Fig. 3

Mechanisms of DC-mediated cross-presentation of LA. a Pathway diagram of DC cross-presenting epitopes. b IFN-γ secretion by ELISPOT assay (n = 3). c–f Monocytes were obtained with a monocyte attachment medium (C-28051, PromoCell) from PBMCs and then cultured in DC generation medium A (C21050, PromoCell) to harvest immature DCs. Immature DCs were treated with 50 μg/ml peptide twice with an interval of 24 h. Maturation of YL/LA-pulsed DCs was promoted (n = 3). c Morphological examination. d Presentation ability. e Uptake capacity. f IL-12p70 secretion by ELISA. g LA was mainly taken up by immature DCs through an active transport (37 °C), and others might directly bind to DCs (4 °C). h LA was mainly taken up by phagocytosis (inhibited with cytochalasin D), partly by receptor-mediated internalization (inhibited with heparin) (n = 3). i Immunofluorescence analysis of co-localization between LA (green, FITC) and different subcellular compartments (red, early endosome, lysosome or endoplasmic reticulum) in immature DCs (blue, DAPI, nuclear) (n = 3). j The trace of LA was mainly concentrated on MHC-I molecular internalization (inhibited with primaquine), rather than vesicle secretion (inhibited with brefeldin A) and TAP transport (inhibited with ICP47) in DCs (n = 3). k The combination of LA-FITC and HLA-A2.1 on DC surface (blue, DAPI, nuclear; red, Dil, membrane; green, LA-FITC). l The presentation of LA (963.6) or YL (912.3) on DC surface by UPLC-QTOF-MS. m Immunoblot analysis of total Zap70 proteins and phosphorylated (p-) Zap70 in whole-cell lysates of epitope/DC-CTLs (n = 3). NC, negative control, water. NP, negative peptide, the peptide (PPGRPSPDN) derived from ECM1 with largest predicted IC₅₀ of affinity with HLA-A2.1. d–f, h, j P values were obtained from independent-samples t-test; error bars denote standard deviation (SD). d–f Compared with NC, ^*P < 0.05, ^**P < 0.01; h Compared with 37 °C blank, ^*P < 0.05, ^**P < 0.01; j ^*P < 0.05, ^**P < 0.01