By 24 h, 4 or 7 days after injury to the gastrocnemius muscle or after sham treatment, OVA was injected into the hind footpads of mice that had received CFSE-labelled T-cells from naive DO11.10 mice 24 h earlier. Three days after OVA administration, single-cell suspensions of pLNs were pooled per group and stained against CD4, KJ1-26, CD25 and CD69. (A) Overview of the experimental design. (B) Representative dot plot of pLN cells. OVA-specific Th-cells were identified as CD4⁺KJ1-26⁺ cells. The number indicates the percentage of OVA-specific Th-cells in the total population of pLN cells. Percentage of (C) CD25⁺ and (D) CD69⁺ cells among gated CD4⁺KJ1-26⁺ OVA-specific Th-cells. Bar graphs show mean ± S.D. of three experiments each with n=3 mice per group. (E) Histograms showing the proliferation of gated OVA-specific Th-cells according to the dilution of CFSE and are representative of three independent experiments. (F) pLN cells were restimulated with pOVA and the concentrations of IFN-γ, IL-2 and IL-10 in the supernatants were determined. Data show mean ± S.D. of triplicate cultures from pLN cells and are representative of three experiments each with n=3 mice per group. Statistical differences between sham-treated mice and injured mice were determined with Student's t-test. *P<0.05; **P<0.01.