CD80 and PD-L2 define functionally distinct memory B cell subsets that are. Griselda V Zuccarino-Catania, Saheli Sadanand, Florian J Weisel, Mary M

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1 Supplementary Figures CD8 and PD-L define functionally distinct memory B cell subsets that are independent of antibody isotype Running title: Memory B Cell Subset Function Griselda V Zuccarino-Catania, Saheli Sadanand, Florian J Weisel, Mary M Tomayko, Hailong Meng, Steven H Kleinstein, Kim L Good-Jacobson, & Mark J Shlomchik Nature Immunology: doi:.8/ni.91

2 Isolate splenic B Cells and transfer NP-CGG in alum i.p. Wait 8 weeks for memory NP-specific Mouse Source of naive B cells (B1-8 +/- Jk +/- KI BALB/c) Irrelevant BCR Recipient (AM1 Tg Vκ8R BALB/c) Memory Mouse with NP-specific MBCs (AM1 Tg Vκ8R BALB/c) Supplementary Figure 1. Experimental design for the generation of MBC subsets using a transfer system. One million NP-specific splenic B cells were transferred into AM1 Tg Vκ8R recipient mice and immunized with NP-CGG in alum. 8 week after immunization these memory mice have NP-specific splenic MBCs. Zuccarino-Catania, et al. Nature Immunology: doi:.8/ni.91

3 a b CD19 CD8 NIP CD9.6e- NIP CD19 Supplementary Figure. Antigen-specific germinal center cells are not found in mice at a memory timepoint. Flow cytometry analysis of splenic cells from a mouse that had received B cells and (a) had been immunized 8 weeks prior or (b) had not been immunized. Left shows gating on Ag-specific B cells (CD19+NIP+) after gating on live cells and right shows staining for germinal center cells (CD9+CD8 ). Numbers indicate percentage of the parent gated cells in the indicated population. Data are from one mouse from one experiment, representative of three independent experiments. Zuccarino-Catania, et al. Nature Immunology: doi:.8/ni.91

4 Supplementary Figure. Example MBC subset staining at 6 weeks post-immunization. Cell transfers, immunizations, and staining were exactly as described in the manuscript. FMO shows the fluorescence minus one staining controls. Right panel is a control of naïve B cells. Nature Immunology: doi:.8/ni.91

5 a b. NIP 1 IgM 1 NIP CD19 IgG1 IgG Supplementary Figure. Expression of IgM, IgG1 or IgG on MBCs. (a) Flow cytometry analysis of MBC IgM or IgG1 isotype expression in memory mice assessed in live splenocytes gated as CD19 + NIP +. Numbers adjacent to outlined area indicate percent gated population. (b) Flow cytometry analysis of splenic cell from memory mice that are CD19 + NIP + IgG +. Numbers adjacent to outlined area indicate percent gated population. Data are from five pooled mice from one experiment, representative of three independent experiments. Zuccarino-Catania, et al. Nature Immunology: doi:.8/ni.91

6 a Nature Immunology: doi:.8/ni.91

7 b Color Key Expression Intensity Naive DN SP DP SIAT7C A7M7R CCND CCBP 911BRI PDLIM1 ASS1 CKB VIM MYADM SGK1 AHNAK RASSF ANXA CD86 ZFP88 SLPI IGH VJ8 FER1L MEFB UHRF1 EDG LOC866 CSRP LRRK BIRC PBK MKI67 119HRI IGHG ZBTB SEMAF FSCN1 SA6 CD8 PRY1 DPP LOC6689 IGHV1S6_M1 A9HRI SLA KCNK MCM SHDA HIST1HAF HIST1HAD HIST1HAH CD7 BCL6 EPHX1 SATB1 BLVRB IGHV1S1_AF IGHV1S_M1 KLF EMP LOC6778 MYL KLHL6 HIST1HAO EG66778 Nature Immunology: doi:.8/ni.91

8 Supplementary Figure. Transcriptional analyis of MBC subsets. (a) Principal component analysis (PCA) of the same dataset, showing that DN, DP, and SP memory B cells differ from each other transcriptionally, but are more related to each other than they are to naive B cells. Analysis was performed on gene expression profiles of most variable probes. DN, DP, SP memory B cell samples and naive B cell samples are projected on the first two PCs, which account for 79% of the variance. (b) Heatmap of differentially expressed genes of the DP subset relative to DN subset. Each column within the labeled cell category is an independent replicate. Genes annotated as transcription factors are colored red. Nature Immunology: doi:.8/ni.91

9 a 8 Replacement Mutations 6 b 8 DN SP DP CDR Replacement Mutations 6 DN SP DP Supplementary Figure 6. Mutation distribution in V regions of MBC of different subsets. Memory B cells prepared and sorted as described in the manuscript were obtained 8 weeks post-infection (DN= double negative, n=6; SP= single positive, n=71; DP= double positive, n=7). Their L chains were sequenced as described. Shown are the distributions of numbers of (a) total Replacement and (b) CDR Replacement mutations for each subset. Each dot is a separate sequence. Nature Immunology: doi:.8/ni.91

