Figure 3

Phenotype of splenocytes from double-transgenic mice cultured with peptides. (A) CD4+ splenocytes from double-transgenic mice cultured with peptide A2, developed a CD62L^hi CD62L^lo memory phenotype, whereas CD4+ cultured with A12 peptide or media alone did not change (%CD44^hiCD62L^loCD4+ T cells = 18 ± 3 for No Ag, 23 ± 5 for A12, and 47 ± 6 for A2; P ≤0.002 for A2 versus No Ag and P ≤0.006 for A2 versus A12). Splenocytes from the double-transgenic mouse cultured with peptide A2 or A12 had no significant differences in numbers of Treg cells (%CD25^hiFoxp3^hiCD4+ T cells = 8 ± 2 for No Ag, 9 ± 2 for A12, and 9 ± 3 for A2). (B) Phenotype of splenocytes from double-transgenic mice tested directly ex vivo: α1(II) was administered in vivo (100 μg/mouse intravenously) alone, or with 100 μg peptide A12. Three days later, CD4+ T cells treated with α1(II) developed a CD62L^hi CD62L^lo memory phenotype, whereas A12 peptide significantly decreased expression of activation markers (%CD44^hiCD62L^loCD4+ T cells = 22 ± 7 for A12 + α1(II)-treated mice, and 37 ± 6 for α1(II)-treated mice; P ≤0.02 for α1(II)-treated versus α1(II) + A12 treated). Staining for intracellular Foxp3 was not different between groups tested directly ex vivo (15 ± 7 for α1(II)-treated mice versus 13 ± 5 for α1(II) + A12-treated mice. Negative control (naïve double-transgenic T cells) had the pattern: (%CD44^hiCD62L^loCD4+ T cells = 16 ± 4 and Foxp3 + CD4 T cells = 12 ± 5). Data representative of three separate experiments (A and B).