8, A and B)
8, A and B). Related results were obtained using TUNEL assays in which we noted that activated primary T cells were more sensitive to apoptosis compared with nonactivated T cells. It is noteworthy that when we cultured the nonactivated T cells in the presence of syngeneic DCs and TCDD, we noted apoptosis in T cells (Fig. 1, B and C). However, we noted highest levels of apoptosis in T cells when activated T cells were cultured in the presence of syngeneic DCs and TCDD (Fig. 1, B and C). It should be noted that in these assays, the lowest concentration at which TCDD brought on significant levels of apoptosis was 10 nM. We did not observe apoptosis in nonactivated or activated T cells with or without DCs when 1 nM TCDD was used in the culture (data not presented). These data exhibited that primary peripheral T cells become more susceptible to TCDD in vitro when they are activated with ConA and especially in the presence of mature syngeneic DCs. Open in a separate window Fig. 1 Effect of TCDD on induction of apoptosis in activated and nonactivated primary T cells in vitro. Purified primary T cells from C57BL/6 mice were cultured in the presence of ConA (activated) or absence (nonactivated) for 24 h followed by LAMB2 antibody an additional (±)-WS75624B culture for 24 h with TCDD. In some cultures, mature syngeneic DCs, generated from bone marrow of C57BL/6 mice, were added to these cultures along with 10 to 1000 nM TCDD or vehicle (DMSO), and (±)-WS75624B the cultures were incubated for an additional 24 h. Apoptosis in T cells was determined by staining the cells with PE-anti-CD3 Abs and FITC-dUTP. The PE+ gated cells were analyzed for apoptosis using flow cytometry (Cytomics FC 500; Beckman Coulter). Data are depicted as viable cellularity (A) and apoptosis as determined by TUNEL assays (B and C). In A and C, the following comparisons made using ANOVA test and Tukey-Kramer multiple comparisons test between various groups were statistically significant ( 0.05): vehicle versus TCDD-treated nonactivated T cells (a versus e, i, m, and q), vehicle versus TCDD-treated nonactivated T cells + DCs (b versus f, j, n, and r), vehicle versus TCDD-treated activated T cells (c versus g, k, o, and s), and vehicle versus TCDD-treated activated T cells + DCs (d versus h, l, p, and t). In addition, the following comparisons made using Students test between different groups after 10 to 1000 nM TCDD treatment were also statistically significant ( 0.05): nonactivated T cells versus nonactivated T cells + DCs (e versus f, i versus j, m versus n, and q versus r), activated T cells versus activated T cells + DCs (g versus h, k versus l, o versus p, and s versus t), nonactivated versus activated T cells (e versus g, i versus k, m versus o, and q versus s), and nonactivated T cells + DCs versus activated T cells + DCs (f versus h, j versus l, n versus p, and r versus t). In B, the top percentage (±)-WS75624B data represent the actual percentage of apoptosis obtained in each histogram, and the bottom percentage data represent the percentage of apoptosis seen after subtracting the background apoptosis in vehicle-treated cells. In A and C, data from five impartial experiments were analyzed and depicted as mean S.E.M. B is usually a representative experiment showing the histograms. TCDD Caused Apoptosis in Antigen-Specific Activated T Cells Normally, professional antigen-presenting cells (APCs) such as DCs present the processed antigens to T cells during an antigen-specific T-cell response. We therefore addressed whether during such T-cell DC conversation, presence of TCDD would promote apoptosis in T cells. To this end, we used purified T cells from OT.II.2a mice, and we cultured them in the presence of mature syngeneic DCs in the absence or presence of specific agonist ovalbumin peptide (Ova323-339: ISQAVHAAHAEINEAGR).