plasmablasts, NP+IgG1+ LZ Fraction 1–3 cells, or DZ cells were single-cell sorted from draining lymph nodes of Bcl6-YFP reporter mice at 12 days after immunization. VH186.2 sequences were analyzed and sub-grouped based on CDR3 sequences, and the numbers of neighbor sequences of plasmablasts or DZ cells found in each LZ Fraction were calculated. (B) The frequency of high-affinity W33L+ variants in NP+IgG1+ LZ Fraction 1–3 cells prepared as in (A). (C) Principal-component analysis of digital RNA-seq data from LZ Fraction 1–3 cells or GC-derived plasmablasts. B cells from B1-8hi Bcl6-YFP reporter mice (CD45.1+) or B1-8hi x S1pr2-ERT2cre x Ai14 mice (CD45.1+) were transferred into C57BL6 mice, followed by immunization with NP-CGG in CFA. The recipients of B1-8hi S1pr2-ERT2cre x Ai14 B cells were treated with tamoxifen at day 6, 7, and 8. Donor-derived (CD45.1+NP+) LZ GC Fraction 1–3 cells or GC-derived plasmablasts (CD45.1+tdTomato+NP+CD44hiCD138hi) were prepared 12 days after immunization. (D) Heatmap illustrating relative RNA expression of selected transcription factors, surface molecules, or cell-cycle related genes in GC-DZ, GC-LZ Fraction 1–3 cells, or GC-derived plasmablasts prepared as in (C). (E) Gene set enrichment analysis showing the enrichment for GC signature genes (left) or plasma cell-related IRF4 target genes (right) in Fraction 1 or Fraction 2 cells. (F) Prdm1 mRNA expression in purified GC LZ Fraction 1–3 cells after 5 hr in vitro culture with or without IL-21. ∗p < 0.05; ∗∗∗p < 0.005; n.s., non-significant; unpaired Student’s t test (A and F) and Fisher’s exact test (B). Bars indicate mean ± SD. Data are from three independent experiments (A, n = 20–30 in each Fraction), pooled from three independent experiments (B), from one RNA-seq analysis (C–E, n = 3 in each cell type), or from at least two independent experiments (F, n = 3). Please also see Figures S2, S3, and S6. Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions

Figure 3 CD40 Regulates Development of Bcl6loCD69hi Plasma Cell Precursors (A) Gene enrichment analysis showing the enrichment of genes upregulated by CD40 (left) or BCR stimulation (right) in GC LZ Fraction 1 or Fraction 2 cells. (B) Schematic illustration of the experimental protocol for assessment of the effect of Cd40 haploinsufficiency in GC B cells on GC homeostasis or plasmablast generation. (C) Expression of CD40 on Cd40+/+ B cells (blue line), Cd40f/+ B cells (red line), or CD3+ cells (gray) after tamoxifen treatment (left histogram) and gMFI (geometric mean fluorescence intensity) of Cd40+/+ or Cd40f/+ B cells after tamoxifen treatment (right graph). (D) The frequency of CD45.1/1 Cd40+/+ cells or CD45.1/2 Cd40f/+ cells among GC B cells (CD38loGL7+) or GC-derived plasmablasts (tdTomato+CD44hiCD138hi) after tamoxifen treatment. (E) DZ/LZ distribution and proliferation status of GC B cells derived from Cd40+/+ or Cd40f/+ B cells after tamoxifen treatment. Proliferation status was assessed by EdU incorporation 30 min after an EdU injection. (F) The frequency of Fraction 1 or Fraction 2 cells in LZ B cells after tamoxifen treatment. Intracellular Bcl6 expression among Cd40+/+ or Cd40f/+ CD69hi LZ cells (left histograms) and the frequency of Fraction 1 (Bcl6loCD69hi) or Fraction 2 (Bcl6hiCD69hi) cells among Cd40+/+ or Cd40f/+ LZ cells (right graphs) are shown. ∗∗p < 0.01; ∗∗∗∗p < 0.001; n.s., non-significant; unpaired Student’s t test or paired Student’s t test (D–F). Bars indicate mean ± SD. Data are from one RNA-seq analysis (A, n = 3) or three independent experiments (C–F; C, n = 3; D, n = 6, E and F, n = 4). Please also see Figures S2, S4, and S5. Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions

Figure 4 The Effects of Stat3 Haploinsufficiency on GC B Cell Maintenance and Plasmablast Generation (A) Schematic illustration of the experimental protocol for assessment of the effect of Stat3 haploinsufficiency in GC B cells on GC homeostasis or plasmablast generation. The expression of Stat3 mRNA in Stat3+/+ or Stat3f/+ GC B cells after tamoxifen treatment is shown in the right graph. (B) The frequency of CD45.1/2 wild-type (+/+) or CD45.2/2 Stat3f/+ (f/+) cells among GC B cells (CD38loGL7+) or plasmablasts (CD44hiCD138hi), which were derived from donor B cells (CD45.1+NP+tdTomato+). (C) The frequency of Fraction 1 or Fraction 2 cells among LZ B cells. Expression of intracellular Bcl6 and CD69 in wild-type (+/+) or Stat3f/+ (f/+) LZ cells (left flow cytometry plots) and the frequency of Fraction 1 (Bcl6loCD69hi) cells or Fraction 2 (Bcl6hiCD69hi) cells in wild-type (+/+) or Stat3f/+ (f/+) LZ cells (right graphs) are shown. ∗p < 0.05; ∗∗∗p < 0.005; n.s., non-significant; unpaired Student’s t test (A) or paired Student’s t test (B and C). Bars indicate mean ± SD. Data are two independent experiments (A, n = 5; B and C; n = 4). Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions

