Immune Cell Profiling of IFN-λ Response Shows pDCs Express Highest Level of IFN-λR1 and Are Directly Responsive via the JAK-STAT Pathway

Abstract

The interferon lambda (IFN-λ) cytokines have well-known antiviral properties, yet their contribution to immune regulation is not well understood. Epithelial cells represent the major target cell of IFN-λ; peripheral blood mononuclear cells are generally considered nonresponsive, with the exception of plasmacytoid dendritic cells (pDCs). In this study we aimed to define the potential for discrete subpopulations of cells to directly respond to IFN-λ. Analysis of peripheral blood leukocytes reveals that, while pDCs uniformly express the highest levels of IFN-λ receptor, a small proportion of B cells and monocytes also express the receptor. Nevertheless, B cells and monocytes respond poorly to IFN-λ stimulation in vitro, with minimal STAT phosphorylation and interferon-stimulated gene (ISG) induction observed. We confirm that pDCs respond to IFN-λ in vitro, upregulating their expression of pSTAT1, pSTAT3, and pSTAT5. However, we found that pDCs do not upregulate pSTAT6 in response to IFN-λ treatment. Our results highlight unique aspects of the response to IFN-λ and confirm that while the IFN-λ receptor is expressed by a small proportion of several different circulating immune cell lineages, under normal conditions only pDCs respond to IFN-λ stimulation with robust STAT phosphorylation and ISG induction. The difference in STAT6 responsiveness of pDCs to type I and type III interferons may help explain the divergence in their biological activities.

Keywords: JAK-STAT; Type III IFNs; dendritic cells; peripheral blood mononuclear cells.

FIG. 1.

Expression of the IFN-λ receptor chains IFN-λR1 and IL-10R2 on peripheral blood lymphocyte populations. Expression of the IFN-λR1 and IL-10R2 receptor chains were measured by flow cytometry on CD3⁺, CD19⁺, and CD56⁺ cells in healthy donor PBMCs, gating on lymphocytes by FSC SSC (n = 5). Graphs show the percentage positive of IFN-λR1 and IL-10R2 expression from n = 5 healthy donors. Gates were set based on isotype and FMO controls. Error bars show SEM. FMO, fluorescence minus one; PBMC, peripheral blood mononuclear cells.

FIG. 2.

Expression of the IFN-λ receptor chains IFN-λR1 and IL-10R2 on peripheral blood monocyte populations. Expression of the IFN-λR1 and IL-10R2 receptor chains were measured by flow cytometry on healthy donor PBMC monocyte populations. Following gating on the monocyte gate by FSC SSC and HLA-DR⁺ cells, the populations were gated to distinguish CD14⁺ classical monocytes, CD14⁺CD16⁺ intermediate monocytes, and CD16⁺ nonclassical monocytes and the % positive IFN-λR1 and IL-10R2 receptor cells shown (n = 5). Error bars show SEM.

FIG. 3.

Expression of IFN-λ receptor on pDCs. (A) IFN-λR1 and IL-10R2 expression was measured on CD123⁺ plasmacytoid and CD11c⁺ myeloid dendritic cells from the fresh whole blood of healthy donors. (i) Following gating on single cells by light scattering, dendritic cells were identified by first gating on Lin1⁻ HLA-DR⁺ population, followed by CD11c and CD123. Gates were set based on FMO controls and isotype controls (shown) (ii) The MFI of IFN-λR1 and IL-10R2 expression from n = 3 healthy donors is presented following subtraction of the isotype control for each cell type. Error bars show SEM. (B) RNA was extracted from total PBMCs or isolated CD3⁺ T cells, CD19⁺ B cells, CD14⁺ monocytes, or BDCA-4⁺ pDCs and IFNLR1 expression was measured by real-time PCR. Results are expressed as mean ± SEM. Statistical analysis was carried out using one-way ANOVA, with Tukey's post-test; ****P < 0.001. pDC, plasmacytoid dendritic cell.

FIG. 4.

IFN-λ induces STAT phosphorylation in pDCs from peripheral blood. (A) Whole blood (200 μL) was treated with 1,000 U/mL IFN-α or 100 ng/mL IFN-λ1 for 30 min. Red cells were then lysed, fixed and stained with the pan-leukocyte marker CD45. Following permeabilization, intracellular staining was performed using antibodies against pSTAT1 and pSTAT3 and cells were acquired immediately on a flow cytometer. (B) STAT phosphorylation was examined in the lymphocyte gate, monocyte/DC gate, and gating on Lin1⁻ BDCA-2⁺ pDCs. (C) Phosphorylation of STAT1 was measured by flow cytometry in pDCs in response to 10 ng/mL and 100 ng/mL IFN-λ1 and 1,000 U/mL IFN-α. Flow plots are representative of at least n = 3 healthy donors.

FIG. 5.

IFN-λ induces phosphorylation of STAT1, 3, and 5, but not STAT6, in pDCs. (A) Whole blood (200 μL) from healthy donors (n = 3) was treated with 100 ng/mL IFN-λ1, IFN-λ2, and IFN-λ3 or 1,000 U/mL IFN-α for 30 min. Following red blood cell lysis, cells were fixed and stained with lin1 and the pDC-specific surface marker BDCA-2. The cells were permeabilized and intracellular staining was carried out using antibodies against phospho-STAT1, STAT3, STAT4, STAT5, and STAT6. (B) The mean percentage STAT phosphorylation is shown for pDCs treated with either (i) IFN-λ1 or (ii) IFN-α at time points of 15–90 min as indicated, with the unstimulated sample gated at less than 3%. Error bars indicate SEM.

FIG. 6.

IFN-λ induces expression of the antiviral genes CXCL10, ISG15, and PKR in peripheral blood immune populations. BDCA-4⁺ pDCs, CD19⁺ B cells, CD14⁺ monocytes, and CD3⁺ T cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic bead separation and cultured either untreated or treated with (A) IFN-λ1 (100 ng/mL) or (B) IFN-α (1,000 U/mL) for 4 h. Following RNA extraction, CXCL10, ISG15, and PKR expression was measured using real-time PCR. Fold change is shown relative to the untreated sample for each cell type (as indicated by dotted line) and values were normalized to the mean of 2 housekeeping genes GAPDH and RPS15 using the ΔΔCt method. Each bar represents the mean of n = 5–6 blood donors for B cells, T cells, and monocytes and n = 3 donors for pDCs. Error bars show SEM.