CD4+ T cell stimulatory capacity of modified peptides.

<p>CD4+CD62L+ splenic T cells (naïve T cells) were purified from 2D2 TCR transgenic mice (A), DO11.10 transgenic mice (B) or Marylin TCR transgenic mice (C). (A,B,C) The proliferative response of naive cells was assayed after 72 h in cocultures with mitomycin C-treated T cell-depleted splenocytes loaded with indicated peptide concentrations. ³H-thymidine was added for the last 12 h of culture. Error bars represent 1 SD. Data representative of three experiments. (D) Conjugate formation. C57BL/6 T cell-depleted splenocytes were stained with DIOC18, preloaded with 1 µM of either natural sequence or peptide modified as indicated, and cultured with CellTrace DDAO-SE far red stained CD4+CD62L+ T cells from Marylin transgenic mice (1/1 ratio). At indicated time-points, cells were gently resuspended, fixed for 10 minutes and analysed by flow cytometry. The percentage of cells forming conjugates was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045366#pone-0045366-g001" target="_blank">Figure 1C</a>. Error bars represent 1 SD. Two tailed <i>p</i> values are derived from unpaired t tests. Data representative of two experiments. (E) Same experiment as in (D), with CD4+CD62L+ T cells from either 2D2 TCR or DO11.10 mice. Conjugates formation was analysed after 30 minutes of culture. Data representative of two experiments. Two tailed <i>p</i> values are derived from unpaired t tests. Error bars represent 1 SD. (F) Kinetics of CD3 or TCR downregulation. CD4+CD62L+ T cells purified from 2D2 transgenic mice were stimulated with T cell depleted splenocytes loaded with 50 µM natural sequence or modified peptide containing a CxxC motif. At indicated time points, cells were resuspended, stained with anti-CD4 and anti-CD3ε antibodies followed by analysis by flow-cytometry. Data set is representative of two experiments.