Figure 3.

Examination of phenotype and suppressive ability of Ts1 cells re-cultured with rIL-12p70 alone or in combination with IL-2. Ts1 cells were washed and re-cultured with alloantigen and either rIL-2 alone, rIL-12p70 alone, or both rIL-2 and rIL-12p70 for 4 days before examination of: (A) CD25 and Foxp3 expression. All three cultures showed that a large proportion of cells were CD25^high and 70–80% expressed Foxp3. (B) Suppressive ability. Cells from all three cultures were tested for ability to suppress naive CD4⁺CD25⁻ T cell proliferation in MLC against PVG (top panel) or third party Lewis (lower panel) stimulator cells. A constant number (1 × 10⁵) of naïve CD4⁺CD25⁻ T cells were cultured with serial dilutions of re-cultured Ts1 cells. Ts1 re-cultured with PVG alloantigen and either rIL-12p70 (●) or rIL-2 and rIL-12p70 (△) had greater suppression compared to those re-stimulated with IL-2 alone (○). Proliferation was suppressed to or below background levels observed with naïve CD4⁺CD25⁻ T cells cultured alone (5.0 ± 1.2 × 10³ cpm) or with syngeneic DA stimulator cells (5.1 ± 2.0 cpm). Ts1 re-cultured with rIL-12p70 showed significant suppression at all dilutions (p < 0.001). Ts1 cells re-cultured with alloantigen and both rIL-2 and rIL-12p70 suppressed at dilutions to 1:256 (p < 0.0001). Ts1 cells re-cultured with rIL-2 and specific alloantigen, only suppressed to 1:32 (p < 0.001) showing no enhancement in suppressive ability of Ts1 cells that suppressed at 1:32–1:64 ratio to naive CD4⁺CD25⁻ T cells, as described (8). Suppression of CD4⁺CD25⁻ T cells response to third party Lewis alloantigen was similar to specific donor PVG. Thus, re-culture of Ts1 cells with specific alloantigen with rIL-12p70 increased their potency to suppress in vitro, whereas re-culture of Ts1 with rIL-2 and alloantigen did not. Similar results in two other experiments.