Figure 3. Treatment with a combination of anti-CD3 and hpIIp increases the number of CD4⁺ T cells with a regulatory phenotype.

The data are from euglycemic animals treated with anti-CD3 (n = 5 experiments with 3–5 mice per experiment) or with the combination therapy (n = 5 experiments with 3–5 mice per experiment). As control, nontreated animals were used (n = 4 experiments with 4–6 mice per experiment). (A) The percentage of CD4⁺CD25⁺ cells is shown. (B) After intracellular staining, the percentage of CD4⁺Foxp3⁺ cells was evaluated. For each group, the mean ± SD is given, with P = 0.016 for the combination treatment group and P = 0.089 for the NM anti-CD3–treated group as compared with the untreated control group (None). (C) CD4⁺CD25⁺ cells expressed high levels of Foxp3. For each sample, cells were lysed in loading buffer containing SDS and β-mercaptoethanol and used in the Western blotting experiment. 5 × 10⁵ purified CD4⁺CD25⁺ and CD4⁺CD25^– cells were obtained from mice protected by treatment with anti-CD3 (α-CD3) alone or in combination with the hpIIp. As positive and negative controls (control+ and control–, respectively), 10⁵ HeLa cells stably expressing or not, respectively, the mouse Foxp3 protein were loaded. Actin was used to evaluate the amount of loaded proteins. (D–I) Long-term protected RIP-LCMV mice were sacrificed and the percentage of Treg markers in pooled splenocytes and PLNs cells determined. The percentages of GITR⁺ and Foxp3⁺ cells was measured in the CD4⁺ population (D and E). The percentage of GITR⁺, Foxp3⁺, CTLA-4⁺, and CD62L⁺ cells were measured in the CD4⁺CD25⁺ population (F–I).