10 a rested effector T cells B cells - Sorted splenic MBC subsets - Naive B cells (control) Secondary Recipient Irrelevant BCR (AM1 KI Vκ8R BALB/c) Day -1 Sort splenic B cells & Transfer cells Day Immunize with NPOVA in alum Day. Analyze for early AFCs Day. Analyze for GCs & early AFCs b + IL- CD KJ1-6 Day 8 Rested effector T cells Nature Immunology: doi:.8/ni CD PD-1 CD CD PD-1 CD CD CD Supplementary Figure 7 Zuccarino-Catania, et al. 9. Rested effector T cells T 6 KJ1-6 T Day Activated T cells 8 Rest alone for days 7. CD # Cells OVA-peptide (-9) 6 CD6L Day Naive T cells CD6L T APC KJ1-6 Irradiated APCs DO11. OVA-specific T cells 71. CD6L Culture for days 71. CD 1 PD-1 1

11 Supplementary Figure 7. Transfer system to evaluate secondary responses and system to generate rested effector T cells. (a) sorted MBCs, or naïve B cells, and rested effector T cells (unless otherwise indicated) are adoptively transferred into recipient mice. One day after transfer, mice are immunized with NP-OVA in alum and harvested at day. post immunization to evaluate early AFC generation or at day. post immunization to evaluate GC B cell generation and AFC formation. (b) Rested effector T cells are generated in vitro by culturing OVA-specific T cells with irradiated antigen presenting cells (APCs) in the presence of IL- and OVA-peptide for four days, followed by culture for four additional days without IL- and OVA-peptide. Flow cytometry analysis of CD, CD, CD6L or PD-1 expression by CD + KJ1-6 + OVAspecific EMA - cells at day, and 8 of culture. Numbers indicate percent of live cells in the indicated gate. Data are from one mouse from one experiment, representative of three independent experiments. Zuccarino-Catania, et al. Nature Immunology: doi:.8/ni.91

12 NP/NP16 (NP + IgG1 + AFCs) ** **** ** DP SP DN IgG1 + Naive No B IgG1 Supplementary Figure 8. AFCs derived from DP IgG1 neg MBCs had the highest relative affinity for NP early after secondary immunization. ELISPOT analysis of NP -specific versus NP 16 -specific IgG1 + splenic AFCs. days post immunization with NP-OVA in alum after transfer of: DP, SP or DN IgG1 MBCs; IgG1 + MBCs; naïve B cells or no B cells. Molar ratios of NP to bovine serum albumin were two (NP -BSA) or sixteen (NP 16 -BSA) as indicated for the capture Ag. ** p<.1, **** p<.1. (Mann Whitney nonparametric, two-tailed test). Data are combined from two independent experiments (error bars represent standard deviation) with three to fifteen mice per group. Zuccarino-Catania, et al. Nature Immunology: doi:.8/ni.91

13 TCR-β + CD+ T Cells (per spleen).7 CD ** 1 *** Memory B cells ** Naive B cells **** No B cells *** d CD TCR-β + CD+ (%) c b 11.9 TCR-β TCR-β a DP DN IgG1 IgG1+ Naive No B DP PBS SP DN IgG1+ Naive No B IgG1 Anti-CD Memory B cells ** 1. 7 Naive B cells *** ** 1. 7 No B cells **** ***. 6. DP DN IgG1 IgG1+ Naive No B PBS DP SP IgG1 DN IgG1+ Naive No B Anti-CD Supplementary Figure 9. Anti-CD Antibody Treatment Depletes Recipient T Cells. Recipient mice were treated with anti-cd Ab or PBS as a control before transfer of B cells. Frequency of CD (RM-)+ TCR-β + T cells in recipient mice treated with (a) anti-cd Ab or (b) PBS. (c) Frequency or (d) number of CD (RM-)+ TCR-β+ T cells after transfer of DP, SP or DN IgG1 MBCs; IgG1+ MBCs or naïve B cells in recipient mice. days post immunization with NP-OVA in alum. Four to twelve mice per group for PBS treated mice, and five to thirteen mice per group for anti-cd treated mice. * p<., ** p<.1, *** p<.1, **** p<.1. (Mann Whitney nonparametric, two-tailed test). Data are combined from two independent experiments (error bars represent standard deviation). Zuccarino-Catania, et al. Nature Immunology: doi:.8/ni.91

14 Supplementary Figure. Quantitative PCR analysis of the expression of zbtb, plk1, cdc and mcm within MBC subsets. MBCs (PI, CD19 +, NIP + ) were sorted from NP-CGG immunized AM1 Tg BALB/c Vκ8R KI BALB/c transfer recipients based on differential expression of CD8 and PD-L (n=, week ). Naive B cells (PI, CD19 +, NIP +, CD9 ) were sorted from B1-8i +/ BALB/c mice. Results were normalized to GAPDH expression and relative changes in gene expression, compared to naive B cells, were calculated using the (-delta delta Ct) method. Error bars are SEM of technical duplicates. Nature Immunology: doi:.8/ni.91