Droplet-Bsed SC-RNA-Seq and Population RNA-Seq (A) Gating Strategy for 10X SC-RNA-Seq

AB C

E FAM101B ● ● ● ● ● TCF7 ● ● ● ● ● LY6A ● ● ● ● ● CXCR5 ● ● ● ● ● TOX2 ● ● ● ● ● SMCO4 ● ● ● ● ● TNFAIP8 ● ● ● ● ● PDCD1 ● ● ● ● ● TBC1D4 ● ● ● ● ● 2310001H17RIK ● ● ● ● ● MAF ● ● ● ● ● S100A11 ● ● ● ● ● SH2D1A ● ● ● ● ● RGS10 ● ● ● ● ● IZUMO1R ● ● ● ● ● SMC4 ● ● ● ● ● FOXP3 ● ● ● ● ● PIM1 ● ● ● ● ● GIMAP7 ● ● ● ● ● IKZF2 ● ● ● ● ● PGLYRP1 ● ● ● ● ● CD81 ● ● ● ● ● TNFRSF18 ● ● ● ● ● CTLA4 ● ● ● ● ● CD74 ● ● ● ● ● CAPG ● ● ● ● ● TNFRSF1B ● ● ● ● ● CST7 ● ● ● ● ● TNFRSF4 ● ● ● ● ● CD83 ● ● ● ● ● TNFRSF9 ● ● ● ● ● scaled mean SDF4 ● ● ● ● ● expression ZFP36L1 ● ● ● ● ● 1.00 ITM2C ● ● ● ● ● ● ● ● ● ● SAMSN1 0.75 BATF ● ● ● ● ● ICOS ● ● ● ● ● SRGN ● ● ● ● ● 0.50 F PRKCA ● ● ● ● ● NFATC1 ● ● ● ● ● 0.25 EEA1 ● ● ● ● ● HSPA5 ● ● ● ● ● 0.00 CD82 ● ● ● ● ● ● ● ● ● ● BCL2A1B ● ● ● ● ● ● ● ZAP70 % detected ● BCL2A1D ● ● ● ● ● ● ● ● ● ● ● TNFSF8 ● ● ● 25% ● ANGPTL2 ● ● ● ● ● ● ● ● ● ● CD200 ● 50% 150 ● ● ● ● ● ● ● SLC29A1 ● ● ● ● ● ●● MARCKSL1 ● 75% ● ● ● ● ● NHP2 ● ● ●● ● IER2 ● ● ● ● ● ● ● ● ● ● ●● SRSF2 ● ● ● ● ● ● ●● ● ● ● ● ● ● ● RANBP1 ● ● ● ● ● ● ● DDX21 ● ● ● ● ● ● ● ● 100 ● ● NAB2 ● ● MAF CYCS ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● NFKBID ● ● ● ● ● NR4A1 ● ● ● ● ● ● ● ● ● ● ● ● ●● ICOS EGR1 BCL2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● EGR2 ● ● ● ● ● ● ● ● ● ● ● KDM6B ● ● ● ● ●● ● ● ● ● ● PDCD1 ● ● ● ● ● ● ● ORAI1 50 ● CCR7 ● ● ● ● ● ● ● ŦNQI $QPHGTTQPKŦCFLWUVGFR ● ● ●● SLC1A5 ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● RILPL2 ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● HIVEP3 ●● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●● ● ● ● TAGAP ● ● ●●●●● ●● ● ●●● ●● ● ● KLF2 ● ●● ● ●●● ● ● ● ● ● ● ● ● ● ●● ● ●●●● ● REL ● ● ● ●● ●●●●●●●● HIF1A ●● ● ● ● ●●● ●● ●●●●● ● ● ● ● ● ●●● ●●●●●●●●●●●●●●●●●●●● ● ● ● ● ●● ● ● ● ● ●●●●●●● ●●● DUSP2 ● ● ● ● ● ●●●●●●●●●●●●●●●●●● ●● ● ● ●●● ●●●● ● ●●●●●●●●●●●●●●●●●●● ● CRIP1 ● ● ● ●●● ● ●● ● ●●●●●●●●●●●●●●●●●● ● NFKBIA ● ● LEF1 ●●● ●●●● ●● ●●●●●●●●●●●●●●●●● ● ● 0 ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● POU2F2 ● ● ● ● ● CXCR5 BCL2 ● ● ● ● ● 0 1 MS4A4B ● ● ● ● ● logFC Se2 vs. Se4 SATB1 ● ● ● ● ● CCR7 ● ● ● ● ● KLF2 ● ● ● ● ● NQI(%6(*XUPQPŦ6(* ARL4C ● ● ● ● ● S1PR1 ● ● ● ● ● Ŧ Ŧ 0.0 0.5 1.0 SELPLG ● ● ● ● ● EMP3 ● ● ● ● ● Se_7_Tfh Se_3_Tfh other Se_4_Treg Se_2_Treg.Tfr

