Figure 6.

CD4⁺CD25⁺ T cells induced by oral tolerance are anergic and suppressive in vitro. T/B monoclonal mice were administered OVA in drinking water and immunized with OVA-HA as represented in Figure 1A. Ten days after immunization, spleen and mLN cells were collected and pooled for the sorting of CD4⁺CD25⁺ and CD4⁺CD25^– populations. Purified CD4⁺CD25^– and CD4⁺CD25⁺ cells from Tol T/B monoclonal mice (Tol) or from BALB/c mice were stimulated in vitro with anti-CD3 antibodies and APCs (2 × 10⁴ CD4 cells + 4 × 10⁴ APC cells/well). For coculture experiments, 2 × 10⁴ CD4⁺CD25^– BALB/c cells (responder cells) were stimulated with anti-CD3 antibodies and APCs in the presence of putative suppressor cells from Tol or BALB/c mice at responder/suppressor ratios of 1.0:1.0, 1.0:0.3, and 1.0:0.1. Proliferation was determined by ³H-thymidine (³H-TdR) incorporation. Results are expressed as mean ± SD of triplicate wells.