CD4+/CD25+ CELLS REGULATE CD8 CELL ANERGY IN TOLERANT MICE

Abstract

947 We previously reported that injection of neonatal BALB/c mice with semiallogeneic CAF1 (BALB/c X A/J) spleen cellns induces antigen specific tolerance that lasts into adulthood in 80% of mice. Neonatal tolerant mice accept fully allogeneic A/J skin grafts for > 60 days, but reject third party C57B6 skin grafts with normal kinetics. Neonatal tolerant mice also show hypoproliferative response to A/J cells, fail to generate CD8 CTL activity in vivo against A/J cells, but contain increased numbers of Th2 memory CD4 cells reactive against A/J stimulator cells. Using in vitro BrdU assays to measure CD8 proliferation, we now demonstrate that A/J specific anergic CD8 cells are present in neonatal primed mice that go on to develop tolerance (NEO-Tol), but not in neonatal primed mice that reject A/J skin grafts (NEO-Rej). The addition of exogenous IL-2 to in vitro cultures of unfractionated spleen cells from NEO-Tol mice fails to restore CD8 cell proliferation to A/J cells or CTL CD8 activity against A/J targets. However, if CD4 cells are removed from the cultures, then exogenous IL-2 restores both the CD8 cell proliferation to A/J and the A/J specific CD8 CTL activity. Therefore, recovery of CD8 CTL effector function of anergic CD8 cells from NEO-Tol mice requires (1) a source of exogenous IL-2 and (2) the removal of CD4 cells from the culture. We further characterized the phenotype of the A/J reactive CD4 cells from NEO-Tol mice. Using four color FACS analysis we determined that stimulation with A/J cells expands a CD25+/CD69− population of CD4+ cells from NEO-Tol mice. When added back into cultures, purified CD4+/CD25+/CD69− cells from NEO-Tol mice inhibit the generation of CD8 CTL against A/J targets, even in the presence of exogenous IL-2. This data indicate that CD4+CD25+ cells from NEO-Tol mice function to maintain CD8 CTL cells in a state of anergy by inhibiting IL-2 rescue of "anergic" CD8 cells.