Plasmacytoid dendritic cells in tolerance

Abstract

Dendritic cells (DC) are professional antigen-presenting cells (APCs) that modulate the outcome of the immune response toward immunity or tolerance. There are a large variety of DC subsets according to surface phenotype, function, and tissue distribution. Murine plasmacytoid DC (pDC) represent a distinctive DC population and are characterized by the expression of CD11c, B220, Gr-1, CD45RA, Ly49Q, BST2, and siglec-H on the cell surface. PDC act as immunogenic cell sentinels by secreting large amounts of type I interferon (IFN) in the lymph nodes in response to viral stimulation. PDC also act as tolerogenic cells when expressing the inducible tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO), the inducible costimulator ligand (ICOS-L), and/or the programmed death 1 ligand (PD-L1), which mediate regulatory T-cell (Treg) development and suppression of self- and alloreactive cells. The PDC ability to induce Treg development is associated with capture and presentation of antigenic peptides associated with major histocompatibility complex (MHC) class I and II. Here, we provide the tools to study PDC development from bone marrow cultures, their antigen presentation properties, and their interactions with Treg under a tolerogenic setting of sterile inflammation.

Fig. 1

Plasmacytoid dendritic cell phenotype. (a) BM cells were isolated from BALB/c mice and cultured for 10 days in the presence of FLT3-L. Data can be analyzed using FlowJo software (TriStar) and BM of BALB/c mice can be divided into two major subpopulations, defined as PDC and mDC. In order to gate on PDCs, 100,000 events should be acquired for each sample. The first step in the analysis is to draw a gate looking at side scatter (SSC) versus forward scatter (FSC). This gate will be gate 1. Cell duplets are excluded in gate 2 (SSC-A versus SSC-H). Dead cells are excluded in gate 3 (SSC-A versus DAPI). Making a drill down of gate 3, we can then identify PDCs within the BM culture, by making a new gate looking at CD11c intermediate versus B220 high. This cell population should be CD11b negative. The PDC population also can be gated on from BM cultures on surface staining for Siglec-H and mPDCA-1.

Fig. 2

Induction of regulatory T cells with BM-derived plasmacytoid dendritic cells. Freshly sorted BM-derived PDC cells (2 × 10⁴ cells/well) isolated from BALB/c mice for 10 days in the presence of FLT3-L, were cultured with CD4⁺CD25⁻ T cells (5 × 10⁴ cells/well) for 96 h with 150 ng/mL of anti-CD3 mAb, 10 ng/mL IL-2, and 10 ng/mL TGFβ. Flow cytometry data indicates percentage of Foxp3⁺ CD4⁺ T cells induced.

Fig. 3

Alloantigen-presenting PDCs circulate systemically through blood. Percentage of alloantigen-presenting YAe⁺ cells from the blood of tolerant mice are shown. Flow cytometry results indicate that only PDC are presenting alloantigen systemically, since only YAe⁺ cells express mPDCA-1. Other cell types on freshly isolated blood do not express YAe in their cell surface. Data are representative results from three independent experiments (n = 3).

Fig. 4

Alloantigen-presenting PDCs are present in secondary lymphoid organs. Images of total alloantigen-presenting YAe⁺ cells from the lymph nodes of tolerized mice at day 5 posttransplant are shown. IHC results indicate that only PDC are presenting alloantigen, since most YAe⁺ cells express mPDCA-1. Data are representative results from three independent experiments (n = 3). Original magnification, ×200.

Fig. 5

PDCs are next to Foxp3 expressing cells in the lymph nodes of tolerized mice. IHC results indicate that Foxp3 expressing cells are in proximity of PDC in the lymph nodes of tolerized mice at day 5 posttransplant. Data are representative results from three independent experiments (n = 3). Original magnification, ×200.