Effect of pronase treatment on the proliferative response of CD4⁺CD80^{acq hi} T cells, expression of IE^k and CD80 on P13.9, function of P13.9 APCs, and impact of possibly contaminated splenic APCs on the proliferation of T cells. (A) Naive CD4⁺ T cells were cocultured with PCC-pulsed P13.9 (CD80^high) APCs for 20 hours. After the separation from the APCs, CD4⁺CD80^{acq hi} T cells were further stripped with 0.01% pronase for 12 or 30 minutes. Expression of CD4, CD80, and IEk on the unstripped or pronase-stripped cells was analyzed by 3-color FACS analysis. (B) Level of IEk, CD80, and CD4 on unstripped or pronase-stripped (treatment for 12 and 30 minutes) CD4⁺CD80^{acq hi} T cells, which had been further cultured in the absence of APCs and PCC peptide for 6 hours, was analyzed by 3-color FACS analysis. (C) CD4⁺CD80^{acq hi} T cells that were not stripped (□) or stripped with pronase for 12 minutes (▪) or 30 minutes (▦) were cultured in the absence of APCs and PCC peptide for 6 hours. Proliferative response of these T cells was examined using 3H uptake assay. (D) Naive CD4⁺ T cells were stimulated with PMA (5 ng/mL) plus ionomycin (250 ng/mL) for 20 hours and further treated with 0.01% pronase for 12 minutes (▪) or 30 minutes (▦). □ represents unstripped. Proliferative response of these T cells was examined using ³H uptake assay. (E) P13.9 APCs were treated with 0.01% pronase for 30 minutes, and IEk and CD80 expression on P13.9 APCs was analyzed by FACS analysis. (F) Naive CD4⁺ T cells were stimulated with either PCC-pulsed P13.9 APCs or pronase-treated PCC-pulsed APCs for 24 hours. Proliferative response of these T cells was examined using 3H uptake assay. CD4⁺: naive T cells only; P13.9: P13.9 APCs pulsed with PCC peptide; pronase-treated APCs: P13.9 APCs pulsed with PCC peptide were further treated with 0.01% pronase for 30 minutes; CD4 + P13.9: naive CD4⁺ T cells were stimulated with P13.9 APCs; CD4⁺ pronase-treated P13.9: naive CD4⁺ T cells were stimulated with pronase-treated P13.9 APCs. (G) Purified naive CD4⁺ T cells were treated with different concentrations (100 ng/mL to 1 pg/mL) of PCC peptide for 24 hours. Proliferative response of purified CD4⁺ T cells was examined using 3H uptake assay. Percentages are the percent positive cells in the respective quadrant. Error bars represent mean ± standard deviation (SD).