Supplemental Information. T Follicular Helper Cell-Germinal Center B Cell. Interaction Strength Regulates Entry into Plasma

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1 Immunity, Volume 48 Supplemental Information T Follicular Helper ell-germinal enter B ell Interaction Strength Regulates Entry into Plasma ell or Recycling Germinal enter ell Fate Wataru Ise, Kentaro Fujii, Katsuyuki Shiroguchi, Ayako Ito, Kohei Kometani, Kiyoshi Takeda, Eiryo Kawakami, Kazuo Yamashita, Kazuhiro Suzuki, Takaharu Okada, and Tomohiro Kurosaki

2 A Lymph node cells NP + D38 + NP-GG/FA s.c. Tamoxifen p.o. Analysis (d5).4 8. Spr2-ERT2cre Tg x Ai4 KI/w Day NP D38 tdtomato B D38 - B22 + NP + D38 lo LZ % of max D38 NP XR4 B22 D38 D86 Bcl6-YFP Bcl6 Spr2 Gpr83 Relative.5..5 ***** Relative.5..5 ****** Relative *. Bcl6hi Bcl6lo. Bcl6hi Bcl6lo Bcl6hi Bcl6lo D Naive Bcl6lo LZ Bcl6hi LZ Pre-G E LZ DZ % of max IRF Ki67 Bcl6-YFP F B22 + NP + D45. + B-8 hi transfer GG/FA D38 lo GL7 + LZ Bcl6 lo IRF4 + Fraction B-8 hi transfer NP-GG/FA B NP.859 D XR IRF Bcl6 hi IRF Fraction 2 Fraction 3 D45. D45. GL7 D86 Bcl6-YFP D69

3 Figure S. Identification of G-derived plasmablasts and each G LZ fraction. Related to Figure. (A) Left; schematic illustration of the experimental protocol for detection of G-derived plasmablasts using Spr2-ERT2-cre Tg x Ai4 mice. Right; flow ytometry plots of tdtomato expression in NP + plasmablasts (NP + D38 hi ) from draining lymph nodes of Spr2-ERT2-cre Tg x Ai4 mice at 5 days after immunization. Data are from at least two independent experiments (n=5). (B) Flow ytometry plots of Bcl6-YFP expression in LZ G B cells (NP + D38 lo XR4 lo D86 hi ) in B22 + D38 - population on day 2 after immunization of Bcl6-YFP reporter mice with NP-GG in FA. Gray histogram, naïve B cells from unimmunized Bcl6-YFP reporter mice. Data are from three independent experiments. (n=4). () Expression of Bcl6, Spr2, or Gpr83 mrna in sorted Bcl6-YFP lo or Bcl6-YFP hi LZ G B cells. G B cells were analyzed on day 2 after immunization of Bcl6-YFP reporter mice. *p<.5, *****p<.5, ******p<., unpaired Student t test. Bars indicate mean± SD. Data are from three independent experiments. (n=4). (D) Ki67 expression in naïve B cells (B22 + D38 hi GL7 - ), pre-g B cells (NP + B22 + D38 hi GL7 + ), Bcl6 lo or Bcl6 hi LZ G B cells (NP + B22 + D38 lo GL7 + XR4 lo D86 hi ). Pre-G B cells or G B cells were analyzed on day5 or day2 after immunization of Bcl6-YFP reporter mice, respectively. Data are from at least two independent experiments (n=3). (E) Flow cytometry plots of Bcl6-YFP and intracellular IRF4 expression in LZ G (NP + D38 lo XR4 lo D86 hi ) or DZ G (NP + D38 lo XR4 hi D86 lo ) on day2 after immunization of Bcl6-YFP reporter mice with NP-GG in FA. Data are from at least two independent experiments (n=3). (F) Flow cytometry plots of Bcl6-YFP, intracellular IRF4, and D69 expression by LZ G B cells (D38 lo GL7 + XR4 lo D86 hi ) derived from transferred D45. + B-8 hi Bcl6-YFP reporter B cells. B-8 hi Bcl6-YFP reporter B cells (D45./D45.2) were transferred into 57BL6 mice, followed by immunization with GG in FA (upper plots) or NP-GG in FA (lower plots). G B cells were analyzed at day 2 after immunization. Data are from at least two independent experiments (n=3).

