Fig. 4. Pik3cd^E1020K/+ T cells intrinsically generate more T_FH cells in ICOS independent manner.

a, Outline for (b-c). Wild-type mice (CD45.1⁺) were transferred with CD45.2⁺ wild-type (n=7) or Pik3cd^E1020K/+ OT-II cells (n=6), followed by i.p. immunization with NP-OVA in alum. Day+8 analyses of spleens. b, Percentage of CD45.2⁺ OT-II cells in live cells. c, Flow plots and summary histogram of percentage of PD-1⁺CXCR5⁺ OT-II T_FH cells (of OT-II cells). d, Outline of anti-ICOS-L in vivo treatment: wild-type or mutant OT-II cells were transferred into wild-type hosts, then immunized as in (a). Mice received isotype control (wild-type OT-II n=5, Pik3cd^E1020K/+ OT-II n=4), or anti-ICOS-L (wild-type OT-II n=4, Pik3cd^E1020K/+ OT-II n=5) on day −1 (i.v.), +1, +3, +5 (i.p.) and were sacrificed on day+7. e, Frequency of PD-1⁺CXCR5⁺ OT-II T_FH cells: flow plots and summary histogram. f, p-AKT^Ser473 and p-S6^Ser235/236 on naïve CD4⁺ T cells ex vivo or after 30 min stimulation with anti-CD3 and anti-CD28, after pretreatment with CAL-101 (PI3Kδ inhibitor), or vehicle. Geom. MFI are indicated. g, FACS plots and histograms of p-FOXO1^Ser256 on day+4 in vitro activated wild-type and Pik3cd^E1020K/+ CD4⁺ T cells, rested for 1 h in RPMI, then stimulated with anti-ICOS or left untreated (f,g, cells pooled from 2–4 mice per group). h, FACS plots and histogram of percentages of splenic T_FH PD-1⁺CXCR5⁺ from wild-type (n=5) or Pik3cd^E1020K/+ OT-II cells (n=4) (gated on transduced GFP⁺ cells), and wild-type (n=5) or Pik3cd^E1020K/+ FOXO1^AAA OT-II (n=3) cells (gated on transduced GFP⁺ HA⁺ cells), transferred into wild-type hosts, and analyzed day+5 post i.p. immunization with NP-OVA. Data are representative of three (a-c, g) and two (d, e, f, h) independent experiments. Data in (b, c, e, h) are expressed as mean ± SEM with each dot indicating one mouse. Significance analyzed by Mann-Whitney U test. *P < 0.05; **P < 0.01.