The acquired MHC II/peptide complexes by T cells can be recognized by fresh responder T cells with the same antigen specificity.

(A) DAP.3-H2-A^k transfectant either pulsed with (DAP3[H2-A^k/HEL]) or without (DAP3[H2-A^k]) HEL_46-61 were used to stimulate IL-2 production by fresh 3A9 responder cells. (B) 3A9 cells were cocultured with CFSE-labeled DAP.3 H2-A^k transfectants pulsed with or without HEL_46-61 peptide. 3A9 populations that had acquired H2-A^k (3A9^II) or H2-A^k:HEL_46-61 complexes (3A9^Iip) and 3A9 cells cultured in medium only (3A9) were isolated as described in “Materials and methods,” fixed, and used to stimulate IL-2 production by fresh responder cells. 2 × 10⁵ fixed 3A9-APCs were cocultured with fresh 3A9 responders. After 24 hours' incubation, 50 μL supernatant was harvested. IL-2 activity in the supernatant was measured by CTLL2 bioassay. (C) DO11.10 CD4⁺ T cells were cocultured with CFSE-labeled BALB/c DCs that had been pulsed either with or without OVA_323-339peptide to allow MHC class II acquisition. After overnight coculture, CFSE-negative DO11.10 CD4⁺ T cells were sorted and used as APCs. The CFSE-negative DO11.10 CD4⁺ T-cell populations that had acquired H2-A^d (■) or H2-A^d/OVA_323-339 complexes (▴) were γ-irradiated to stimulate proliferation of fresh responder cells. DO11.10 CD4⁺ T cells cultured in medium only (♦) and peptide-pulsed DCs (○), cell numbers corresponding to 1% of the number of DO11.10^IIp cells used in the same experiment, were used as controls. (D) The purity of the isolated cells was assessed by flow cytometry. CPM indicates counts per minute. Error bars indicate SD of triplicates in the same experiment.