CD4⁺CD25⁺ T cells suppress B-cell proliferation. (A) Preactivated, but not fresh, CD4⁺CD25⁺ T cells suppress LPS-primed B-cell proliferation in the presence of anti-CD3. B cells (5 × 10⁴) from C57BL/6 mice were stimulated with LPS (3 μg/mL) and cocultured with the graded numbers of freshly explanted CD4⁺CD25⁺ T cells (▴, ▵), or preactivated CD4⁺CD25⁺ T cells (▪, □) in the presence (▪, ▴) or absence (▵, □) of soluble anti-CD3 (2 μg/mL). Note that preactivated CD4⁺CD25⁺ T cells cultured in the presence of LPS and anti-CD3, but in the absence of B cells, incorporated little if any radioactivity (○). *P < .01 between the absence and presence of anti-CD3 with preactivated CD4⁺CD25⁺ T cells. The statistical analysis was based on the Student t test. (B) The suppression of B-cell proliferation is specific to CD4⁺CD25⁺ T cells. B cells were stimulated with LPS (3 μg/mL) and cultured with irradiated activated CD4⁺CD25⁺ T cells (▪, □) or irradiated activated CD4⁺CD25^– T cells (▴, ▵) in the presence (▴, ▪) or absence (▵, □) of soluble anti-CD3. *P < .01 between the CD4⁺CD25⁺ and CD4⁺CD25^– cells in the presence of anti-CD3. (C) Activated CD4⁺CD25⁺ T cells suppress B-cell proliferation in response to restimulation with HA peptide. B cells stimulated with LPS were cultured with activated CD4⁺CD25⁺ T cells from HA-TCR transgenic mice in the absence (□) or presence (▪) of HA peptide (8 μM), or in the presence of anti-CD3 (▾). ³H-TdR incorporation was measured after the cells were pulsed for 6 hours during a total of 72 hours of culture. *P < .01 between the absence and presence of HA peptide with preactivated CD4⁺CD25⁺ cells. Results are expressed as the mean of triplicate cultures and are representative of at least 3 experiments.