Background. Injection of neonatal BALB/c mice with semi-allogeneic splenocy
tes leads to antigen-specific tolerance lasting into adulthood. Tolerant mi
ce 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 toler
ant mice, and CD4 regulatory cells function to maintain CD8 cell anergy.
Methods. Neonatal BALB/c mice were injected with 10(8) live CAF(1) splenocy
tes, and mice were deemed tolerant by accepting A/J grafts over 40 days. CD
8 cell proliferation was measured by in vitro incorporation of bromodeoxyur
idine coupled with fluorescence-activated cell sorter analysis. Alloantigen
-specific cytotoxicity was tested using Cr-51 release assays of A/J or thir
d-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 proliferatio
n 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 targe
ts. However, addition of IL-2 60 CD4-depleted cultures restores A/J-specifi
c 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 CD
4(+)/CD25(+) cells from tolerant mice inhibit the generation of CD8 CTL aga
inst A/J targets.
Conclusion. These data indicate that CDS anergy is associated with the stat
e of tolerance, and that CD4(+)CD25(+) cells from tolerant mice function to
maintain A/J-specific CD8 cell anergy in vitro.