Monitoring in vitro immune suppressive and immune stimulatory responses in a naive donor to several self-antigens presented using CD14+ derived dendritic cells.

D. Hopkins, R. Doll, K. Puorro, B. L. Roberts and S. Shankara,Genzyme Molecular Oncology, 5 MTN Road Framingham, MA 01701.

In cancer immuno-therapy our goal is to identify antigens, either alone or in combination with adjuvants, cytokines or professional antigen presenting cells (APCs) that are capable of mobilizing a directed immune response to tumor. Understanding the quality of immune responses generated by the antigens as well as the effects of different adjuvants and APCs in-vitro studies with human cells may provide a lead in selecting an antigen for clinical applications. In the following experiments, we have demonstrated that with carefully characterized T cells and Dendritic cells (DCs) derived from healthy human volunteer apheresis products we can prime a CD3+ lymphocyte population to recognize both classical recall and self antigens. We used adenovirus vectors encoding a putative tumor antigen (melan A/MART-1, gp100, NY-ESO-1, HER2, and cytokeratin 18) or peptides of known HLA-A2 epitopes to deliver the antigen/epitope to APC. Our analysis incorporates cell surface marker profiling of APCs and lymphocytes followed by cytokine release and cytolytic activity of T cells generated from in-vitro priming (IVP). Further analysis of T cell subsets and their specific cytokine secretion profile revealed a more complete picture of the type of priming that occurs with each antigen or antigen/adjuvant combination. In order to detect significant variation in T cell phenotype in bulk populations we found it was important to link it with cytokine production, by selecting those cells secreting the cytokine (IFN-, IL-10) of interest and co-labeling them with antibody for cell surface markers. Using Adenoviral vectors to express tumor antigens, gp100 MART-1 and NY-ESO-1 we have demonstrated an antigen dependent variation in both cytokine profile and TH1 vs. TH2 T cell subset proportions. Specifically, we have seen a demonstrable shift from a high CD4+ population to a predominantly IFN+CD8+CD56- population within three re- stimulations for gp100. This was not observed with MART-1, where the IFN+ were evenly divided between CD4+ and the CD8+CD56-. It is clear from our observations that analyzing antigens and antigen presenting cells for development of novel immuno-therapy approaches in cancer, infectious disease and autoimmune disorders requires a comprehensive and yet dissectible approach that takes into account pro-inflammatory and anti-inflammatory cytokines, cytolytic activity as well as phenotypic characterization of T cells.