A method for directly determining the number of dendritic cells and for evaluation of their function in small amounts of human peripheral blood.

Bone marrow-derived dendritic cells (DC) are highly potent antigen-presenting cells (APC) capable of initiating primary responses of naive T lymphocytes to antigen. Studies on DC in disease have been impeded by the lack of a defined method for accurate DC counting and for evaluation of their function in a small amount of blood. In order to detect and enumerate DC in whole peripheral blood preparations, we applied a direct two-color immunofluorescence method. Blood from healthy donors was stained with a mixture of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) recognizing lineage-associated molecules (CD3, CD14, CD16, CD20, CD57) and phycoerythrin (PE)-conjugated anti-HLA-DR mAb. DC were identified as lineage marker negative (lin-), HLA-DR highly positive cells. The mean percentage of these cells present in peripheral blood leukocytes (PBL) was 0.54%, and the mean absolute DC count was 31.4 x 10(6)/l of blood. DC stained directly in whole blood were heterogeneous with regard to their expression of CD2 and CD4 molecules, and did not express CD80 and CD83 molecules. Expression of CD80 and CD83 on DC was induced following a multistep isolation procedure, including overnight culture. We demonstrated a significant primary proliferative response to keyhole limpet hemocyanin (KLH) in cultures of peripheral blood mononuclear cells (PBMNC). Since primary proliferative response to neoantigens is entirely dependent on DC as APC, the cultures of unseparated PBMNC stimulated with KLH can be used to evaluate DC function in a relatively simple test. This test does not require previous isolation of DC and T lymphocytes and, therefore, can be performed on a small amount of blood. The elaborated flow cytometric method of DC counting in blood and the proliferative test of DC-dependent primary response to neoantigen are currently being applied in an ongoing study on the effect of chemotherapy on DC number and function in cancer patients.