GW0742 Treated DCs. (C) Gram-negative stimulated DCs were cultured after being carefully washed with allogenic PBLs (ratio 1:20) for 7 days. The of proliferating cells was measured by CFSE dilution using flow cytometry. Significant allo-response inhibition of E. coli dex-DC (inhibition 28 1379592 ; p,0.05) compared to control DCs. IFN-c secretion was analyzed in the supernatant by standard ELISA. Results represent the mean and standard deviation of three independent donors. Student’s t-test: *p,0.05, **p,0.001. doi:10.1371/journal.pone.0052456.gintermediate CD80, CD83, CCR7, MHC class I and MHC class II expression. The high levels of CD86 on DCs can be explainedby the presence either of human serum or steroids in the culture [37]. Indeed, dexamethasone has been shown to increase CDTolerogenic Dendritic Cells Response to BacteriaFigure 6. Gram negative E. coli induces tolerogenic activation on Tol-DCs. DCs were carefully washed to eliminate cytokines and dexamethasone at day 7, and viable DCs were further re-challenged with E. coli (ratio 1:10) without cytokines or dexamethasone. (A) GSK2126458 tol-DCs (dexTolerogenic Dendritic Cells Response to Bacteriamatured-DCs) produced significant higher levels of IL-10 whereas levels of pro-inflammatory cytokines were very low compared with mDCs or iDCs in response to E. coli (n = 4, from each donor, iDCs, mDCs and tol-DCs were generated in parallel). (B) The production of IFN-c was evaluated in the supernatant of allogenic T cells cultured for 7 days with E. coli stimulated mDCs or tol-DCs. IFN-c production was significantly (p = 0.024) reduced in T cells stimulated with tol-DCs plus E. coli. IL-10 was not detected in any condition (data not included). Student’s t-test: *p,0.05, **p,0.001. doi:10.1371/journal.pone.0052456.gexpression through GILZ (glucocorticoid-induced leucine zipper) induction [38]. Furthermore, interactions involving CD80/86 are needed in order to expand Tregs, as was revealed when Treg expansion was inhibited via the use of CD86-blocking antibodies [39]. CCR7 mediates the migration of peripheral DCs to lymph nodes [40]. Although CCR7 expression is induced on DCs by PGE2 [41], we were unable to detect CCR7 expression in tol-DCs by increasing PGE2 concentration (unpublished results). Our data clearly demonstrate that a phenotypic description alone without functional studies appears insufficient for ascertaining the nature of tol-DCs. Comparisons between different tolerogenic agents have revealed the differences among these so-called tol-DCs [11,33]. The cytokine balance determines the type of T-cell effector response when DC-T cell interaction occurs. Pro-inflammatory cytokines like IL-12p70 and IL-23 were absent in tol-DCs at both the protein and mRNA transcripts levels. Interestingly, levels ofIL-10 in response to maturation stimuli, which is one of the most important anti-inflammatory cytokines having powerful tolerogenic properties, were significantly higher in tol-DCs compared with mDCs. The balance between IL-12/IL-10 might be crucial both for the induction of tolerance and for Th1 inhibition. Tol-DCs exhibited a low stimulatory capacity in an allogeneicmixed leucocyte reaction, as well as skewed T-cell polarization toward an anti-inflammatory phenotype. Importantly, this immunosuppressive function was also observed in autologous settings when superantigen TSST-1 or TT antigens were used as recall antigens. DCs can be manipulated to induce T-cell anergy and regulatory T-cell activity depending.Treated DCs. (C) Gram-negative stimulated DCs were cultured after being carefully washed with allogenic PBLs (ratio 1:20) for 7 days. The of proliferating cells was measured by CFSE dilution using flow cytometry. Significant allo-response inhibition of E. coli dex-DC (inhibition 28 1379592 ; p,0.05) compared to control DCs. IFN-c secretion was analyzed in the supernatant by standard ELISA. Results represent the mean and standard deviation of three independent donors. Student’s t-test: *p,0.05, **p,0.001. doi:10.1371/journal.pone.0052456.gintermediate CD80, CD83, CCR7, MHC class I and MHC class II expression. The high levels of CD86 on DCs can be explainedby the presence either of human serum or steroids in the culture [37]. Indeed, dexamethasone has been shown to increase CDTolerogenic Dendritic Cells Response to BacteriaFigure 6. Gram negative E. coli induces tolerogenic activation on Tol-DCs. DCs were carefully washed to eliminate cytokines and dexamethasone at day 7, and viable DCs were further re-challenged with E. coli (ratio 1:10) without cytokines or dexamethasone. (A) Tol-DCs (dexTolerogenic Dendritic Cells Response to Bacteriamatured-DCs) produced significant higher levels of IL-10 whereas levels of pro-inflammatory cytokines were very low compared with mDCs or iDCs in response to E. coli (n = 4, from each donor, iDCs, mDCs and tol-DCs were generated in parallel). (B) The production of IFN-c was evaluated in the supernatant of allogenic T cells cultured for 7 days with E. coli stimulated mDCs or tol-DCs. IFN-c production was significantly (p = 0.024) reduced in T cells stimulated with tol-DCs plus E. coli. IL-10 was not detected in any condition (data not included). Student’s t-test: *p,0.05, **p,0.001. doi:10.1371/journal.pone.0052456.gexpression through GILZ (glucocorticoid-induced leucine zipper) induction [38]. Furthermore, interactions involving CD80/86 are needed in order to expand Tregs, as was revealed when Treg expansion was inhibited via the use of CD86-blocking antibodies [39]. CCR7 mediates the migration of peripheral DCs to lymph nodes [40]. Although CCR7 expression is induced on DCs by PGE2 [41], we were unable to detect CCR7 expression in tol-DCs by increasing PGE2 concentration (unpublished results). Our data clearly demonstrate that a phenotypic description alone without functional studies appears insufficient for ascertaining the nature of tol-DCs. Comparisons between different tolerogenic agents have revealed the differences among these so-called tol-DCs [11,33]. The cytokine balance determines the type of T-cell effector response when DC-T cell interaction occurs. Pro-inflammatory cytokines like IL-12p70 and IL-23 were absent in tol-DCs at both the protein and mRNA transcripts levels. Interestingly, levels ofIL-10 in response to maturation stimuli, which is one of the most important anti-inflammatory cytokines having powerful tolerogenic properties, were significantly higher in tol-DCs compared with mDCs. The balance between IL-12/IL-10 might be crucial both for the induction of tolerance and for Th1 inhibition. Tol-DCs exhibited a low stimulatory capacity in an allogeneicmixed leucocyte reaction, as well as skewed T-cell polarization toward an anti-inflammatory phenotype. Importantly, this immunosuppressive function was also observed in autologous settings when superantigen TSST-1 or TT antigens were used as recall antigens. DCs can be manipulated to induce T-cell anergy and regulatory T-cell activity depending.
