CD4 CELLS MAINTAIN CD8 T CELL ANERGY IN NEONATAL TOLERANT MICE

Abstract

668 Neonatal tolerance occurs via a number of mechanisms, including clonal deletion and immunedeviation. We previously demonstrated that the injection of semi-allogeneic CAF1 spleen cells → neo-natal BALB/c mice(NEO-primed) established A/J specific tolerance (TOL) in 70% of mice. TOL mice accept A/J skin grafts but reject third party grafts, demonstrate negative MLR proliferative responses against A/J cells in vitro, fail to generate IFNγ producing CD8 cells or CD8 CTL responses against A/J in vitro, but develop enhanced Th2 CD4 responses. We examined the possibility that neonatal tolerance involves anergy mechanisms. Using in vitro BrdU assays to measure CD8 subset proliferation, we now demonstrate that CD8 cells from TOL mice show negative MLR proliferative responses to stimulation with A/J-bearing cells, but normal proliferation to either third party B6 or to IL-2 alone. The addition of IL-2 to A/J stimulated cultures failed to restore CD8 proliferation, ruling out the presence of the traditional type of anergic CD8 cell. Interestingly, CD8 cells from TOL mice proliferate more to stimulation with IL-2 alone than to stimulation with IL-2 plus A/J. But the addition of IL-2 to B6 stimulated cultures augments TOL CD8 cell proliferation. In contrast, the addition of IL-2 to A/J stimulated cultures augments proliferation of CD8 cells from either naïve mice or control mice that rejected A/J grafts or NEO-primed mice that rejected A/J grafts (non-TOL). These results suggest that the ability of A/J cells to suppress IL-2 induced proliferation of CD8 cells is related to the tolerant state, and that A/J reactive CD8 cells are present, but suppressed in TOL mice. We speculated that an A/J-reactive CD4 cell maintains tolerance by inhibiting A/J-reactive CD8 cells. To test this possibility we compared the proliferative responses and the effector functions of the CD8 T cells within unfractionated cultures to CD4-depleted cultures. Depletion of CD4 cells alone fails to restore anti-A/J proliferative responses and effector function of CD8 cells from TOL mice. However, the addition of IL-2 to CD4-depleted cultures restores the ability of CD8 cells from TOL mice to proliferate to A/J and to generate anti-A/J specific CTL and IFNγ producing CD8 cells. Thus, tolerogen-reactive CD8 cells are present, but non-functional in neonatal tolerant mice. CD4 regulatory cells may function to maintain tolerance in TOL mice by inhibiting IL-2 rescue of “anergic” CD8 cells. The presence of regulatory CD4 cells may explain why anergy is so difficult to demonstrate in different models of in vivo tolerance.