Fig 2: Effect of electroacupuncture on lymphocyte subsets and inflammatory cytokines in sedentary mice after ischemic stroke. (a) Representative fluorescence activated cell sorting plots of F4/80+CD11b+ macrophages gated on CD45+ cells, CD4+ cells gated on CD45+CD3+ cells, CD8+ cells gated on CD45+CD3+ cells, and CD3-CD49b+ cells gated on CD45+CD49b+ cells in the peripheral blood. Bar graph showing the percentage of F4/80+CD11b+ cells in CD45+ cells [F (2,10) = 9.48, P = 0.008] (b), CD4+ cells in CD45+CD3+ cells [F (2,10) = 10.24, P = 0.008] (c), CD8+ cells in CD45+CD3+ cells [F (2,10) = 6.05, P = 0.013] (d), the ratio of CD4+ to CD8+ cells [F (2,10) = 30.25, P < 0.001] (e), and CD3-CD49b+ cells in CD45+CD49b+ cells [F (2,10) = 35.34, P < 0.001] (f). (g) Electroacupuncture significantly increased the level of IL-1β in the ischemic hippocampus. (h) Electroacupuncture significantly increased the level of IL-6 in the ischemic hippocampus. (i) Electroacupuncture significantly increased the level of TNF-α in the ischemic hippocampus. All values are presented as means ± SD. *P < 0.05 and **P < 0.01 as determined by one-way ANOVA (Tukey’s multiple comparison test, n = 4). ANOVA, analysis of variance.

Fig 3: M1-type macrophages are increased in HTX-offspring’s spleen of both sexes, while M2-type macrophages and NK cells remain similar among experimental groups. Splenic innate immune cells were quantified by flow cytometry (see Materials and Methods). (A) Representative dot-plots of CD68-positive cells, (B) The graph shows the absolute number of CD68-positive cells, (C) Representative dot-plots of CD206/CD163 double-positive cells, (D) the graph shows the absolute number of CD206/CD163 double-positive cells, (E) Representative dot-plots of NK1.1/CD49b double-positive cells, and (F) the graph shows the absolute number of NK1.1/CD49b double-positive cells. N = 15 per group and sex. Data are presented as mean ± S.E.M. Multiple comparisons between experimental groups and both sexes were analyzed by Mixed-effects model and Tukey’s post-test. (***p<0.001, ****p<0.0001). Control-offspring: blue circles, HTX-offspring: red squares, and HTX+T4-offspring: green triangles.

Fig 4: Effect of electroacupuncture on lymphocyte subsets and inflammatory cytokines in sedentary mice after ischemic stroke. (a) Representative fluorescence activated cell sorting plots of F4/80+CD11b+ macrophages gated on CD45+ cells, CD4+ cells gated on CD45+CD3+ cells, CD8+ cells gated on CD45+CD3+ cells, and CD3-CD49b+ cells gated on CD45+CD49b+ cells in the peripheral blood. Bar graph showing the percentage of F4/80+CD11b+ cells in CD45+ cells [F (2,10) = 9.48, P = 0.008] (b), CD4+ cells in CD45+CD3+ cells [F (2,10) = 10.24, P = 0.008] (c), CD8+ cells in CD45+CD3+ cells [F (2,10) = 6.05, P = 0.013] (d), the ratio of CD4+ to CD8+ cells [F (2,10) = 30.25, P < 0.001] (e), and CD3-CD49b+ cells in CD45+CD49b+ cells [F (2,10) = 35.34, P < 0.001] (f). (g) Electroacupuncture significantly increased the level of IL-1β in the ischemic hippocampus. (h) Electroacupuncture significantly increased the level of IL-6 in the ischemic hippocampus. (i) Electroacupuncture significantly increased the level of TNF-α in the ischemic hippocampus. All values are presented as means ± SD. *P < 0.05 and **P < 0.01 as determined by one-way ANOVA (Tukey’s multiple comparison test, n = 4). ANOVA, analysis of variance.

Fig 5: In vivo anti-tumor efficacy and immune evaluation(A) Schematic illustration of the in vivo treatment procedure.(B) Average tumor growth curves of each group.(C) H&E and Ki-67 staining of tumor sections (scale bar: H&E, 200 μm; Ki-67, 100 μm).(D) Immunofluorescence staining of NLRP3, C-Cas-1, and GSDMD-N in tumor sections (scale bar: 100 μm).(E) Schematic illustration of the immune evaluation procedure.(F–M) Flow cytometry analysis of CD80+ CD86+ DCs in CD11c+ cells (F), CD8+ CD3+ T cells in CD45+ cells (G), CD4+ CD3+ T cells in CD45+ cells (H), IFN-γ+ CD8+ T cells in CD3+ CD45+ cells (I), CD49b+ NK cells in CD3− CD45+ cells (J), the ratio of M1 to M2 macrophages (K), Foxp3+ CD4+ Tregs in CD3+ CD45+ cells (L), and Gr-1+ CD11b+ MDSCs in CD45+ cells (M).(N) ELISA assay for the detection of the cytokines TNF-α, IL-1β, IFN-γ, and IL-12p70 within the tumor.(O) Schematic illustration of the treatment and evaluation procedure in combination with VX-765.(P) Average tumor growth curves of each group.(Q–V) Flow cytometry analysis of DCs (Q), CD8+ T cells (R), CD4+ T cells (S), NK cells (T), MDSCs (U), and Arg-1+ MDSCs (V).(W) Schematic illustration of the treatment procedure in combination with αLy6G/Ly6C (Gr-1).(X) Average tumor growth curves of each group.(Y) Immunofluorescence staining of CD8 and CD4 in tumor sections (scale bar: 100 μm).The data were presented as mean ± SD from n = 5. Statistical significance was calculated by two-way ANOVA comparison test in (B) by day 10 and in (P) and (X), one-way ANOVA with Dunnett’s comparison test in (F)–(M), and with Tukey’s comparison test in (Q)–(V).