Cross‐presentation of a human tumor antigen delivered to dendritic cells by HSV VP22‐mediated protein translocation

Abstract

Dendritic cells (DC) capture antigens from apoptotic and/or necrotic tumor cells and crosspresent them to T cells, and various ways of delivering tumor antigens to DC in vitro and in vivo are being pursued. Since fusions of antigenic proteins with the HSV integument protein VP22 are capable of intercellular trafficking, this approach has been exploited for delivery of antigens to antigen-presenting cells. Adenoviral vectors were used to express the tumorassociated-but-self-antigen MART-1 fused to HSV VP22 in MART-1-negative A375 melanoma cells and in DC. When expressed in A375 cells and allowed to spread to DC across a transwell barrier, the VP22-MART-1 fusion protein localized to both early and late endosomal structures of the DC. The DC loaded with the VP22-MART-1 fusion by intercellular trafficking efficiently presented the MART-1 27-35 epitope to MART-1 27-35 -specific CTL. Furthermore, transloaded DC were capable of expanding the population of MART-1 27-35 -specific CTL. Thus, a tumor antigen acquired by intercellular trafficking can be crosspresented by DC. This experimental approach should therefore be useful not only for studying the mechanism of cross-presentation but also for vaccine development.

Activation‐induced cell death of human melanoma specific cytotoxic T lymphocytes is mediated by apoptosis‐inducing factor

“…1 recapitulates our basic observation of the protective affect of the JNK inhibitor SP600125 on AICD in MART-1 [27][28][29][30][31][32][33][34][35] epitope-specific CTL [12]. A fraction of in vitro expanded CTL dies upon encountering the cognate antigen for the second time (Fig.…”

“…Culture medium, and other reagents used to generate MART-1 [27][28][29][30][31][32][33][34][35] peptidespecific CTL and to assess AICD have been described previously [12]. Jurkat cells were obtained from ATCC and cultured in RPMI 1640 with 10% FCS.…”

“…AICD in the human primary CTL primed and expanded specifically for melanoma epitope MART-1 [27][28][29][30][31][32][33][34][35] was induced by re-exposure of the CTL to MART-1 [27][28][29][30][31][32][33][34][35] peptide (1 lg/mL) -pulsed T2 cells as reported previously [12]. Staurosporinemediated death was induced by exposure of CTL to 1 lM staurosporine for 4 h. Quantitation of AICD and the effects of various inhibitors on AICD of CTL, were carried out by annexin-V staining by FACS analyses as reported previously [12].…”

“…Western blot analysis was done as described previously, using the enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ) [28]. Briefly, protein extractions were performed in RIPA buffer; samples were separated on 15% SDS polyacrylamide gels and electrophoretically transferred to PVDF membranes (Millipore, USA).…”

“…The cells were fixed with 4% para-formaldehyde in PBS and stained with appropriate antibodies for co-localization of proteins by fluorescence microscopy as described previously [28]. The anti-AIF antibody used for immunofluorescence staining was purchased from Santa Cruz Biotechnologies.…”