Dendritic cells are uniquely potent accessory cells for presenting antigen and initiating primary T cell responses. This project will investigate the capacity of human bone marrow-derived dendritic cells [DC] to generate MHC- restricted cytotoxic T cell [CTL] responses against an endogenously expressed viral antigen. A gene encoding the influenza virus nucleoprotein [NP] that contains epitopes for CTL generation will be introduced into CD34+ DC progenitors by retroviral gene transfer. Recent identification of CD34+ DC progenitors in marrow and cord blood as well as cytokines that foster their growth and differentiation render gene transfer a promising approach. Transduced DC progeny whose surface class l MHC molecules are charged with endogenously expressed viral peptides will be evaluated for their ability to stimulate MHC-restricted NP-specific CTL. This approach avoids the need to utilize synthetic peptides or live infectious viruses. This in vitro model also eliminates the complex effects of bystander APCs and local inflammatory cytokines present in in vivo systems. The project will focus on the following specific aims: l. determine the optimal culture conditions and cytokine combination for efficient retroviral transduction of DC progenitors while supporting their differentiation into DCs utilizing semiquantitative PCR to assess integration and Northern blot analysis immunoprecipitation [lP] and immunofluorescence [IF] to demonstrate expression of NP in purified dendritic cells: 2. determine the capacity of the transduced dendritic cells [DC/NP] to stimulate resting syngeneic T cell response based on 3HTdR incorporation and cytotoxic T cell assays; 3. characterize the T cell responders with regard to immunophenotype requirement for CD4+ help, and MHC-restriction. Retrovirally transduced DCs may be utilized in systems other than anti viral CTL generation. This system however could provide a valuable model to evaluate the immunogenicity of presumed CTL epitopes derived from products of transplantation antigens oncogenes or other chromosomal aberrations responsible far cellular transformation. These studies could therefore lead to efficient generation of highly specific antiviral [HIV,CMV,EBV, etc.] and antitumor CTL.
| Szabolcs, P; Gallardo, H F; Ciocon, D H et al. (1997) Retrovirally transduced human dendritic cells express a normal phenotype and potent T-cell stimulatory capacity. Blood 90:2160-7 |
