FIGURE 2.

Regulatory function of CD4⁺ subsets sorted by CD25 and LAP cell surface markers in vitro. A, Proliferation of CD4⁺ populations sorted by CD25 and LAP cell surface markers. Sorted CD4⁺CD25⁻LAP⁺, CD4⁺CD25⁺LAP⁺, CD4⁺CD25⁻LAP⁻, or CD4⁺CD25⁺LAP⁻ cells (1 × 10⁵) were cultured together with irradiated (3000 rads) syngeneic splenocytes in the presence of 1 μg/ml anti-CD3, and proliferation was assessed by scintillation counting after a pulse with [³H]thymidine for the last 16 h of a 72-h incubation period. Data are presented as means ± 95% confidence intervals for the mean. B, Suppressive function of CD4⁺ populations sorted by CD25 and LAP cell surface markers. The proliferation of CFSE-labeled responder CD4⁺ CD25⁻LAP⁻ cells was analyzed by FACS after being cultured with sorted CD4⁺CD25⁻LAP⁺, CD4⁺CD25⁺LAP⁺, CD4⁺CD25⁻LAP⁻, or CD4⁺CD25⁺LAP⁻ cells for 72 h. C, Transwell assay. Responder CD4⁺CD25⁻LAP⁻ cells (1 × 10⁵) were stimulated with anti-CD3 alone or in the presence of an equal number of sorted CD4⁺CD25⁻LAP⁺, CD4⁺CD25⁺LAP⁺, CD4⁺CD25⁻LAP⁻, or CD4⁺CD25⁺LAP⁻ cells, either in direct contact or separated by a transwell inset (Transwell) as described in Materials and Methods. Number next to each bar, percentage of suppression. Data are representative of at least two independent experiments and are presented as means ± 95% confidence intervals for the mean. D, IFN-γ production of responder CD4⁺CD25⁻LAP⁻ cells in vitro. CD4⁺CD25⁻LAP⁻ responder cells were cultured with CD4⁺CD25⁺LAP⁺ or CD4⁺CD25⁺LAP⁻ populations in the presence of anti-CD3 Ab (1 μg/ml) and irradiated (3000 rad) syngeneic splenic APCs for 60 h, IFN-γ production by responder cells were then determined by intracellular cytokine staining. Data were expressed as the percent suppression of IFN-γ production.