In contrast to DC, uptake of AFP by HepG2 cells was mediated by pinocytosis and several scavenger receptors, including LOX-1 and CD36. Vaccines, including malignancy vaccines, have utilized many approaches to convey antigen to stimulatory antigen presenting cells. AFP (nAFP) and tumor-derived AFP (tAFP) proteins. While in healthy donors, nAFP and tAFP were cross-presented to CD8+ T cells similarly and CD4+ T cell responses were dependent upon MR-mediated uptake. In CEP-32496 HCC patient cells, tAFP was more immunogenic, and CD4+ T cell responses were not MR-dependent. Conclusions Secreted, cytoplasmically retained, and endocytosed forms of AFP utilize unique uptake and processing pathways, resulting in different immunologic responses from your induced antigen-specific CD4+ and CD8+ T cells and between healthy donors and HCC patients. Collectively, these data elucidate pathways of spontaneous and induced anti-tumor immunity in HCC patients to this secreted antigen. Electronic supplementary material The online version of this article (doi:10.1186/s40425-015-0077-x) contains supplementary material, which is available to authorized users. [20]. At least three clinical trials have tested AFP-based vaccine regimens: i) four immunodominant HLA-A*0201-restricted AFP peptides emulsified in Montanide adjuvant [21], ii) AFP peptide-pulsed autologous DC [22], and iii) a DNA-prime/adenovirus (AdV)-boost genetic immunization [23]. Although no objective clinical responses were observed in the small numbers of vaccinated patients, AFP-specific T cell responses were either developed or expanded in the majority of patients. The association between AFP secretion and poor clinical outcome, HCC stemness [24] and tumor growth rate supports further screening of AFP as an immunogenic tumor-associated antigen target. Because of the inherent variability in human self-tumor antigen responses and the small size of most cancer vaccine clinical trials, it is not yet clear how to weight DC with antigen optimally for CTL induction. Clinical trials continue to utilize a wide array of antigen sources and uptake pathways to attempt to promote antitumor immunity. It is also increasingly clear that there is considerable tumor-immune crosstalk before tumors become clinically evident, and many patients have spontaneous immune responses to tumor antigens without vaccination or other therapy. In this CEP-32496 study, we examined different forms CEP-32496 of AFP antigen to identify how the antigen is usually taken up, processed, and offered by DC. By investigating Mouse monoclonal to EhpB1 the fetal and tumor-induced immunity to this secreted antigen and examining the subsequent impact on T cell responses, we inform the design of future vaccination strategies targeting this oncofetal antigen. Results and conversation AdV-transduction induces partial maturation of DC We have previously utilized adenoviral vectors for genetic engineering of DC due to their ability to express full length antigens within DC and positively impact some aspects of DC function [25C29]. To further characterize the maturation effects of AdV on DC, we first transduced healthy donor (HD) DC with an AFP-encoding AdV (AdVhAFP) and monitored the expression of several maturation markers over the course of 3?days. Compared to immature DC (iDC) and LPS/IFN–matured DC (mDC), AdV-transduced DC exhibited intermediate expression levels of antigen presentation molecules (HLA-ABC, HLA-DR) and costimulatory molecules (CD40, CD83, CD80, CD86) (Fig.?1a). We also analyzed expression of the endocytic receptors MR and CD36 following AdV-transduction (Fig.?1b). Unlike mDC, which highly downregulate these receptors, AdV-transduced DC express levels much like iDC, suggesting that AdV contamination does not compromise the endocytic function of DC. Open in a separate windows Fig. 1 Phenotype of AdV-transduced DC. a and (b) Immature DC (iDC) from healthy donors (n?=?3) were left untreated, matured with LPS/IFN- (mDC), or transduced with AdVhAFP, and then cultured in DC media for 24, 48, or 72?hr. Cells were stained for (a) antigen presentation and costimulatory markers and (b) endocytic receptors, and analyzed by circulation cytometry. Mean fluorescence intensity (MFI) is usually reported as the mean??SD Adenovirally-expressed AFP localizes to the Golgi apparatus and related compartments in DC To determine the intracellular expression patterns of adenovirally-expressed AFP, DC were transduced for 3?hr and AFP localization was examined by fluorescent microscopy for 24, 48, or 72?hr post-infection. Throughout the observation period, the AFP transgene was detected almost exclusively in the perinuclear space (Fig.?2). Adenovirally-expressed AFP is only transiently present in early endosomes (EEA-1) at 24?h, and not detected in late endosomes/lysosomes (LAMP-1), or the endoplasmic reticulum CEP-32496 (KDEL). Some colocalization was observed with ERGIC-53 (ER-Golgi intermediate complex), a compartment which has been implicated in cross presentation [30]. However, the AFP expressed in the beginning in the cytoplasm from your AdV construct colocalizes extensively with Golgi (golgin-97) and cultures are screening the loaded DC ability to boost these T cells. Indeed, the frequencies of AFP-specific CD4+ T cells are.