4 A DZ Fr.3 Fr.2 Fr. Plasmablast Normalized expression level B Fraction Fraction 2 Fraction 3 S G2-M G-G 8 Myc 8 Batf EdU % Relative 4 2 Relative 4 2 5K K 5K 2K 25K 7-AAD 5K K 5K 2K 25K 5K K 5K 2K 25K Fr. Fr.2 Fr.3 Fr. Fr.2 Fr.3 Fr. Fr.2 Fr.3 D E Enrichment Score.6.3. Up-regulated by D4-stimulation % of max Da D2 LPAM- Naive B cell Fraction Fraction 2 Fraction 3 Ranked list metric (Signal2Noise) 5. Fraction (positively correlated). Fraction 2 (negatively correlated) -5. Fraction 2 Fraction 3 p-value<. FDR q-value= % of max D48 D84 Ly8

5 Figure S2. Gene expression, cell cycle status, and surface molecule expression in each LZ G fraction. Related to Figure 2, Figure 3, Figure 5, and Figure 6. (A) Heat map of 53 genes differentially expressed between Fraction and Fraction 2 as determined using DESeq2. LZ Fraction, Fraction 2, Fraction 3 cells, DZ cells, and G-derived plasmablats were prepared on day 2 after immunization for digital RNA seq analysis. Genes were considered differentially expressed when they had log 2 fold changes of >.5 or <.5, FDR of <., and p-value of <.. (B) ell cycle status of LZ G B cells assessed by EdU incorporation and 7-AAD staining. Bcl6-YFP reporter mice were immunized with NP-GG in FA, followed by injection of EdU 3 min before analysis on day2. Data are from at least two independent experiments (n=3). () Myc or Batf mrna expression in G LZ Fraction-3 cells. G LZ Fraction, Fraction 2, or Fraction 3 cells in draining lymph nodes from Bcl6-YFP reporter mice were sorted 2 days after immunization with NP-GG in FA. Expression levels of Myc or Batf mrna were determined by real-time PR. Data are from at least two independent experiments (n=3). (D) Gene enrichment analysis showing the enrichment of genes up-regulated by D4-stimulation in the G LZ Fraction 2 or Fraction 3. (E) Expression of Da (LFA), D2 (IAM-2), LPAM- (Integrin α4β7), D48 (SLAMF2), D84 (SLAMF5), or Ly8 (SLAMF6) in G LZ Fraction (red line), Fraction 2 (blue line), or Fraction 3 (green line) from draining lymph nodes of Bcl6-YFP reporter mice 2 days after immunization with NP-GG in FA. Expression of these molecules in naïve B cells from unimmunized Bcl6-YFP reporter mice is shown as a gray histogram. Data are from two independent experiments (n=3).

6 A B-8 hi Irf4 f/- Spr2-ERT2cre D45.2/2 NP-GG/FA s.c. Tamoxifen p.o. Analysis (d5) D45./ Day- 8 9 B G D NP + tdtomato 76. D38 D38 GL B GFP Plasmablast.962 GFP GFP + (%) G **** Plasmablast D G GFP n.s. 3.7 EdU + (%) 2 GFP GFP+ XR GFP- GFP+ D86 EdU D D69 hi LZ G GFP- GFP+..2 Bcl6 (i.c.) ells in LZ (%) 3 2 Fraction ** GFP- GFP+ ells in LZ (%) Fraction 2 n.s. GFP- GFP+

