Analysis of lymphocytes from MBPβ transgenic mice cultured with the AC1-11 peptide.

<p>Total lymphocytes from MBPβ-only transgenic mice were bulk cultured with the MBP-derived AC1-11 peptide. Several additional rounds of activation were done by culturing with T cell depleted B10.PL splenocytes loaded with the AC1-11 peptide. FACS was carried out seven days after the final activation. (<b>A</b>) Nearly all remaining cells were CD4⁺ and expressed the transgene-encoded Vβ8 TCRβ chain. Across several experiments, approximately 30% of the T cells were not Vα2 positive. (<b>B</b>) The Vα2⁺ and Vα2⁻ T cells were sorted and then stimulated by the addition of irradiated, T cell depleted B10.PL splenocytes and titrated amounts of the AC1-11 peptide. Cells were pulsed with 3[H]thymidine after 48 hours and harvested after a total of 72 hours. Approximately 5×10⁴ T cells and 1×10⁵ APCs were used per well. Assays were carried out in triplicate and averaged. Results shown are representative of three independent experiments. (<b>C</b>) Predicted amino acid sequence of the CDR3α region of the Vα6 (TRAV6-7/DV9*02) – Jα13 (TRAJ13*01) TCR cloned from the Vα2⁻ population. Gene nomeclature based on the IMTG Marie-Paule convention <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017702#pone.0017702-Lefranc1" target="_blank">[46]</a>.