Figure 5.

Blocking IFN-γ or iNOS had no effect on alloantigen/IL-12p70 mediated enhancement of suppressive ability of Ts1 cells: Ts1 cells generated by culture of nTreg with PVG alloantigen and rIL-2 for 3 days were re-cultured with PVG alloantigen and either rIL-12p70 alone, rIL-12p70, and anti-IFN-γ (DB-1) or rIL-12p70 and L-NIL. After culture (A) Foxp3 expression was maintained in all cultures with 75–81% expressing both CD25 and Foxp3. The starting nTreg and Ts1 populations had 70–80% Foxp3⁺ with nearly all were CD4⁺CD25⁺. (B) Blocking either IFN-γ or iNOS had no effect on the capacity of Ts1 cells re-cultured with rIL-12p70 and PVG stimulators to suppress naïve CD4⁺ T cell proliferation in MLC against specific PVG stimulators as measured by ³H-thymidine incorporation (cpm ×10³). MLC was significantly suppressed to or below background proliferation of control CD4⁺ T cells with no allogeneic stimulator (1.69 + 1.2 × 10³ cpm). Ts1 cells re-cultured with specific PVG stimulators and rIL-12p70 alone (●) had significantly suppressed proliferation of CD4⁺ T cells to PVG stimulators at all ratios of 1:4 through to 1:1024 (p < 0.02); for Ts1 cells re-cultured with rIL-12p70 and anti-IFN-γ (○), p < 0.015 to 1:512, and p = 0.02 at 1:1024. Ts1 re-cultured with rIL-12p70 and L-NIL (△) significant differences were from 1:4 through to 1:512; p < 0.004 except for 1:64 and 1:128. These results suggested rIL-12p70 directly acted on Ts1 cells, not via induction of IFN-γ and iNOS.