Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage−  cells

Abstract

SUMMARYDendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric ow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic pro®le and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II /lineage À were identi®ed on the basis of their distinct phenotypic characteristics: one DC subset was CD33 strong and CD123 dim (0´16 6 0´06% of the PB nucleated cells and 55´9 6 11´9% of all PB DC) and the other, CD33 dim and CD123 strong (0´12 6 0´04% of PB nucleated cells and 44´53 6 11´5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor a-chain (CD126) and for CD38. In contrast, the CD123 strong /CD33 dim DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33 strong /CD123 dim DC are able to produce signi®cant amounts of in¯ammatory cytokines, such as IL-1b (97 6 5% of positive cells), IL-6 (96 6 1´1% of positive cells), IL-12 (81´5 6 15´5% of positive cells) and tumour necrosis factor-alpha (TNF-a) (84 6 22´1% of positive cells) as well as chemokines such as IL-8 (99 6 1% of positive cells), the functional ability of the CD123 strong /CD33 dim DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.

Altered maturation of peripheral blood dendritic cells in patients with breast cancer

“…Owing to the important role of regulatory cytokines of DC origin in the induction and activation of immune responses, we evaluated the cytofluorimetric DC expression of IL-12 and IL-10, both in basal and stimulated conditions. We used LPS as the DC stimulator because LPS, with or without IFN-g, has been reported to be the optimal stimulus for analysis of cytokine expression by DCs in whole-blood samples (Almeida et al, 1999;Bueno et al, 2001), and because TLR4, which is required for the response to LPS, is selectively expressed on myeloid CD11c þ DCs (Krug et al, 2001). We found that DC constitutive production of IL-10 was increased in patients with invasive cancer, while that of IL-12 was similar to controls.…”

“…At the end of the incubation period, samples were aliquoted and labelled with appropriate combinations of mAbs for staining of surface markers. Cells were then fixed, permeabilised and stained with cytokine-directed mAbs anti-IL-12 PE and anti-IL-10 PE (Caltag Laboratoires), using the Fix & Perm reagent (Caltag Laboratoires) (Almeida et al, 1999;Bueno et al, 2001). Cytokine-directed mAbs were used at saturating concentrations and conditions.…”

Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement

“…19 Importantly, we and many others have reported the existence of acute leukemias and plasmacytoid dendritic cell (pDC) leukemias (450%) lacking MLL rearrangements but expressing NG2 (Table 1). [60][61][62] Based on the controversial data about the clinical relevance of NG2 expression together with the existence of NG2-expressing acute leukemias lacking MLL rearrangements, in particular pDC leukemias, 60,61 we wanted to gain further insight into the biological association between NG2 expression and MLL rearrangements. We analyzed whether the expression of NG2 may depend on the particular gene(s) paired to MLL when it is rearranged and we also explored the hypothesis that the expression of NG2 in leukemias lacking MLL rearrangements, such as NG2 þ pDC leukemias, may be due to the existence of a minor subset of CD34 þ hematopoietic stem/progenitor cells readily coexpressing NG2 wherein the leukomogenesis process may be initially triggered.…”

Sequential analysis of CD34+ and CD34− cell subsets in peripheral blood and leukapheresis products from breast cancer patients mobilized with SCF plus G-CSF and cyclophosphamide

“…In this step, a minimum of 3 ϫ 10 5 events from the total PB cellularity was measured in order to obtain information on at least 1000 dendritic cells defined as being MHC II ϩ /lineage Ϫ cells (non-T, non-B, non-NK and non-monocytic cells) for further immunophenotypic analysis. 26 For data analysis, the Paint-A-Gate PRO software was used. Identification and enumeration of the relative numbers of the different CD34 Ϫ cell subsets present in PB was performed as shown in Figure 1.…”