Reduced capacity of TE and F<10 KDa to prime allogeneic responses <i>in vitro</i>.

<p>(A) DC from BALB/c mice treated with medium, LPS (1 µg/ml), TE (80 µg/ml) or TE plus LPS for 18 h were co-cultured for 5 days with C57BL/6splenocytes at a 1∶10 ratio in the presence or absence of PMA. Allogeneic response was determined by IFN-γ production by ELISA. (B) DC from BALB/c mice, treated as described in A, without PMA, were cultured with a sorted EGFP negative cell population from splenocytes of Foxp3EGFP mice for 5 days. Dot plots show the CD4 vs CD25 profile of culture cells (upper panel) and the Foxp3 vs CD25 on the CD25 high gated cell population (lower panel). The numbers represent the percentage of Foxp3 positive and negative cells gated on the CD25 high cell population. (C) IL-10 was measured in the supernatant of allogeneic cultures as described in A in the presence of PMA for the last 18 h of culture, p<0.006 versus T/L-DC (ANOVA Test). (D) IL-27 mRNA was measured in DC treated with medium, LPS (1 µg/ml), TE (80 µg/ml) or TE plus LPS for 6 h by RT-PCR or IL-10 was detected in culture supernatant by ELISA after 18 h of culture, p<0.001 versus T/L-DC (ANOVA Test). (E). IFN-γ or IL-17 were detected in the supernatant of DC from BALB/c mice treated with medium, LPS, TE or TE plus LPS for 18 h, and cultured with splenocytes from C57BL/6 at a 1∶10 ratio for 5 days in the presence or absence of anti IL-10 R, anti-IL-27 or the control isotype Abs. Data are representative of three experiments, with p representing a significant difference in the Student's t-test. (F) IFN-γ was measured by ELISA in culture supernatants of splenocytes (C57BL/6) and DC (BALB/c mice), previously pulsed for 18 h with medium, or F<10 kDa (20 µg/ml) in the presence or absence of LPS (1 µg/ml) for 5 days. On day 4, the cells were stimulated with PMA for the last 18 h. (G) DC from BALB/c mice treated with medium or F<10 kDa (20 µg/ml) for 18 h were cultured with a sorted EGFP negative cell population from splenocytes of Foxp3EGFP in the presence or absence of anti- TGF β or anti IL-10R for 5 days. Dot plots show the Foxp3 <i>vs</i> CD25 profile of splenocytes gated on the CD4 population. (H) IFN-γ and IL-17 were detected in the supernatant of cultures as described in F, in the presence or absence of anti IL-27 or anti IL-10R blocking Abs. Data are representative of 2 experiments with similar results. The bar graphs shown one experiment representative of three with the mean ± SD. p represents a significant difference in the Student's t-test.