7 Figure S3. Irf4 ablation in G B cells blocks generation of plasma cell precursors. Related to Figure 2. (A) Schematic illustration of the experimental protocol for assessment of the effect of Irf4 deletion from G B cells on G homeostasis and plasmablast generation. (B) The presence of GFP+ (=Irf4 -/- ) cells among G B cells (D38 lo GL7 + ) or plasmablasts (D38 + ) which were derived from donor B cells (D NP + tdtomato + ). () DZ/LZ distribution and proliferation status of GFP- or GFP+ G B cells. DZ and LZ were defined by XR4 and D86 expression. Proliferation status of total G B cells was assessed by EdU incorporation 3 min after an EdU injection. GFP- or GFP+ G B cells were FAS-sorted for assessment of EdU incorporation. (D) The frequency of Fraction or Fraction 2 among Irf4 -/- LZ B cells. Intracellular Bcl6 expression in GFP- or GFP+ D69 hi LZ cells (left histograms) and the frequency of Fraction (Bcl6 lo D69 hi ) or Fraction 2 (Bcl6 hi D69 hi ) among GFP- or GFP+ LZ cells (right graphs) are shown. GFP- or GFP+ G B cells were FAS-sorted for assessment of intracellular Bcl6 expression. **p<., ****p<., n.s., non-significant, paired Student t test. Bars indicate mean± SD. All data are from at least two independent experiments. (B-D, n=4).

8 A wt allele d4 locus Ex7 Ex8 Ex9 targeted allele LoxP Ex7 Ex8 Ex9 neo r floxed allele Ex7 Ex8 Ex9 deleted allele B B-8 hi d4 Spr2-ERT2cre D45./2 (5%) B-8 hi d4 Spr2-ERT2cre D45./ (5%) 57B6J Tamoxifen p.o. NP-GG/FA s.c. Day- 8 9 Analysis (d5) D45. + NP + tdtomato 89. D38 D38 GL7 D D45.2 D45.2 G D45. Plasmablast D45. ells in G (%) Plasmablast (%) (45./) (45./) n.s. n.s. (45./2) (45./2) B-8 hi d4 f/+ Spr2-ERT2cre D45./2 (5%) B-8 hi d4 Spr2-ERT2cre D45./ (5%) 57B6J Tamoxifen p.o. NP-GG/FA s.c. Day- 8 9 Analysis (d5 and d2) ells in G (%) 6 n.s. n.s Days after Immunization ells in tomato+ PB (%) 8 6 * * Days after Immunization f/+

9 Figure S4. The effect of d4 haploinsufficiency on G B cell maintenance and plasmablast generation. Related to Figure 3. (A) Schematic diagram of d4 flox mice. The loxp sites are indicated as solid triangles. The exons 7, 8 and 9 are flanked with two loxp sites. Neomycin resistance cassette (Neor) of targeted allele was removed by the transient transfection of a re expression vector. (B) Upper; schematic illustration of the experimental protocol of co-transfer of B cells with different congenic marker for assessment of G B cell maintenance and plasmablast generation. Lower; the frequency of D45./ d4 cells or D45./2 d4 cells among G B cells (D38 lo GL7 + ) or G-derived plasmablasts (tdtomato + D44 hi D38 hi ). n.s., non-significant, paired Student t test. Data are from three independent experiments (n=4). () Upper; schematic illustration of the experimental protocol for assessment of the effect of d4 haploinsufficiency in G B cells on G homeostasis or plasmablast generation on day 5 or day 2. Lower; the frequency of d4 cells or d4 f/+ cells among G B cells (left graph) or G-derived plasmablast (right graph) on day 5 or 2 after immunization with NP-GG in FA. *p<.5, n.s., non-significant, paired Student t test. Bars indicate mean± SD. Data are from two independent experiments. (n=4).