Figure 5 Bcl6loCD69hi GC B Cells Form Most Stable Conjugates with Tfh Cells (A) Expression of CD40, ICAM-1, or SLAM in the LZ GC B cell Fraction. Expression of CD40, ICAM-1, or SLAM among naive B cells (black line), GC LZ Fraction 1 cells (red line), Fraction 2 cells (blue line), or Fraction 3 cells (green line) (flow cytometry histograms) or gMFI of CD40, ICAM-1, or SLAM of Fraction 1–3 cells (right graphs) is shown. GC B cells in draining lymph nodes were analyzed 12 days after immunization with NP-CGG in CFA. (B) Expression of CD40, ICAM-1, or SLAM on high- or low-affinity LZ GC B cells. Expression of CD40, ICAM-1, or SLAM in IgG1+LZ GC B cells with high-affinity (red line) or low-affinity (blue line) BCRs defined by NP binding or gMFI of CD40, ICAM-1, or SLAM of high- or low-affinity LZ GC B cells is shown. (C) Schematic illustration of in vitro conjugate formation assay to examine the efficiency of Tfh-GC-B cell conjugate formation. (D) In vitro conjugate formation between Tfh cells and GC LZ cells. Conjugate formation between CXCR5+ OT-II cells and (OVA323–339-pulsed) purified Fraction 1–3 cells was analyzed after 30 min incubation. The frequencies of CD4+ cells in the LZ Fraction after incubation are shown. (E) In vivo conjugate formation. CD4 expression on GC LZ Fraction 1–3 cells derived from donor B1-8hi Bcl6-reporter B cells (upper panel), FSC (forward scatter) of B220+CD4+ cells (solid line, lower panel) or B220+CD4− cells (dotted line, lower panel), and the frequency of CD4+ cells in LZ Fraction 1–3 (graph) are shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005; n.s., non-significant; unpaired Student’s t test. Bars indicate mean ± SD. All data are from at least two independent experiments (A, B, and D, n = 3; E, n = 4). Please also see Figure S2. Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions

Figure 6 Importance of Stability of Tfh-GC B Cell Contact in IRF4 Induction and Generation of Bcl6loCD69hi Cells (A) Increased expression of ICAM-1, SLAM, or CD40 by GC LZ cells after CD40 stimulation. Histogram of ICAM-1 or SLAM expression by untreated (blue line) or CD40-stimulated (red line) GC LZ cells, gMFI of ICAM-1 or SLAM, or CD40 mRNA expression by untreated or CD40-stimulated LZ cells is shown. Sorted GC LZ cells were stimulated with rCD40L for 5 hr, and expression of surface ICAM-1 or SLAM or expression of Cd40 mRNA was examined. (B) Increased conjugate formation with Tfh cells by CD40-stimulated GC B cells. Sorted GC LZ cells were stimulated with rCD40L for 2 hr, pulsed with OVA323–339 for 2 hr, and mixed with sorted CXCR5+ OT-II cells for 30 min, and conjugate formation (CD4 expression in B220+ GC B cells) was analyzed. (C) Inhibition of conjugate formation and IRF4 expression by ICAM-1 blocking. Sorted GC LZ cells were pulsed with OVA323–339 for 2 hr, incubated with anti-ICAM-1 mAb (monoclonal antibody) for 30 min, and mixed with OT-II Tfh cells for 30 min for assessment of conjugate formation (upper panel) or for 4 hr for measurement of IRF4 expression (lower panel). (D) Inhibition of generation of GC LZ Fraction 1 cells by ICAM-1 blocking in vivo. Schematic illustration of the experimental protocol for assessment of the effect of ICAM-1 blocking on maintenance of GC B cells is shown in upper panel. In the lower panel, the expression of CD38 and GL7 by donor B cells (left plots) and Bcl6-YFP expression in CD69hi LZ GC B cells (right histograms) are shown. The number of GC B cells, the frequency of Fraction 1 or 2 cells in LZ cells, or Irf4 mRNA expression in CD69hi LZ cells (graphs) is also shown. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005; n.s., non-significant; unpaired Student’s t test. Bars indicate mean ± SD. All data are from at least two independent experiments (A–C, n = 3; D, n = 4). Please also see Figure S2. Immunity 2018 48, 702-715.e4DOI: (10.1016/j.immuni.2018.03.027) Copyright © 2018 Terms and Conditions