Supplementary Figure 5: B cell intrinsic roles of hyperactivated PI3Kδ | Nature Immunology

Supplementary Figure 5: B cell intrinsic roles of hyperactivated PI3Kδ

From: Hyperactivated PI3Kδ promotes self and commensal reactivity at the expense of optimal humoral immunity

Supplementary Figure 5

a, Related to Figure 5a. Frequency of wild-type OT-II and wild-type OT-II TFH cells (PD-1+CXCR5+CD4+B220) in wild-type hosts together with wild-type (n=8) or mutant (n=7) MD4 B cells immunized i.p. with HEL-OVA 323-339 in alum. b, Schematic experimental layout for panels (c-f). Wild-type OT-II (CD45.1+) and wild-type (n=6) or Pik3cdE1020K/+ (n=8) polyclonal B cells (CD45.2+) were adoptively transferred into MD4 hosts (CD45.1/2+) and immunized i.p. with NP-OVA in alum. Analysis in the spleen on day+8. c, Frequency of transferred polyclonal CD45.2+B220+CD19+ B cells (of live cells). d, Numbers of transferred CD45.2+B220+CD19+GL-7+FAS+ GC B cells. e, Frequency of polyclonal CD138+B220int/lo plasma cells/blasts (of transferred CD45.2+ cells). f, Representative contour plots of frequency of NP+ GC B cells within transferred B cells, histogram of NP+ GC B cells, and ratio between numbers of NP+ and NP GC B cells. g, Analysis of CD86 and CD69 on wild-type and Pik3cdE1020K/+ follicular (FO) MD4 B cells activated in vitro with HEL for 20 h (pool of 2-3 mice per group). h, Analysis of in vitro differentiated plasma cells (BLIMP-1-YFP+CD138+) generated from FO B cells stimulated with LPS and IL-4 or IL-21 for 3 days (gated on live B cells) (pool of 2-3 mice per group). i, Experimental outline of mixed BM chimeras between different ratios (20:80 n=6, 80:20 n=10, 50:50 n=2) of wild-type (CD45.1+) and Pik3cdE1020K/+ (CD45.2+) bone marrow cells, transferred into irradiated wild-type hosts (CD45.1/2+). j, Frequency of B220+CD19+GL-7+FAS+ GC B cells within wild-type and Pik3cdE1020K/+ cells, and live GC B cells (Annexin viability dye). k, FO B cells were kept in vitro without stimulation and analyzed for viability at different time points (wild-type open circles, mutant closed circles). l, m, FO B cells were activated in vitro for 3 days with the indicated stimuli and assessed for CTV dilution and viability. n, FO B cells were stimulated in vitro with LPS+IL-4, treated with or without CAL-101 (PI3Kδ inhibitor) and analyzed for CTV dilution and viability on day+3 (k-n, pool of 2-3 mice per group). Data are representative of 2 (a, b-g, i-k, n), and 3 (h, l, m) independent experiments. Data are expressed as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. **P < 0.01