The emergence of CD4⁺CD8⁻T cells in the PB of the developing mouse correlates with allograft rejection. (A) T cells were measured as a percentage of all PB mononuclear cells after birth at 19 to 21 DPC. (B) CD4 and CD8 expression among PB T cells was then measured at 19 to 21 DPC. Allogeneic transplantation was performed by injecting litters of newborn pups via the facial vein (C) with TCD BM cells at 19 to 21 DPC, with 1 to 3 uninjected pups from each litter euthanized for T-cell analysis (D). As maternal sensitization only occurs with prenatal injection, pups injected postnatally were not fostered. The frequency of macroengraftment (PB chimerism > 1% at 4 weeks of age) among injected animals was correlated with the frequency of CD4⁺CD8⁻ cells among PB T cells in uninjected littermates (E) and the developmental age of the litter (F). (G) Finally, to determine the kinetics of graft rejection in this model, several litters were injected at 20 DPC, and the frequency of mice with chimerism higher than 1% was measured weekly for 4 weeks, demonstrating that the majority of animals reject the allograft between 1 and 3 weeks posttransplant consistent with an adaptive immune response.