Fig. 3.

ApoVs directly interact with T cells via phosphatidylserine to inhibit TCR signaling. (A) Representative images of flow cytometric analysis of CD11b expression on CD11b-DTR mice treated with or without diphtheria toxin (DT). (B) Frequency of CD25⁺CD4⁺ T cells in circulation and spleen of CD11b-DTR mice at 24 h after treatment with or without apoVs. N = 3 per group. (C–E) The experimental scheme, representative flow cytometric analyses and quantification of CD25 expression from naïve CD4⁺ T cell cultures that were unstimulated (Unstim) or anti-CD3/CD28 stimulated with or without 0.2x apoVs. N = 3 per group. (F) Activation fold change of naïve CD4⁺ T cells stimulated in the presence of PBS (Ctr), 0.2x apoVs (ApoVs) or 0.2x annexin V (A5) pre-treated apoVs. N = 4 per group. (G) IL-2 levels in supernatants of naïve CD4⁺ T cells stimulated in the presence of PBS (Ctr), 0.2 x apoVs (ApoVs) or 0.2 x annexin V (A5) pre-treated apoVs. N = 4 per group. (H, I) Representative flow cytometric analyses and quantification of CD25 expression on CD4⁺ T cells from splenocyte cultures that were unstimulated (Unstim), anti-CD3/CD28 stimulated in the presence of PBS (Stim), apoVs, exosome or soluble fraction of apoVs (sApoVs). N = 3–4 per group. (J) Frequency of CD25⁺CD4⁺ T cells measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of 1x apoV (1 apoV: 1 T cell) derived from BMMSCs or T cells from C57bl6 (WT) and MRL/lpr (LPR) mice. N = 3–6 per group. (K) Frequency of activated CD4⁺ T cells in healthy human peripheral blood mononuclear cells (PBMC) measured in vitro after 3 days with or without anti-CD3/CD28 antibody stimulation in the presence of different doses of apoVs derived from mouse BMMSCs. N = 3–5 per group. (L) Representative immunofluorescence staining of CD3⁺ T cells and PKH26-labelled apoVs with or without A5 pre-treatment. Scale bar = 10 μm. (M) Frequency of apoVs⁺ T cells after 15, 30 and 60 min of coculture measured by immunofluorescence staining. N = 3 per group. (N) Frequency of apoVs⁺ T cells after 60 min of coculture with or without A5 pre-treatment measured by immunofluorescence staining. N = 3 per group. (O) Western blotting shows expression of proximal TCR signaling proteins p-CD3, p-ZAP70, p-LCK, p-PLCγ and NFAT1 in naïve CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies in the presence of PBS (Ctr), 0.2 x apoVs (ApoVs) or 0.2 x annexin V (A5) pre-treated apoVs for 1 h. Mann-Whitney test and student's t-test was used for comparison between two groups when appropriate. Kruskal-Wallis test and ANOVA was used for comparison among three groups when appropriate. Data are shown as mean ± standard deviation. **, p < 0.01. ***, p < 0.001. ****, p < 0.0001.