Figure 7. Immunological effects of hPLA2G1B on mouse CD4⁺ T cells in vitro and in vivo.

(A–G) FACS analysis of the effect of hPLA2G1B on mouse CD4⁺ T cells after anti-CD3/CD28 and IL-2 stimulation (5 days, see gating strategy on Supplemental Figure 6C). (A–E) mCD4⁺ T cells were pretreated with WT or H48Q hPLA2G1B. (A) CD25 expression after treatment with 125 nM hPLA2G1B. (B) CD25 expression (MFI) and (C) cell survival (n = 3, 10 mice). (D) mCD4⁺ T cell proliferation profile after treatment with 125 nM hPLA2G1B. (E) Percentage of live mCD4⁺ T cells per cell generation (Go to G5; n = 3, 9 mice). (F and G) Effects of mAb anti-PLA2G1B 14G9 in vitro treatment on 125 nM hPLA2G1B action on CD4⁺ T cell survival and CD25 expression (n = 4, 11 mice). (H–L) In vivo effects of hPLA2G1B on CD4⁺ T cell response to IL-7. Spleen CD4⁺ T cells were isolated after intraperitoneal injection into C57BL/6 mice and the ex vivo p-STAT5 NT response to IL-7 was evaluated by confocal microscopy, with an average of 200 cells examined for each condition. Effect of hPLA2G1B injection at several doses of PLA2G1B for 3 hours (H, 6 mice, 2 experiments) and at several times after injection (I, 3 mice, 1 experiment; J, 8 mice, 2 experiments). (K) Effects of mAb anti-hPLA2G1B 14G9 injected in vivo on the hPLA2G1B (100 μg, 3 hours) response (5 mice, 1 experiment). (L) Inhibition of the effects of hPLA2G1B after injection into hPLA2G1B/BSA–immunized mice (5 mice, 1 experiment). Results are shown as the mean ± SEM (B, C, and E–G) or mean ± SD (H–L). *P < 0.05; **P < 0.01; ***P < 0.001 adjusted for multiple comparisons by Kruskal-Wallis test P < 0.001, followed by the Mann-Whitney test (B) and 2-way ANOVA with correction for multiple comparisons by Tukey’s (C, H, and J–L), Dunnett’s for the condition without PLA2G1B as a control group (E), or Sidak’s (F, G, and I) post hoc test.