Characterization of CD56+ dendritic-like cells: a normal counterpart of blastic plasmacytoid dendritic cell neoplasm? - PubMed (original) (raw)

. 2013 Nov 29;8(11):e81722.

doi: 10.1371/journal.pone.0081722. eCollection 2013.

Akihiko Yokohama, Akio Saito, Kenichi Tahara, Kunio Yanagisawa, Yoshiyuki Ogawa, Takuma Ishizaki, Takeki Mitsui, Hiromi Koiso, Makiko Takizawa, Hideki Uchiumi, Takayuki Saitoh, Hiroshi Handa, Hirokazu Murakami, Norifumi Tsukamoto, Yoshihisa Nojima

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Characterization of CD56+ dendritic-like cells: a normal counterpart of blastic plasmacytoid dendritic cell neoplasm?

Yohei Osaki et al. PLoS One. 2013.

Abstract

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy. Plasmacytoid DCs (pDCs), which are defined as lineage marker (Lin)(-)HLA-DR(+)CD56(-)CD123(+)CD11c(-) cells, are considered to be the normal counterpart of BPDCNs. However, BPDCN can be distinguished from pDCs by uniform expression of CD56. In this study, to identify a normal counterpart of BPDCN, we searched for a Lin(-)HLA-DR(+)CD56(+) population and focused on a minor subpopulation of Lin(-)DR(+)CD56(+)CD123(+)CD11c(-) cells that we designated as pDC-like cells (pDLCs). pDLC constituted 0.03% of peripheral blood mononuclear cells (PBMCs), and the pDLC/pDC ratio was higher in bone marrow cells than in PBMCs. pDLC clearly expressed BDCA2, BDCA4, and myeloid antigens, which are frequently expressed by BPDCN. pDLCs exhibited modest expression of Toll-like receptors and produced less interferon-α after CpG stimulation, but presented very low endocytic ability unlike mDCs. These functional differences were attributed to the expression profile of transcriptional factors. After in vitro culture with Flt3-ligand and GM-CSF, pDLCs expressed CD11c and BDCA1. These data suggested that pDLCs are a distinct subpopulation, with an immunophenotype similar to BPDCNs. Moreover, our results indicate that pDLCs might be immature DCs and might contribute to the immunophenotypical diversity of BPDCNs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. pDLCs are a rare DC population that are morphologically similar to pDCs.

(A) PBMNCs were analyzed using flow cytometry. The gated Lin-HLA−DR+ population (left) was divided according to CD56 expression (middle), and these two subpopulations were analyzed according to CD123 and CD11c expression to define pDLCs, mDLCs, pDCs, and mDCs (right). The results of one representative experiment are presented. (B) The frequency of pDLC, mDLCs, pDCs, and mDCs in PBMCs (n = 5). pDLCs comprised a very small (0.03%) proportion in PBMCs. (C) The pDLC to pDC ratio was compared between peripheral blood (PB) and bone marrow (BM) cells (n=5). *P < 0.05 compared to PB. (D) MFI of CD123 (left panel) and CD11c (right panel) expression was measured on pDLCs, pDCs, and mDCs in PBMCs (n = 5). *P < 0.05 compared to pDLC. (E) Sorted cells were stained with May–Giemsa solution and observed by light microscopy at 1000× magnification.

Figure 2

Figure 2. Immunophenotypic characteristics of pDLCs.

Expression of cell-surface and intracellular markers in pDLCs, mDLCs, pDCs, and mDCs were measured by flow cytometry. Upper graph showed the results of one representative experiment. pDLCs (solid line), pDCs (dotted line), mDCs (dashed line) and isotype control (filled histogram). Lower graph showed mean fluorescence intensity of pDLC, mDLC, pDC and mDC (n=5). * P < 0.05 compared to pDLC.

Figure 3

Figure 3. TLR, cytokine, and transcription factor expression levels in pDLCs are distinct from pDCs and mDCs.

qRT-PCR analyses were performed using cDNA from pDLCs, pDCs, and mDCs. Results were standardized using PBMC cDNA (n=5). * P < 0.05 compared to pDLC. (A) TLR expression analysis revealed that mDCs expressed TLR2 and TLR4, whereas pDCs expressed TLR7 and TLR9. (B) Cytokine production was analyzed after CpG or LPS stimulation. (C) Transcripts of E2-2, Id2, Irf8, PU.1, and SpiB were analyzed in pDLCs, pDCs, and mDCs.

Figure 4

Figure 4. pDLCs exhibit lower endocytic activity than that in mDCs, and also induce less proliferation of CD4+T cells than that in pDCs.

* P < 0.05 compared to pDLC. (A) PBMCs were incubated with FITC–dextran and analyzed by flow cytometry. The open histograms represent 37°C- and the filled histograms represent 4°C-cultured cells. Results of one representative experiment are presented (left). The average frequencies of FITC–dextran positive cells are indicated (n = 5, right). (B) Sorted DCs were co-cultured for 72 h with CFSE-labeled CD4+ T cells under CpG stimulation (1µM). The average frequencies of divided CD4 positive cells are indicated (n = 5).

Figure 5

Figure 5. pDLCs expressed mDC specific antigens.

pDLCs were cultured with Flt3-Ligand GM-CSF for six days. (A) CD123, CD11c and CD56 expressions were analyzed at day 0 and 6. CD123highCD11cdim and CD123dimCD11chigh populations were observed at day 6. Results of one representative experiment are presented (left). The average frequencies of CD123highCD11cdim and CD123dimCD11chigh populations are indicated (n = 5, middle). The average MFI of CD56 at day 0 and 6 are indicated (n = 5, right). * P < 0.05 compared to day0. (B) BDCA1 expression was compared by flow cytometry. pDLCs did not express BDCA1 at day 0, but they did express BDCA1 at day 6 especially CD123dimCD11chigh population. * P < 0.05 compared to day0 (n = 5). (C) Sorted cells were CFSE-labeled and incubated for six days with Flt3-L and GM-CSF. The filled histograms represent PHA-stimulated CFSE-labeled CD4+ T cells that were included as a control for cell division (left). The average frequencies of divided cells are indicated (n = 5, right).

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This work was supported in part by the National Cancer Center Research and Development Fund (23-A-17). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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