Figure 3.

In vitro generation and functional analysis of spleen regulatory T cells from CD6^−/− mice. (A) Purified CD4⁺ T cells (10⁵ cells/well) from CD6^−/− (solid bars) or CD6^+/+ (empty bars) spleens were cocultured with irradiated bm12 splenocytes (bm12^#) at a 2:1 ratio for different periods of time for further flow cytometry analysis. Data are shown as percentage (mean ± SD) of CD25⁺FoxP3⁺ cells on gated CD4⁺ cells from duplicates of one representative experiment of four performed. Control means the results of coculturing responder CD6^−/− or CD6^+/+ cells with irradiated splenocytes from the same responder. (B) Same 72 h cocultures as in (A) in the absence (bm12^#) or the presence of exogenously added IL-2 (+IL-2; 150 pg/mL), blocking anti-CD25 monoclonal antibody (mAb) (+αIL-2, 30 µg/mL) or agonistic anti-CD28 mAb (+ αCD28; 5 µg/mL). (C) Sorted (10⁵ cells/well) conventional CD4⁺CD25⁻ T cells (T_conv) from CD6^−/− (solid bars) or CD6^+/+ (empty bars) spleens were cultured for 3 days with plastic-bound anti-CD3 mAb (2 µg/mL) plus soluble anti-CD28 mAb (5 µg/mL) in the presence or absence of TGF-β (2 ng/mL) alone or plus IL-2 (20 ng/mL). (D) Flow cytometry analysis of latency-associated peptide (LAP) surface expression on CD4⁺ gated CD25⁺FoxP3⁺ T cells generated at 72 h as in (A) (left) or as in (C) (right). Shown is a representative experiment of four performed. n.s., not significant (Student’s t-test).