CD2 distinguishes two subsets of human plasmacytoid dendritic cells with distinct phenotype and functions - PubMed (original) (raw)

. 2009 Jun 1;182(11):6815-23.

doi: 10.4049/jimmunol.0802008.

John E Connolly, Mark Michnevitz, Damien Chaussabel, Chun-I Yu, Casey Glaser, Sasha Tindle, Marc Pypaert, Heidi Freitas, Bernard Piqueras, Jacques Banchereau, A Karolina Palucka

Affiliations

CD2 distinguishes two subsets of human plasmacytoid dendritic cells with distinct phenotype and functions

Toshimichi Matsui et al. J Immunol. 2009.

Abstract

Plasmacytoid dendritic cells (pDCs) are key regulators of antiviral immunity. They rapidly secrete IFN-alpha and cross-present viral Ags, thereby launching adaptive immunity. In this study, we show that activated human pDCs inhibit replication of cancer cells and kill them in a contact-dependent fashion. Expression of CD2 distinguishes two pDC subsets with distinct phenotype and function. Both subsets secrete IFN-alpha and express granzyme B and TRAIL. CD2(high) pDCs uniquely express lysozyme and can be found in tonsils and in tumors. Both subsets launch recall T cell responses. However, CD2(high) pDCs secrete higher levels of IL12p40, express higher levels of costimulatory molecule CD80, and are more efficient in triggering proliferation of naive allogeneic T cells. Thus, human blood pDCs are composed of subsets with specific phenotype and functions.

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Figures

Figure 1

Figure 1. pDCs kill malignant cells

(A-B) Killing of K562 cells analyzed in an 18hrs 51Cr release assay with pDCs activated with IL-3CD&40-L (A), Influenza virus, or CpG (B). Specific lysis (ordinate). Paired Wilcoxon test. Color code indicates experiments with cells from the same donor. Insert plot shows killing by NK cells isolated from the same donors. (C) Killing of breast cancer (1806) and melanoma (Colo8290) cells by CpG activated pDC and mDC; 18hrs 51Cr release assay; specific lysis (ordinate). Duplicate wells from one experiment; representative of two experiments. (D) Inhibition of malignant cell replication on 3-day co-culture. Thymidine incorporation (ordinate). Duplicate (or triplicate) wells from four different experiments. (E) Fluorescence microscopy analysis of conjugates between CD45PE labeled K562 cells (red) and CD123FITC/HLA-DRFITC labeled pDCs (green) (1:2 ratio; 45 min). (F) Flow cytometry analysis of conjugate formation between CD45PE labeled K562 cells (red) and CD123FITC/HLA-DRFITC labeled pDCs (green) (1:2 ratio; 45 min) (upper right quadrant).

Figure 2

Figure 2. CD2 expression discriminates two subsets of pDCs

(A) PBMCs staining shows the lack of CD2 expression by blood CD19+ B cells (upper plots), expected peak of expression on CD3+ T cells (middle plots) and a subset of BDCA2+ pDC that show CD2 staining within the range of staining on T cells (lower plots). Representative of the analysis of PBMCs from three donors. (B) pDC subsets distinguished by CD2 expression can also be defined among HLA-DR+BDCA2+ (left panel) CD11cneg (middle panel) cells in pre-enriched PBMCs. (C) Frequency of two subsets within the total pDCs population. (D) BDCA-2+CD2+ pDCs in single cell suspension prepared from tonsil; representative of three independent experiments.

Figure 3

Figure 3. CD2high pDCs and CD2low pDCs show similar ultrastructure

(A) CD2low (left panels) and CD2high (right panels) pDCs were fixed and processed for transmission electron microscopy. Note the abundance of endoplasmic reticulum in both subsets of cells (arrows). N: nucleus. The bar: 2 micrometers. (B) CD2 staining in flow cytometry on pDC subsets pre-sort, after sort and after 18hrs activation with CpG. (C) Sorted CD2low (upper panels) and CD2high (lower panels) were cultured overnight at 30,000 cells /well with either live PR8 virus 1HA unit/well or CpG 6 μg/ml. Harvested cells were analyzed by flow cytometry for their surface expression of CD2. Upper panels: CD2low pDCs isotype control, CD2 staining after sort, CD2 staining after PR8 exposure and CD2 staining after CpG exposure. Lower panels: CD2high pDCs after PR8 exposure. Representative experiment of four performed testing different conditions of pDC activation.

Figure 4

Figure 4. CD2high pDCs and CD2low pDCs show distinct transcriptional profiles

(A) Top ten genes overexpressed in CD2high pDCs as compared to CD2low pDCs (determined by fold change). Microarray scaled expression data; two experiments with pDC subsets sorted from two different donors. (B) Sorted pDCs subsets are stained with FITC conjugated anti-Lysozyme mAb and either biotin conjugated anti-BDCA2 or anti-Granzyme-B mAb followed by treatment with either Alexa 568 labeled streptavidin or goat-anti-mouse IgG2a, respectively. Images are acquired on a Leica DMIRBE microscope with the 63x Plan APO objective, using Leica TCS v 2.1 software. (C) staining of melanoma tumor. (D) Top ten genes overexpressed in CD2low pDCs as compared to CD2high pDCs (determined by fold change). Microarray scaled expression data; two experiments with pDC subsets sorted from two different donors. Insert plot shows genes with expression values <1000.

Figure 5

Figure 5. CD2high pDCs strongly bind target cells

(A) Activated CD2high or CD2low pDCs were mixed with K562 cells at 2:1 ratio and monitored in 37°C 5% CO2 live cell imaging chamber. The percentage of pDCs interacting with K562 cells was determined through an analysis of time course image stacks. Percent interaction is expressed as the ratio of bound pDCs over the total number of pDCs in the frame for that time point. (B) K562 cell (green) surrounded by tightly bound CD2+ pDCs.

Figure 6

Figure 6. CD2 expression discriminates two subsets of pDCs one of which is immunogenic

(A) Sorted pDC subsets are pulsed with heat-inactivated Influenza virus and used in co-cultures with CFSE-labeled autologous T cells. T cell proliferation (CFSE dilution) is measured at day 6. (B-D) Sorted pDC subsets are activated with CpG and plated with naïve allogeneic T cells. T cell proliferation is measured by thymidine incorporation (B; representative experiment of three performed) or by CFSE dilution (C; representative experiment) and (D; four independent experiments). (E) Supernatants of pDC/T cell cocultures are analyzed at day 5 for cytokine secretion.

Figure 7

Figure 7. Activated CD2high pDCs and CD2low pDCs show unique and distinct features

(A-B) Cytokine secretion (ng/ml, ordinate) by pDC subsets after activation with Influenza virus (Luminex analysis). (A) Representative of eight experiments; (B) five different donors, color indicates pDC subsets isolated from the same donor; 25,000-30,000 cells per well. (C-E) Flow cytometry analysis of protein expression by activated pDCs subsets: MHC class I and class II molecules (B; two different donors); CD80 (C; two different donors) and CD86 (D; representative of four different donors). Values indicate mean fluorescence intensity.

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