Figure 6. CD4⁺ CD25⁺ T lymphocytes generated by combination therapy exert dominant tolerance and block autoimmune diabetes after adoptive transfer.

(A) Adoptive transfer of 5 × 10⁶ CD4⁺ T cells from nondiabetic donors, either nontreated RIP-LCMV mice (control, n = 5 mice) or mice treated with the anti-CD3 alone (n = 4 mice) or in combination with the proinsulin peptide (n = 6 mice). A 3-day antigenic stimulation of splenocytes derived from protected mice was performed for each group (treated as well as control) to expand the proinsulin-specific Treg population prior to adoptive transfers. The putative Tregs were transferred into nonimmunodeficient RIP-LCMV-NP recipient mice day 5 after LCMV infection. (B) CD4⁺CD25⁺ and CD4⁺CD25^– T cells were purified from treated RIP-LCMV mice protected by the anti-CD3 alone (lower panel) or in combination with the proinsulin peptide (upper quadrant). Those cells (10⁶) were transferred into nonimmunodeficient RIP-LCMV-NP recipient mice 5 days after LCMV infection (n = 6 mice per group). (C) Proliferation of CD4⁺CD25⁺ lymphocytes in the spleens and PLNs of RIP-LCMV recipient mice. CD4⁺CD25⁺ T lymphocytes were purified from treated RIP-LCMV mice protected by the anti-CD3 alone (left panels) or in combination with the hpIIp (right panels). These cells were CFSE labeled and transferred into wild-type RIP-LCMV recipient mice day 5 after infection. Spleens and PLNs were removed from recipients 2 days after transfer and stained with a CD4-specific antibody. The data are representative of 2 independent experiments. Numbers correspond to the mean ± SD of CFSE-diluted CD4⁺CD25⁺ lymphocytes (n = 2 mice per group).