Flow cytometric detection of circulating dendritic cells in healthy subjects

Abstract

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c–) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123– cells, do identify the myeloidderived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123–, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5±0.2% and 30±11 cells/?L for DCs; 0.2±0.1% and 15±6 cells/?L for DC1; 0.2±0.1% and 15±7 cells/?L for DC2. The same values were: 0.2±0.1% and 16±7 cells/?L for BDCA.1; 0.2±0.1% and 12±7 cells/?L for BDCA.2; 0.02±0.01% and 2±1 cells/?L for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects

A new marine-derived sulfoglycolipid triggers dendritic cell activation and immune adjuvant response

“…In this study, human MoDCs were placed in 96 multiwell plates and treated with methanol extracts from a collection of marine organisms including invertebrates and photosynthetic protists (microalgae, diatoms and dinoflagellates). Evolution from immature antigen-capturing cells to mature, immunologically competent DCs was assessed by measuring the expression of MHC-Class II receptor HLA-DR and the co-stimulatory molecule CD86 by flow cytometry 22 . When mature DCs were detected, we also measured production of IL-12, a specific mediator of Th1/CTL activation 23–25 , by quantitative PCR.…”

Flow cytometric analysis of peripheral blood dendritic cells in patients with severe sepsis

“…In a previous article, we compared the number of DC obtained (1) by calculating the number of DC when gated as lineage-negative/ HLA-DRþ and by identifying the two subsets as CD11cþ (mDC) and CD123þ (pDC), and (2) by using three specific markers: BDCA1 and BDCA3 for mDC, and BDCA2 for pDC. No statistically significant difference was found between the sum of the CD11cþ and CD123þ cell populations versus the sum of the BDCA1þBD-CA2þBDCA3 cell populations (40). Despite the fact that each one of the BDCA markers clearly do not identify exactly and exclusively mDC or pDC cell populations, both analytical methods are therefore able to identify DC compartment in a clinical setting.…”

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

“…There are two major subpopulations of DCs in the human peripheral blood, CD11c + CD123 − (mDC, also known as myeloid DC or DC1) and CD11c − CD123 + DC (known as plasmacytoid DC, pDC, or also DC2). DCs occur in very low frequency in the peripheral blood of both humans and mice (<1%) [1], and the homeostatic mechanism regulating DC frequency has not been fully elucidated. It is established that DC progenitors require factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF: mDC) and FMS-like tyrosine kinase 3 ligand (FL: mDC, pDC) to expand and differentiate [2, 3].…”