Figure 4. hpIIp–specific T cells from mice treated with anti-CD3 and i.

. proinsulin peptide exhibit a regulatory cytokine profile after in vitro stimulation. (A) Splenocytes and PLNs from animals treated and cured by the anti-CD3 alone or in combination with the hpIIp were used in vitro in a cytokine secretion assay. Cells were stimulated with a mixture of anti-CD3 and anti-CD28 Abs, various peptides (hpIIp, insB9–23, and LCMV-specific GP61–80 and NP118–126) or remained nonstimulated. After 72 hours, the supernatants were analyzed by ELISA as well as using the Luminex 100 LabMAP System. The data represent the levels of cytokine above the background observed without stimulation (NM-anti-CD3 [IL-4: 17 pg/ml, IL-5: 430 pg/ml, IL-10: 10 pg/ml, TGF-β1: 530 pg/ml, and IFN-γ: 2,500 pg/ml] or combination therapy [IL-4: 41.5 pg/ml, IL-5: 380 pg/ml, IL-10: 200 pg/ml, TGF-β1: 580 pg/ml, and IFN-γ: 3,000 pg/ml]). Data for an average of 4 mice per group are shown. *P < 0.05 compared with the nonstimulated control. (B) To confirm the cytokine profile found in the supernatants, intracellular cytokine stainings were performed. The cells were stimulated specifically with the hpIIp or unspecifically with a mixture PMA/ionomycin or remained unstimulated (nonstimulated) for 6 hours in the presence of an inhibitor of intracellular protein transport (monensin). The cells were recovered and stained at the surface for CD4 and intracellularly for Foxp3 and the cytokines TGF-β, IL-10, and IL-4. The cytokine expression was analyzed in the CD4⁺Foxp3⁺ (upper panel) and CD4⁺Foxp3^neg (lower panel) populations.