Figure 9.

Functional assessment of the suppressive activity of in vivo isolated CD4⁺ CD25⁺ spleen T cells from CD6^−/− and CD6^+/+ mice. (A) Density plot histogram showing the fluorescence intensity of conventional CD4⁺CD25⁻ (T_conv) and regulatory CD4⁺CD25⁺ [regulatory T cells (T_reg)] spleen T cells from CD6^+/+ mice subjected to further FACS sorting. (B) Contour plot and percentages (mean ± SD) of FoxP3 expression in sorted CD4⁺CD25⁺ T cells 5 weeks after chronic graft-versus-host disease induction. (C) Flow cytometry analysis and percentages (mean ± SD) of latency-associated peptide (LAP) surface expression on CD4⁺ gated CD25⁺FoxP3⁺ T cells from spleens of untreated CD6^−/− and CD6^+/+ mice. (D) Sorted T_conv cells from untreated CD6^+/+ mice were cocultured at a 2:1 ratio with irradiated bm12 splenocytes alone or in the presence of sorted T_reg from CD6^−/− (solid circles) or CD6^+/+ (empty circles) mice at week 5 post GvHD induction, added at the indicated T_conv:T_reg ratios. Shown is the [³H]-thymidine incorporation (in cpm) after 5 days of coculture. (E) Left, density plot histogram showing the sorting strategy to isolate B220⁺ cells from CD6^+/+ mice (E) Right. Sorted spleen B220⁺ cells from wild-type (CD6^+/+) mice were CFSE-labeled and cultured for 3 days in the presence of F(ab’)₂ anti-IgM fragments (10 µg/mL) alone or plus sorted CD4⁺CD25^hi (T_reg) splenocytes from CD6^−/− (solid circles) or CD6^+/+ (empty circles) mice, at the indicated B:T_reg ratios. The line chart show the percentage (mean ± SD) CFSE^low cells from a representative experiment of two performed. Data are mean ± SD of triplicate samples from one representative experiment of four performed. *p < 0.05; **p < 0.01 (Student’s t-test).