Droplet-bsed SC-RNA-seq and population RNA-seq (A) Gating strategy for 10x SC-RNA-seq. Wild-type mice were immunized with NP-OVA/CFA(s.c.) for 7days before cells from draining lymph nodes were sorted based on as CD4+CD19-CXCR5+PD-1+ with permissive gating on CXCR5 and PD-1. (B) Gating strategy for Tfh/Tfr population RNA-seq. Three Foxp3-IRES-eGFP reporter mice were immunized. Foxp3- Tfh cells were sorted as CD4+CD19-CXCR5+PD-1+Foxp3- and Tfr cells were sorted as CD4+CD19-CXCR5+PD-1+Foxp3+. (C) Volcano plot showing differentially expressed genes between Tfh and Tfr cells from population RNA-seq data (D) Heatmap showing soluble molecules differentially elevated in Tfr cells (E) Genes that are differentially expressed in a comparison of the four highlighted clusters to the other cells (grey in Fig.1 a) or in comparisons between the highlighted clusters. For each gene, its mean expression and the ratio of non-zero detections in each cluster were computed, and the means were subsequently scaled to [0,1] range for visualization purposes. (F) A volcano plot of differentially expressed genes in a comparison of Se2 vs. Se4, colored by the log fold-chage (logFC) of a comparison between Se3 and Se7 vs. the “other” cluster.

Fig. S3

A Anti-IgM Condition

***

PBS

Fgl2

C B LPS Condition Anti-CD40 Condition

D E ! PBS ** * Fgl2

NP-OVA KLH NP-OVA KLH NP-OVA KLH KLH NP-OVA

Effects of Fgl2 on B cell viability (A) – (C) Sorted total B cells (CD19+CD4-) from wild-type mice were cultured under different conditions with or without Fgl2 treatment. Viable cells were analyzed by FACS. In (A), proliferation was measured using CFSE. (D) Viability of B cells from Tfh/Tfr/B co-culture assay measured by FACS (E) Viability of B cells from Tfh/B co-culture assay from mice immunized with either NP-OVA/CFA or KLH/CFA for 7 days with or without Fgl2 treatment. The results are representative of 3 independent experiments. Differences between two groups were compared using two-tailed unpaired T tests (n.s; * p <0.05, ** p<0.01 and *** p<0.001). All plots show the means ± SD. Fig. S4

LPS+IL-4 condition LPS+IFN condition

PBS Fgl2 PBS Fgl2 PBS Fgl2

B cells from non- immunized mice IgE IgG1 IgG2b

B cells from NP- OVA/CFA- immunized mice

CD19 B

C IgG1 IgE IgG2b 600 *** 400 * 1000

300 800 400 600 200 400 Bead MFI Bead MFI 200 Bead MFI 100 200

0 0 0 PBS Fgl2 PBS Fgl2 PBS Fgl2 PBS Fgl2 PBS Fgl2 PBS Fgl2

Fc Block Fc Block Fc Block

Fgl2 directly influences CSR in B cells (A) FACS plots showing intracellular staining of corresponding Ig isotypes in sorted total CD19+CD4- B cells under cytokine-polarizing conditions. The sorted cells were cultured in the presence of LPS with or without recombinant Fgl2 for 4 days. (B) The culture media samples from (A) were analyzed for secreted Ig using cytometric bead array assay. (C) Secreted Ig from the culture media with or without Fc Block using cytometric bead array assay. Fig. S5

A 24hrs 6hrs 512 ** PBS 256 256 PBS Fgl2 128 128 * 64 Fgl2 64 * 32 * 32 16 16 8 8 ** 4 4

.d.n

.d.n .d.n .d .d 2 scale) (log2 pg/mL

. .n

.d. . 2 pg/mL (log2 scale) (log2 pg/mL

n 1 d.n

n 1 0.5 IL-5 IL-2 IL-4 IFN-g IL-10 IL-21 IL-13 IL-5 IL-2 IL-4 IL-17A IFN-g IL-10 IL-21 IL-13 IL-17A 96hrs 48hrs 72hrs ** 32768 ** 32768 32768 ** PBS * PBS ** PBS * Fgl2 Fgl2 Fgl2 1024 1024 ** * ** * * 1024 * * 32 32 **

32 1 1

.d.

