ABSTRACT
Dendritic cells (DC) originate from a bone marrow (BM) precursor and circulate via the blood to most body tissues where they fulfill a role in antigen surveillance. Little is known about DC numbers in disease, although the reported increase in tissue DC turnover due to inflammatory stimuli suggests that blood DC numbers may be altered in some clinical situations. The lack of a defined method for counting DC has limited patient studies. We therefore developed a method suitable for routine monitoring of blood DC numbers, using the CMRF44 monoclonal antibody (MoAb) and flow cytometry to identify DC. A normal range was determined from samples drawn from 103 healthy adults. The mean percentage of DC present in blood mononuclear cells (MNC) was 0.42%, and the mean absolute DC count was 10 x 10(6) DC/L blood. The normal ranges for DC (mean +/- 1.96 standard deviation [SD]) were 0.15% to 0.70% MNC or 3 to 17 x 10(6) DC/L blood. This method has applications for monitoring attempts to mobilize DC into the blood to facilitate their collection for immunotherapeutic purposes and for counting blood DC in other patients. In preliminary studies, we have found a statistically significant decrease in the blood DC counts in individuals at the time of blood stem cell harvest and in patients with acute illnesses, including allogeneic bone marrow transplant (BMT) recipients with acute graft-versus-host disease (aGVHD).
Subject(s)
Cell Count , Dendritic Cells , Hematopoietic Stem Cell Transplantation , Adolescent , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal , Blood Cells , Female , Flow Cytometry , Humans , Male , Middle Aged , Reference ValuesABSTRACT
The CMRF-44 monoclonal antibody (MoAb) recognizes an intermediate stage of blood dendritic cell (DC) differentiation as well as mature CD83+ blood DC. Here we describe the use of the CMRF-44 MoAb to monitor the in vitro development of DC-like cells from peripheral blood mononuclear cells. Neither granulocyte-macrophage colony-stimulating factor (GM-CSF) nor GM-CSF plus tumor necrosis factor-alpha (TNF-alpha) supported the development of CMRF-44+ cells. However, GM-CSF plus interleukin (IL)-4 generated a substantial number of CMRF-44+ cells among the heterogeneous CD14- myeloid cell population, produced after 7 or 10 days of culture. The addition of TNF-alpha to GM-CSF+IL-4 on the fifth day of culture enhanced the generation of CMRF-44+ cells from days 7 to 14. A concentration of 50 U/mL of IL-4 was sufficient to allow the development of CMRF-44+ cells. The presence of GM-CSF was essential, but a wide range of concentrations (50-800 U/mL) was effective for supporting IL-4-induced generation of CMRF-44+ cells. TNF-alpha at concentrations of 20 or 50 ng/mL induced a maximal increase in the number of CMRF-44+ cells. The CMRF-44+ DCs generated in the presence of GM-CSF+IL-4 were large, irregularly shaped cells with variable CD1a expression and have CD83 transcripts but no CD83 surface expression. Additional TNF-alpha treatment induced prominent dendritic processes and surface expression of CD83 on CMRF-44+ DCs. The CMRF-44+ DCs generated in GM-CSF+IL-4 showed higher allostimulatory activity than CMRF-44 cells but were less efficient at processing and presenting soluble antigen to T-lymphocyte lines. TNF-alpha treatment reduced antigen uptake but increased the allostimulatory activity of CMRF-44+ DCs. CMRF-44+ DC differentiation from blood CD14+ monocytes was not radiosensitive and thus does not involve cell division. We conclude that the MoAb CMRF-44 identifies both intermediate and fully mature stages of monocyte-DC differentiation and may be a useful marker in establishing the optimal timing for antigen loading of in vitro-generated monocyte-derived DCs.
