CD4+CD25+ cells regulate CD8 cell anergy in neonatal tolerant mice

Background. Injection of neonatal BALB/c mice with semi-allogeneic splenocytes leads to antigen-specific tolerance lasting into adulthood. Tolerant mice accept A/J skin grafts and fail to generate CD8 cytotoxic T lymphocyte (CTL) activity against A/J targets. Anergic CD8 T cells are present in tolerant mice, and CD4 regulatory cells function to maintain CD8 cell anergy. Methods. Neonatal BALB/c mice were injected with 10(8) live CAF(1) splenocytes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD8 cell proliferation was measured by in vitro incorporation of bromodeoxyuridine coupled with fluorescence-activated cell sorter analysis. Alloantigen-specific cytotoxicity was tested using Cr-51 release assays of A/J or third-party targets. Results. We demonstrate that A/J-specific anergic CD8 cells are present in neonatal primed mice that develop tolerance but not in neonatal primed mice that reject A/J skin grafts. Anergic CD8 cells show decreased proliferation and no CTL activity against A/J targets. Addition of interleukin-2 (IL-2) to unfractionated cultures fails to restore CTL activity against A/J targets. However, addition of IL-2 60 CD4-depleted cultures restores A/J-specific CD8 CTL activity. Removal of CD4(+)/CD25(+) cells, but not CD4(+)/CD25(-) cells, also restores CD8 CTL activity against A/J in the presence, but not the absence, of IL-2, Moreover, when added back into cultures, purified CD4(+)/CD25(+) cells from tolerant mice inhibit the generation of CD8 CTL against A/J targets. Conclusion. These data indicate that CDS anergy is associated with the state of tolerance, and that CD4(+)CD25(+) cells from tolerant mice function to maintain A/J-specific CD8 cell anergy in vitro.