View All Abstracts for Session 37
199 Enhanced Maturation of Clinical Grade Human Dendritic Cells Using Different Plastic Surfaces and Cryopreservation Techniques
M. Marovich, M. Eller*, B. Tassaneetrithep, A. Herring, D. Birx, and S. Frankel
US Military HIV Res. Program, Rockville, MD, USA
Background: Ex vivo targeting of human dendritic cells might enhance the immunogenicity of HIV vaccines candidates. However, recent literature suggests that injection of immature antigen-loaded DC may in fact lead to specific inhibition of T-cell responses. Using monocyte-conditioned media (MCM) as a maturational signal for dendritic cells in an ongoing preventative HIV vaccine trial, we optimized maturation conditions.
Methods: We generate leukopack derived DC to support a human clinical trial proposed to enhance the immunogenicity of vCP205, a canarypox vaccine candidate. A maturation signal is delivered using autologous MCM. The conditions under which MCM is generated were tested. Cryopreservation media were studied and various plastic surfaces were tested for their ability to generate potent MCM. The potency was evaluated by flow cytometry of the matured DC (CD25, 40, 83, and 86) and the MCM was tested for the presence of known maturation signals: IL-1 beta IL-6, PGE2, and TNF-alpha by ELISA. Functional maturation of DC was tested by alloreaction.
Results: Fresh or cryopreserved peripheral mononuclear cells (PBMC) can be used to generate MCM. More potent MCM was made using nontissue culture treated plates, regardless of whether the cell source was fresh or frozen. Higher levels of cytokine proteins were detected in MCM made from nontissue culture-treated plates. MCM generated from previously frozen cells induced the most mature DC as determined by the highest levels of maturational markers (CD25, 40, 83, and 86) and greatest proliferation in alloreactions.
Conclusions: The quality of the MCM is influenced by the type of plastic used for adherence. Frozen cells are the best cell source for MCM generation. TNF, IL-1beta, IL-6, and PGE2, cytokines detected in MCM, may determine the potency of MCM. Use of frozen PBMCs and non tissue culture treated plates may be used to enhance the maturation of clinical grade human DC.
Contact Author about this Abstract