10 A B B-8 hi d4 f/f Spr2-ERT2cre D45./ (5%) NP-GG/FA s.c. Tamoxifen p.o. Analysis (d5) % of max D3 + D4 D4 f/f B-8 hi d4 Spr2-ERT2cre D45./2 (5%) 57B6J Day- 8 9 D4 -Tamoxifen 98.2 tdtomato D38 GL D45.2 G B cell 48.5 f/f 45. D45. ells in G (%) n.s. f/f +Tamoxifen tdtomato 8.3 D38 D38 GL7.43 G B cell D45.2 D45.2 f/f.5 D45. G-derived plasmablast 9.2 f/f 7.25 ells in G (%) Plasmablast (%) *** f/f *** D44 D45. f/f D LZ G B cell f/f D ells in LZ (%) Fraction Fraction 2 *** 2 *** ells in LZ (%) 5 5 f/f f/f

11 Figure S5. The effect of inducible complete d4 deletion on G B cell maintenance and plasmablast generation. Related to Figure 3. (A) Schematic illustration of the experimental protocol for assessment of the effect of d4 deletion in G B cells on G homeostasis or plasmablast generation. (B) Expression of D4 on d4 B cells (blue line), d4 f/f B cells (red line), or D3 + cells (gray) after tamoxifen treatment. () Upper; the frequency of D45./2 d4 cells or D45./ d4 f/f cells among G B cells (D38 lo GL7 + ) in the absence of tamoxifen treatment. Lower; the frequency of D45./2 d4 cells or D45./ d4 f/f cells among G B cells (D38 lo GL7 + ) or G-derived plasmablasts (tdtomato + D44 hi D38 hi ) after tamoxifen treatment. (D) The frequency of Fraction or Fraction 2 among d4-deleted LZ G B cells. Expression of D69 in LZ d4 or d4 f/f cells after tamoxifen (left histogram) and the frequency of Fraction or Fraction 2 among LZ cells defined by expression of D69 and intracellular Bcl6 (data not shown) are shown. ***p<.5, n.s., non-significant, paired Student t test. Bars indicate mean± SD. Data are from at least two independent experiments. (B, n=5;, n=3 or 4; D, n=4).

12 A.5 * B-8hi Irf4 f/+ Spr2-ERT2cre D45.2/2 D45./ Tamoxifen p.o. NP-GG/FA s.c. Day- 8 9 Analysis (d5) Irf4 mrna (relative)..5. GFP- GFP+ B D69 hi LZ G GFP- GFP+ Fraction 3 * 25 Fraction 2 n.s ells in LZ (%) 2 ells in LZ (%) Bcl6 (i.c.) GFP- GFP+ GFP- GFP+.5 Bcl6 mrna * Spr2 mrna 2. ** Gpr83 mrna.5 * relative..5 relative.5..5 relative..5. GFP- GFP+. GFP- GFP+. GFP- GFP+

13 Figure S6. The effect of Irf4 haploinsufficiency on G B cells. Related to Figure 2. (A) Schematic illustration of the experimental protocol for assessment of the effect of Irf4 haploinsufficiency in G B cells. The expression levels of Irf4 mrna in GFP- or GFP+ G B cells after tamoxifen treatment is shown in the right graph. (B) The frequency of Fraction or Fraction 2 in Irf4 f/- LZ B cells. Intracellular Bcl6 expression in GFP- or GFP+ D69 hi LZ cells (left histograms) and the frequency of Fraction (Bcl6 lo D69 hi ) or Fraction 2 (Bcl6 hi D69 hi ) among GFP- or GFP+ LZ cells (right graphs) are shown. GFP- or GFP+ G B cells were FAS-sorted for assessment of intracellular Bcl6 expression. () The expression of Bcl6, Spr2, or Gpr83 mrna in purified D69 hi LZ cells from GFP- or GFP+ G B cells. *p<.5, **p<., n.s., non-significant, unpaired Student t test (A and ) or paired Student t test (B). Bars indicate mean± SD. Data are from two independent experiments. (A and B, n=4;, n=5).