CD4 regulatory cells in immune tolerance.

We have used the classic model of neonatal tolerance to investigate the hypothesis that acquired tolerance depends on the generation of regulatory CD4 cells. Injection of neonatal BALB/c mice with semi-allogeneic CAF1 (BALB/c X A/J) spleen cells induces antigen-specific tolerance (TOL) in 80% of mice. TOL mice accept fully allogeneic A/J skin grafts for >60 days. TOL mice show diminished Th1 CD4 and CD8 cell immunity against A/J in vitro. In contrast, TOL mice show increased levels of anti-A/J Th2 CD4 responses. Thus tolerance is associated with the inhibition of Th1 CD4 and TC1 CD8 responses and the enhancement of Th2 CD4 responses. Because of this relationship, we hypothesized that regulatory Th2 CD4 cells in TOL mice maintain tolerance by blocking activation of A/J-reactive TC1-CD8 cells. Using in vitro BrdU assays to measure CD8 proliferation within unfractionated cell cultures, we showed that CD8 cells from TOL mice proliferate normally to exogenous interleukin-2 (IL-2) but fail to proliferate in response to A/J cells. The addition of exogenous IL-2 does not restore CD8 proliferation to A/J, ruling out simple CD8 cell anergy. However, when CD4 cells are depleted from the cultures, IL-2 could restore the ability of A/J-reactive CD8 cells to proliferate and to secrete IFN-gamma. Thus CD4 cells from TOL mice inhibit IL-2 rescue of "anergic" A/J-reactive CD8 cells. The results demonstrate a novel link between two major mechanisms of tolerance, immunoredirection and anergy.