The Nature of the Principal Type 1 Interferon-Producing Cells in Human Blood

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Science  11 Jun 1999:
Vol. 284, Issue 5421, pp. 1835-1837
DOI: 10.1126/science.284.5421.1835


Interferons (IFNs) are the most important cytokines in antiviral immune responses. “Natural IFN-producing cells” (IPCs) in human blood express CD4 and major histocompatibility complex class II proteins, but have not been isolated and further characterized because of their rarity, rapid apoptosis, and lack of lineage markers. Purified IPCs are here shown to be the CD4+CD11c type 2 dendritic cell precursors (pDC2s), which produce 200 to 1000 times more IFN than other blood cells after microbial challenge. pDC2s are thus an effector cell type of the immune system, critical for antiviral and antitumor immune responses.

Interferons were discovered in the 1950s as factors rapidly produced by virus-infected cells that enable neighboring cells to resist virus infection (1). IFN-α (leukocyte IFN) and IFN-β (fibroblast IFN), the two type 1 antiviral IFNs, are distinct from type 2 IFN-γ produced by effector T cells. Specialized leukocytes, the “natural IFN-producing cells” (IPCs), were shown to be the chief IFN-α producers in response to enveloped viruses, bacteria, and tumor cells (2–14). IPCs express CD4 and major histocompatibility complex (MHC) class II, but lack hematopoietic-lineage markers (2–14). The nature of IPCs—whether they represent dendritic cells (6,12, 14) or cells of a distinct lineage (7, 9)—has been controversial. There is a progressive loss of CD4+ T lymphocytes and functional IPCs during human immunodeficiency virus (HIV) infection (15, 16). Preservation of IPCs is associated with protection from opportunistic infections, suggesting the importance of IPCs in the host defense (16).

A plasmacytoid cell type from human tonsils and blood that lacks lineage markers also expresses CD4 and MHC class II (17–21). These cells differentiate into type 2 dendritic cells (DC2s) when cultured with interleukin-3 (IL-3) and CD40 ligand (19, 21). Unlike monocyte-derived type 1 dendritic cells (DC1s) that induced type 1 T helper cell (TH1) differentiation, DC2s induced type 2 T helper cell (TH2) differentiation (21). Here we investigated whether DC2 precursors (pDC2s) represent IPCs. Human peripheral blood cells were separated into the following populations (19, 21): (i) monocytes (over 90% purity), obtained by centrifugation through 52% Percoll, then magnetic bead depletion of B, T, and natural killer (NK) cells; (ii) CD4+CD3CD11c+ immature DCs (99% purity) and (iii) CD4+CD3CD11c pDC2s (99% purity), obtained by magnetic bead depletion of B, T, NK cells, and monocytes, followed by fluorescence-activated cell sorting (FACS) (Fig. 1, A and B); (iv) pDC2-depleted blood mononuclear cells; and (v) pDC2-enriched blood mononuclear cells (19). pDC2s have a plasmacytoid morphology, with rough endoplasmic reticulum and Golgi apparatus (Fig. 1, C and D). The CD11c+ blood immature DCs display short dendrites (Fig. 1, E and F). The frequency of pDC2s in human blood mononuclear cells is less than 0.5% and increased to 3 to 10% after magnetic bead depletion of lineage-positive cells. The pDC2-depleted population contains B, T, NK cells, monocytes, and DCs. These cell populations were exposed to ultraviolet (UV)-irradiated herpes simplex virus (HSV) for 24 hours (22), and IFN in the culture supernatant was measured by a bioassay (23).

Figure 1

Tracing and isolation of IPCs/pDC2s from human peripheral blood. CD3+ T cells, CD19+ B cells, CD16+ and CD56+ NK cells, and CD14+monocytes were depleted from blood mononuclear cells by immunomagnetic beads (Dynabeads M-450; Dynal, Oslo, Norway). The cells were stained with anti-CD4-Tricolor (Immunotech, Marseille, France), anti-CD11c-PE (Becton Dickinson, San Jose, California), and a mixture of fluorescein isothiocyanate–labeled antibodies to CD3, CD15, CD16, CD20, CD57 (Becton Dickinson), CD14 (Coulter, Miami, Florida), and CD34 (Immunotech). Within the lineage-negative population (A), CD4+CD11c IPCs and CD11c+immature DCs were isolated (B). IPCs are plasmacytoid by Giemsa staining (C) and contain rough endoplasmic reticulum and Golgi apparatus under transmission electron microscopy (D). The CD11c+ blood immature DCs display dendrites (E and F).

IFN production by total peripheral blood mononuclear cells (PBMCs) from three donors was 40, 500, and 700 international units (IU) per 2 × 105 cells (Table 1). There was a four to six times increase in IFN production from pDC2-enriched blood mononuclear cells (180 to 2800 IU per 105 cells) and a 180 to 911 times increase in IFN generation from purified pDC2s (20,000 to 638,000 IU per 105 cells). pDC2-depleted PBMCs, immature CD11c+ DCs, monocytes, and monocyte-derived DC1s produced little or low levels of IFN. The ability of pDC2s to produce IFN was decreased after maturation into DC2s by culture with IL-3 or IL-3 plus CD40L for 6 days (Table 1). The geometric mean IFN-α generation by pDC2s was ∼1 IU per cell, similar to previous estimates (2). Immunoperoxidase staining for human IFN-α confirmed that most pDC2s contained IFN-α protein after 6 hours of exposure to HSV (Fig. 2) (22). Analysis of IFN-α and IFN-β mRNA by polymerase chain reaction (PCR) showed that among human blood cells, pDC2s were making the most IFN-α and IFN-β mRNA (Fig. 3) (24). Thus, the blood cells responsible for IFN generation in response to HSV, previously known as the IPCs, are actually the DC2 precursors. These cells can be traced and isolated by their expression of CD4 or IL-3 receptor after depletion of cells expressing lineage markers and CD11c (19, 21).

Figure 2

Immunoperoxidase staining for IFN-α (21, 22). (A) Purified CD4+CD11clinIPCs were stimulated with HSV for 6 hours. (B) IPCs cultured in medium for 6 hours. The isotype controls for the primary antibody in both cell preparations show no staining.

Figure 3

RT-PCR amplification of IFN-α and IFN-β. PCR products amplified from each cell population after 20, 25, 30, or 35 cycles were separated on a 2% agarose gel containing ethidium bromide. Negative controls contained no cDNA. Marker is 1-kb DNA Ladder (Life Technologies, Grand Island, New York). IFN-α mRNA is apparent in PBMCs, is increased in the DC2 precursors (pDC2s) with enrichment and purification, and is diminished in the monocyte fraction. IFN-β mRNA is visualized only in the most highly purified DC2 precursors.

Table 1

Precursor DC2 cells are the natural IFN-producing cells. Cells (2 × 105) were cultured for 24 hours with HSV. Without HSV, IFN activity from different cell types was less than 12.5 U/ml (23). PBMC: total blood mononuclear cells; pDC2-dep: blood mononuclear cells positively selected for expressing CD3, CD11c, CD19, CD14, and CD56; pDC2-enrich: blood mononuclear cells that were depleted of cells expressing CD3, CD19, CD14, and CD56; pDC2: FACS-sorted CD4+CD11clin cells; CD11c+DC: FACS-sorted CD11c+lin immature DCs; Mo: monocytes; DC1: monocyte-derived DCs after 6 days of culture with either granulocyte-macrophage colony-stimulating factor (GM-CSF) + IL-4 or GM-CSF + IL-4 + CD40 ligand (21); DC2: pDC2-derived DCs after 6 days of culture with IL-3 or IL-3 + CD40 ligand (23). ND, not determined.

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The purified IPCs also produced high levels of IFN in response to Sendai virus and heat-killed Staphylococcus aureus, confirming the previous studies on PBMCs and partially purified IPCs (2–14). The ability of UV-irradiated virus and heat-killed bacteria to induce IFN production by IPCs suggests that viral infection is not required for triggering IFN production. The rapid production of IFN by IPCs in the absence of other cells suggests that IPCs represent an effector cell type of the innate immune system. We propose that the IPCs/pDC2s should be included in the hematopoietic developmental chart as a distinct cell lineage. They function as professional IFN-producing cells at the precursor stages and as professional antigen-presenting type 2 DCs upon terminal differentiation.

Type 1 IFNs have pleiotropic effects on the immune system, including up-regulation of MHC class I on all cell types and activation of macrophage and NK cells (2). IFNs are also critical in the activation and survival of both CD4+ and CD8+ T cells (25, 26). Now with the ability to trace and isolate IPCs, it should be possible to directly study the interaction between IPCs and other cell types within the immune system. IFN-α has been widely used for treating hepatitis B and C as well as various cancers. A progressive loss of IPCs has been observed during HIV infection, suggesting that IPCs may represent targets for HIV-mediated infection and deletion. The present study provides an approach to directly monitor the number and functional state of IPCs in these patients. The ability to purify and culture IPCs in vitro will allow further studies on the molecular mechanisms that control the survival and growth of IPCs and their production of IFN, which may lead to novel therapies for patients with viral infections and cancer.


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