A peptide, designated human CDR1 (hCDR1), that is based on the CDR1 of an anti-DNA Ab ameliorates systemic lupus erythematosus (SLE) in murine models via the induction of CD4(+)CD25(+) regulatory T cells (Tregs). In the present study, the involvement of CD8 Tregs in the mode of action of hCDR1 was investigated in SLE-afflicted (NZB x NZW)F1 mice and in SJL mice following immunization with the lupus-inducing anti-DNA mAb that bears a common Id, 16/6Id. Treatment with hCDR1 up-regulated Foxp3-expressing CD8(+)CD28(-) Tregs in association with clinical amelioration of lupus manifestations. Furthermore, the in vivo depletion of the latter cells diminished the clinical improvement and the inhibitory effects of hCDR1 on the secretion of IFN-gamma and resulted in the up-regulation of IL-10. However, the stimulatory effect of hCDR1 on the secretion of TGF-beta was not affected by the CD8 Tregs. In the absence of CD8 Tregs, CD4(+)CD25(+) Tregs were unable to expand in the hCDR1-treated mice, and the expression of Foxp3 was reduced, thereby interfering further with the suppressive function of CD4(+)CD25(+) Tregs as determined in the in vitro assays. However, CD8 cells from hCDR1-treated mice that were adoptively transferred into SLE-afflicted mice led to up-regulation of CD4(+)CD25(+) cells with intensified Foxp3 expression in the recipient mice. Thus, a functional link between two subsets of Tregs is demonstrated in which CD8(+)CD28(-) Tregs are required for both the optimal expansion and function of lupus ameliorating hCDR1-induced CD4(+)CD25(+) Tregs.