Figure 4

Indirect alloactivation of unsensitized and sensitized CD4⁺T cells by autologous APCs in the presence of sonicates derived from allogeneic EC, fibroblasts or RPTEC. PBMC were cultured either alone on plastic or with confluent monolayers of IFN-treated EC, fibroblasts or RPTEC for 7days. Subsequently, CD4⁺ T cells were isolated from cultures by positive selection and, after a 5day rest period, were used as responders with irradiated autologous APCs in the presence of sonicate. Sonicate was derived from IFN-treated EC, fibroblasts or RPTEC, as indicated, and the identical stimulator cell was used in primary and secondary cultures. Panel A, proliferation of unsensitized (white bars) and sensitized (black bars) CD4⁺ T cells as assessed by [³H] thymidine incorporation for the last 18h of a 6day culture. Results are representative of at least 3 different experiments performed in triplicate 1SD using different donors of PBMC and different EC, fibroblasts and RPTEC. Panel B, representative ELISpot analysis of IFN, IL-4, IL-2 and IL-5 following 48h culture of unsensitized or sensitized CD4⁺ T cells with sonicate derived from allogeneic EC. The results are representative of 3 different experiments. Similar cytokine production was found following indirect alloactivation of T cells by fibroblasts and RPTEC, but the response in general was less pronounced than that found using EC sonicates (not shown)