Iferation was assessed by the incorporation of Hthymidine. HERsensitized T cells coincubated with HER, tyrosise [tyr]pulsed or unpulsed DCs, specifically proliferated in response only for the HERneu epitope (Table ). ProT and proTmatured DCs showed fairly high imply stimulation indices (S.Is) (. and respectively), comparable to those recorded for TNFmatured DCs. Addition of mAb to MHC class II molecules decreased mean S.Is in all groups (. for TNF;. for proT;. for proT). These results recommend that following our in vitro culture protocol, peptidereactive T cells are generated, which 
proliferate only inside a HERdependent MHC class IIrestricted manner.ProT and proT induce the maturation of DCs via triggering TLRWe have previously reported that stimulation of human monocytes with proT upregulated IRAK, a protein kise involved in TLR downstream sigling, whereas Mosoian et al. showed that proT ligates TLR and sigls via each TRIF and MyDdependent pathways. To identify no matter whether TLR is triggered by our peptides, we studied the kinetics of TLR surface expression on proT and proTstimulated DCs. Immature DCs (iDCs) and DCs maturedwith LPS (a identified TLR ligand; ), proT or proT for min, min, h, h and h have been alyzed by flow cytometry. The percentage of surface TLR expression more than time is presented in Figure. Maturation PubMed ID:http://jpet.aspetjournals.org/content/120/2/261 of DCs with LPS led to an early ( and min) decrease of TLR expression (by ) due to interlization, in addition to a subsequent raise from to h. At h postLPS addition, TLR expression was lower and comparable to that of iDCs ( h). ProT and proT, margilly downregulated TLR expression at min and, similarly to LPS, transiently increased it from to h. As with LPSmatured DCs, basal levels of TLR expression had been detected at h FT011 postmaturation. The similar kinetics of TLR expression in LPS, proTand proTmatured DCs is consistent with all the notion that the two peptides interact with TLR. To extend these findings, we subsequent investigated the intracellular expression levels of 3 adaptor molecules that take part in sigling pathways downstream of TLR, mely TRIF, an adaptor molecule common 
to TLR and sigling; TIRAP, a sigling adaptor widespread to TLR, and ; and MyD, a molecule upregulated upon ligation of all TLRs except TLR. We particularly chosen these 3 adaptors mainly because this constellation is special to TLR activation. Total cell extracts from iDCs and DCs matured with LPS, proT or proT for h and h have been immunoblotted (Figure A). Upon densitometric quantification of each protein band detected, expression relative to GAPDH was calculated. As shown in Figure B, addition of LPS led to a substantial fold enhance with the expression of all three adaptors within h (. for TRIF for TIRAP and. for MyD) relative to iDCs (. for TRIF for TIRAP and. for MyD). At h postaddition ofTable T cells stimulated with proT or proTmatured DCs proliferate in the presence of HERpulsed DCsDCs matured with TNF DCs pulsed with HER tyr HER + KS176 antiMHC class II ProT HER tyr HER + antiMHC class II ProT HER tyr HER + antiMHC class II Mean cpm, S.I. SD from donors.Mean counts per minute (cpm) SD Stimulation index (S.I.) SD………Ioannou et al. BMC Immunology, : biomedcentral.comPage ofFigure Expression of TLR on DCs matured with LPS, proT or pro. iDCs differentiated from human monocytes ( h) were matured with LPS, pro and pro for min, min, h, h and h, and alyzed for TLR surface expression. Data show imply TLR surface expression SD from donors.LPS, expression of all adaptors was decreased and a.Iferation was assessed by the incorporation of Hthymidine. HERsensitized T cells coincubated with HER, tyrosise [tyr]pulsed or unpulsed DCs, specifically proliferated in response only towards the HERneu epitope (Table ). ProT and proTmatured DCs showed comparatively higher imply stimulation indices (S.Is) (. and respectively), comparable to those recorded for TNFmatured DCs. Addition of mAb to MHC class II molecules decreased mean S.Is in all groups (. for TNF;. for proT;. for proT). These benefits recommend that following our in vitro culture protocol, peptidereactive T cells are generated, which proliferate only in a HERdependent MHC class IIrestricted manner.ProT and proT induce the maturation of DCs by way of triggering TLRWe have previously reported that stimulation of human monocytes with proT upregulated IRAK, a protein kise involved in TLR downstream sigling, whereas Mosoian et al. showed that proT ligates TLR and sigls by means of both TRIF and MyDdependent pathways. To identify whether TLR is triggered by our peptides, we studied the kinetics of TLR surface expression on proT and proTstimulated DCs. Immature DCs (iDCs) and DCs maturedwith LPS (a recognized TLR ligand; ), proT or proT for min, min, h, h and h had been alyzed by flow cytometry. The percentage of surface TLR expression more than time is presented in Figure. Maturation PubMed ID:http://jpet.aspetjournals.org/content/120/2/261 of DCs with LPS led to an early ( and min) lower of TLR expression (by ) on account of interlization, in addition to a subsequent increase from to h. At h postLPS addition, TLR expression was lower and comparable to that of iDCs ( h). ProT and proT, margilly downregulated TLR expression at min and, similarly to LPS, transiently increased it from to h. As with LPSmatured DCs, basal levels of TLR expression had been detected at h postmaturation. The related kinetics of TLR expression in LPS, proTand proTmatured DCs is constant together with the notion that the two peptides interact with TLR. To extend these findings, we subsequent investigated the intracellular expression levels of three adaptor molecules that participate in sigling pathways downstream of TLR, mely TRIF, an adaptor molecule widespread to TLR and sigling; TIRAP, a sigling adaptor typical to TLR, and ; and MyD, a molecule upregulated upon ligation of all TLRs except TLR. We particularly selected these 3 adaptors mainly because this constellation is one of a kind to TLR activation. Total cell extracts from iDCs and DCs matured with LPS, proT or proT for h and h have been immunoblotted (Figure A). Upon densitometric quantification of each protein band detected, expression relative to GAPDH was calculated. As shown in Figure B, addition of LPS led to a substantial fold improve of the expression of all 3 adaptors inside h (. for TRIF for TIRAP and. for MyD) relative to iDCs (. for TRIF for TIRAP and. for MyD). At h postaddition ofTable T cells stimulated with proT or proTmatured DCs proliferate in the presence of HERpulsed DCsDCs matured with TNF DCs pulsed with HER tyr HER + antiMHC class II ProT HER tyr HER + antiMHC class II ProT HER tyr HER + antiMHC class II Imply cpm, S.I. SD from donors.Imply counts per minute (cpm) SD Stimulation index (S.I.) SD………Ioannou et al. BMC Immunology, : biomedcentral.comPage ofFigure Expression of TLR on DCs matured with LPS, proT or pro. iDCs differentiated from human monocytes ( h) were matured with LPS, pro and pro for min, min, h, h and h, and alyzed for TLR surface expression. Information show imply TLR surface expression SD from donors.LPS, expression of all adaptors was decreased in addition to a.