Fig. 4

SC MSC injection expands activated Tregs in the LN. (A) CD45⁺Ly6G⁺SSC-A^hi neutrophils, Ly6C^lo/hiCD11b⁺ monocytes, CD11c⁺MHCII⁺ DCs and CD11b⁺MerTk⁺ macrophages were purified from the LNs 24 h after SC MSC injection, and then added to CTV-labelled, anti-CD3/anti-CD28 activated splenic CD4⁺CD25⁻ T cells at 1:5 ratio for 72 h. (B) Flow cytometric profile of CTV dilution (indicating T cell proliferation) in the presence of innate immune cells purified from LN of untreated mice (UNT LN) or LNs from SC MSC-injected mice (MSC LN). (C) Division index of T cell proliferation. Data expressed as mean ± SEM; n = 3 mice per group. (D) Representative flow cytometry plot showing Treg gating. (E) Relative change in Treg proportion in the presence of LN neutrophils, monocytes, DCs or macrophages after standardising to T cells alone group). Data expressed as mean ± SEM; n = 3 mice per group. (F) Draining (dLN; white bar) and non-draining LNs (ndLN; shaded bar) were harvested from mice that received SC injection of viable or apoptotic MSCs into the inflamed hock. Flow cytometry plots showing viability of MSCs treated with DMSO (mock) or BH3 mimetics prior to SC injection. (G) Treg cellularity in the dLN (white bars) and ndLNs (shaded bars) following LPS and SC MSC injection. (H) Changes in the PD-1 expression on Tregs after SC MSC injection, and Pearson correlation analysis of Treg PD-1 expression level and Treg proportion in the dLNs from LPS and MSC groups (Pearson r=-0.861, p = 0.00137). (I) CD44 expression on activated Tregs, and changes in CD44^hi Tregs in dLNs and ndLNs after SC MSC injection. (J) Changes in PD-1 expression on CD44^hiTregs after SC MSC injection, and Pearson correlation analysis of CD44^hi Treg PD-1 expression level and Treg proportion in the dLNs from LPS and MSC groups (Pearson r=-0.883, p = 0.001). Data expressed as mean ± SEM; n = 5 mice per group. One-way ANOVA, Tukey’s multiple comparison, comparing UNT, LPS and MSC dLNs; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (K) Treg cellularity in dLNs and ndLNs following SC injection with ApoMSC. (L) Changes in PD-1 expression on Tregs after SC injection with ApoMSC, and Pearson correlation analysis of Treg PD-1 expression level and Treg proportion in the dLNs from LPS and ApoMSC groups (Pearson r=-0.239, p = 0.392). Data expressed as mean ± SEM; n = 5–10 mice per group. One-way ANOVA, Tukey’s multiple comparison, comparing UNT, LPS and ApoMSC dLNs; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant