Supplementary Figure 4: T cell intrinsic roles of hyperactivated PI3Kδ. | Nature Immunology

Supplementary Figure 4: T cell intrinsic roles of hyperactivated PI3Kδ.

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

Supplementary Figure 4

(a-b, c, j) Experimental setting described in Figure 4a. a, Representative CD44 histogram on wild-type and Pik3cdE1020K/+ OT-II cells and endogenous CD4+ T cells. b, Representative flow plots and histograms of GC-TFH, pre-TFH and non-TFH cells (gated on transferred OT-II cells in the spleen) (wild-type n=7, Pik3cdE1020K/+ n=6). c, Intracellular staining for IL-21 after in vitro re-stimulation with PMA/Ionomycin on FACS sorted wild-type or Pik3cdE1020K/+ OT-II non-TFH cells (PD1CXCR5CD4+B220) and TFH cells (PD1+ CXCR5+CD4+B220), isolated on day +7 post immunization, as described in Fig. 4a (pool of wild-type OT-II (n=7) and Pik3cdE1020K/+ OT-II cells (n=6)). d, Analysis of IgG supernatant on in vitro culture between wild-type B cells and wild-type or Pik3cdE1020K/+ polyclonal TFH cells stimulated with anti-IgM and anti-CD3 for 7 days (pool of 4-5 mice per group). e, Frequency of endogenous PD-1+CXCR5+ of CD4+ B220 T cells in wild-type mice adoptively transferred with wild-type or Pik3cdE1020K/+ OT-II cells and treated with isotype control (wild-type OT-II n=5, Pik3cdE1020K/+ OT-II n=4), or anti-ICOS-L (wild-type OT-II n=4, Pik3cdE1020K/+ OT-II n=5) (experiment described in Fig. 4d). f, Experiment outline of (g, h). Naïve wild-type and Pik3cdE1020K/+ mice received isotype control (wild-type n=5, Pik3cdE1020K/+ n=5), or anti-ICOS-L (wild-type n=6, Pik3cdE1020K/+ n=5) on day 0 (i.v.), +2, +4, +6 (i.p.) and were sacrificed on day+7 post treatment. g, Representative contour plots and histogram of endogenous PD-1+CXCR5+ T cells (of CD4+B220). h, Histogram of FAS+GL-7+ GC B cells (of B220+CD19+ B cells). i, Analysis of CD69 and CD25 on wild-type and Pik3cdE1020K/+ naïve OT-II cells activated in vitro with CD11c+ DC and OVA323-339 for 20 h (pool of 2-3 mice per group). j, Frequency of FAS+GL-7+ GC B cells (of B220+CD19+ B cells) related to Fig. 4a (wild-type n=7, Pik3cdE1020K/+ n=6). Data are representative of two independent experiments. Data are expressed as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01