.d.n

. pg/mL (log2 scale) (log2 pg/mL pg/mL (log2 scale) (log2 pg/mL

.d.n

.d.n pg/mL (log2 scale) (log2 pg/mL

d.n

n 0.03125 0.03125 1 IL-5 IL-2 IL-4 IL-5 IL-2 IL-4 IFN-g IL-10 IL-21 IL-13 IFN-g IL-10 IL-21 IL-13 IL-17A IL-17A IL-5 IL-2 IL-4 IFN-g IL-10 IL-21 IL-13 IL-17A B Bcl6 Maf Cxcr5 Icos Prdm1 800 ** * * 6000 *** 4000 200 * 1000 800 600 3000 150 4000 600 400 2000 100 400 2000 200 1000 50 200 Relative Expression Expression Relative Relative Expression Expression Relative Relative Expression Expression Relative Relative Expression Expression Relative Relative Expression Expression Relative

0 0 0 0 0

Ex-vivo Ex-vivo Ex-vivo Ex-vivo Ex-vivo Cultured Cultured Cultured Cultured Cultured

C D 2000 0.1408 1000 15000 5000 200 5000 WT Il10 800 4000 4000 -/- 1500 150 Fgl2 Dock7 10000 600 3000 3000 4930452B06Rik 1000 100 FPKM FPKM FPKM FPKM Cc2d2a 400 2000 FPKM FPKM 2000 5000 500 50 Mctp1 200 1000 1000 Gnaq 0 0 0 0 0 0 Kif18a Il21 Il4 Itgal Sh2d1a Icos Cd40lg 2 Cd83 10000 10000 6000 0.0662 15000 0.0197

Prdm1 1 scoreZ 8000 8000 Il1r2 0 4000 10000 6000 6000 Gtpbp8 -1 FPKM Rapgef5 FPKM 4000 4000 FPKM FPKM -2 2000 5000 Axl 2000 2000 Cxxc5 0 0 0 0 Setx Cxcr5 Pdcd1 Btla Slamf6 Psme4 2500 250 0.0486 2500 4000 Ngly1 2000 200 2000 Galnt11 3000

Kras 1500 150 1500 2000 FPKM FPKM FPKM 1000 100 FPKM 1000 1 2 3 0 0 0 02 03 - - _0 -/ _001 -/- _0 -/ _0 1000 WT_0 WT_0 WT l2 2 l2 500 50 500 Fg Fgl Fg 0 0 0 0 Bcl6 Ascl2 Tox Tox2

Effects of Fgl2 on Tfh cells (A) Sorted Tfh cells were cultured under plate-bound anti-CD3/CD28 with Fgl2 or PBS control. Culture media samples were collected at different time points and cytokines were analyzed by LegendPlex. (B) Taqman gene expression analysis on ex vivo Tfh cells vs. Tfh cells after the culture as in (A) for 96 hours. The results are representative of 2 independent experiments. Differences between two groups were compared using two-tailed unpaired T tests (n.s; * p <0.05, ** p<0.01 and *** p<0.001). All plots show the means ± SD. (C) Heatmap summarizing the top differentially-expressed comparing WT vs Fgl2-/- Tfh cells. (D) Normalised expression (FPKM:fragments per kilobase of exon model per million) of selected Tfh-related and effector genes in both groups. Fig. S6

A B C Gated on CD19+CD4-

WT Tfh + B WT Tfh + B + Fgl2 IgG+ B cells

5 Secreted IgG1 105 10 5.66 0.019 4 10 * 500 n.s. 104 10 ** ** 3 8 400 * 103 10 : IgG1 :

ns IgG1 :

6 300 0 0

2 3 4 5 0 102 103 104 105 0 10 10 10 10 4 MFI 200 : CD19 : CD19

Prdm1-/- Tfh + B Prdm1-/- Tfh + B + Fgl2 2 100

5 5

% of CD4-CD19+ cells CD4-CD19+ of % 10 10 0 0 0.77 0.022 4 10 104

3 10 103 : IgG1 : : IgG1 :

0 0 WT Tfh + WT B WT Tfh + WT B

IgG1 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 WT Tfh + WT BFcgr2b-/- + Fgl2 Tfh + WT B WT Tfh + WT BFcgr2b-/- + Fgl2 Tfh + WT B : CD19 : CD19 Fcgr2b-/- Tfh + WT B + Fgl2 Fcgr2b-/- Tfh + WT B + Fgl2 CD19 D E F G Secreted IgG1 + IgG1 B cells n.s. + Secreted IgG1 IgG1 B cells 30 *** 8 1500 *** 6000 ***

6 1000 20 4000 4

Bead MFI Bead 500 10 Bead MFI Bead

Bead MFI Bead 2000 2

0 % of total CD19+CD4- cells CD19+CD4- total of % 0 0 0

WT Tfh + WT B WT Tfh + WT B WT Tfh + WT B WT Tfh + WT B WT Tfh + WT BPrdm1-/- + Fgl2 Tfh + WT B Prdm1-/- Tfh + WT B Prdm1+/- Tfh + WT B WT Tfh + WT B + Fgl2 WT Tfh + WT B + Fgl2 WT Tfh + WT BPrdm1+/- + Fgl2 Tfh + WT B Prdm1-/- Tfh + WT B + Fgl2 Prdm1-/- Tfh + WT B + Fgl2 Prdm1+/- Tfh + WT B + Fgl2 Prdm1+/- Tfh + WT B + Fgl2 H Gated on CD19-CD4+ IgG1 + B cells K 8 I 50 0.0019

