Figure 2

In vitro assays of phenotype and function do not predict differences in immunogenicity. (A) Cell surface marker staining. Selected or Adherence DCs made from the same donor were stained with CD14, CD83, HLA-DR, CD86, CD40 and CCR7 antibody. This is representative of 3 repeated experiments. (B) Phagocytosis assay. HLA-DR stained Selected or Adherence iDCs were cultured with PKH26 stained apoptotic lymphocytes with or without EDTA for 24 hours. Cells are gated on HLA-DR. Cells staining positive for both HLA-DR and PKH26 indicate phagocytosis of apoptotic cells by DC groups. The data shown is representative of 3 repeated experiments. (C) Allo-MLR. Selected and Adherence DCs were made from cells from 3 donors. Average CPMs are shown for syngeneic and allogeneic responses at the DC:T cell ratio of 1:30. Solid black line represents the mean. NS = not statistically significant. (D) Lymphocyte proliferation. Adherence or Selected DCs co-cultured with apoptotic 3T3 cells (DC/ctrl) or with influenza-infected 3T3 cells (DC/flu), were cultured with syngeneic CD14- cells. The cultures were assessed for proliferation by ³H thymidine incorporation and the data shown is representative of 3 repeated experiments. NS = not statistically significant. (E) IFNγ ELISPOT. Purified syngeneic CD8 or CD4 T cells were plated in an ELISPOT with either non-infected DCs (DC) or influenza-infected (DCF) made by the 2 methods. The data shown is an average of triplicate wells and representative of 3 repeated experiments. NS = not statistically significant.