40 6

30 4

FPKM 20 2 % of IgG1+ B cells 10 0 0 Il10 WT Tfh + + + + TIM-3

PD-1 WT B + + + + CD4 WT Fgl2 - + - + CD4 -/- J Fgl2 anti-IL-10 - - + + 150 1500 Secreted IgG1

100 1000

50 Bead MFI 500

0 mRNA normalized to control 0 il10

TIGIT TIGIT WT Tfh + + + + LAG-3 WT B + + + + CD4 CD4 Tfh+PBS Tfh+Fgl2 Fgl2 - + - + anti-IL-10 - - + +

Effects of Fgl2 on TFH cells in the context of B cell presence -/- (A) – (B) Wild-type TFH cells or Fcgr2b TFH cells were co-cultured with wild-type B cells from immunized mice in cells in the presence of soluble ĮCD3 and ĮIgM with or without Fgl2 for 3 days. IgG1 in B cells was detected by flow cytometry (A) and secreted IgG1 in the media was detected by bead-based mouse immunoglobulin isotyping kit (B).

(C) – (E) IgG1+ B cell (C,D) and secreted IgG1 in media (E) from wild-type B cells co-cultured with control TFH cells fl/+ - -/- fl/fl + (Prdm1fl/fl or Prdm1 with CD4Cre ) or Prdm1 (Prdm1 with CD4Cre ) T FH cells with or without Fgl2 for 3 days.

(F) – (G) IgG1+ B cell (F) and secreted IgG1 in media (G) from wild-type B cells co-cultured with control TFH cells fl/fl fl/+ - +/- fl/+ + (Prdm1 or Prdm1 with CD4Cre ) or Prdm1 (Prdm1 with CD4Cre ) T FH cells with or without Fgl2 for 3days.

(H) Wild-type TFH cells were co-cultured with wild-type B cells from immunized mice in cells in the presence of soluble soluble ĮCD3 and ĮIgM with or without Fgl2 for 3 days. Protein expression was detected by flow cytometry for PD-1, TIM-3, LAG-3 and TIGIT. (I) FPKM of IL-10 from Tfh from wild-type or Fgl2 -/- mice. (J) Relative expression of IL-10 of Tfh treated with PBS or Fgl2. (K) Percentage of IgG1+ B cells and MFI of secreted IgG1 from culture media with or without Fgl2 and anti-IL-10 blockade antibody. Fig. S7 B C A MFI 1000 2000 3000 4000 5000 0

% of CD19+CD4- cells PI Bacteria IgA-coated 0 1 2 3

1 MFI 1000 2000 3000 4000 5000 WT were comparedusingtwo-tailed unpaired T tests(n.s and*p<0.05). plotsshowthemeans ± All results in(B)arerepresentative of3independentexperiments.Differences betweentwogroups arerepresentativeof2independentdetectionsindifferentThe resultsin(A) cohorts. The and cytometricbeadarrayassayrespectively. bacteriaandfreefecalIgAFecal IgA-coated (B) FACS from3-month-oldmicewere analyzedby (A) Total serumantibodyisotypesin8-week-oldmicemeasuredbycytometricbeadarrayassay homeostaticstateorimmunized conditionFgl2-/- micein SD. IgM unpaired T tests. plots showthemeans±SD. All independent experiments. Differences betweentwogroups werecomparedusingtwo-tailed nodes wereanalyzedforGC Bcells, Tfh cellsand Tfr cells. The resultsarerepresentative of4 (E)Wild-typeof Fgl2-/-micewereimmunizedwithNP-OVA(C) – for10days anddraininglymph 0 1 WT GC B cells

ns 1 1 Fgl2 IgG1

1 -/- Fgl2-/- WT IgA

MFI 1 1000 2000 3000 0

Fgl2 1 Fgl2-/- WT g D -/- IgA Gated onCD4+CD19-CXCR5+Bcl6+Foxp3 Gated onCD4+CD19-CXCR5+Bcl6+Foxp3 MFI 2000 4000 6000

1 0

1 Fgl2-/- WT WT

MFI IgG2a 1000 2000 3000 MFI 0 1000 1500 500

0 1

1 1 Fgl2 Total IgA Total Fgl2-/- WT IgG3 -/- * + -

MFI 10000

1 2000 4000 6000 8000 1 0

1 Fgl2-/- WT E IgG2b Fgl2-/- WT MFI 100 110 70 80 90

1 IgE Fgl2-/- WT

1 Fgl2-/- WT