Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin/CD209 is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction

Abstract

The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.

FIGURE 1

Reactivity of mAbs with DC-SIGN in transfected cells and DCs. A, Reactivity of anti-DC-SIGN mAb with DCs and transfectants expressing full-length DC-SIGN or L-SIGN. B, Reactivity by immunoblot of anti-DC-SIGN mAb 2. The indicated cells were lysates in RIPA buffer, equivalent to 3 × 10⁵ cells. After separation on SDS-PAGE, the lysates were blotted with anti-DC-SIGN mAb 2.

FIGURE 2

DC-SIGN staining is restricted to macrophages in the medullary sinuses (S) of lymph nodes. Two-color staining is shown, with red being the anti-DC-SIGN reagent, and green the other markers. First row, Clone 1 anti-DC-SIGN stains macrophages in the sinuses of the medulla and anti-CD3 stains T cells in the medullary cords (C). Second row, Clone 1 and anti-CD68 colabel medullary macrophages. Third row, Clone 1 and anti-macrophage mannose receptor (MMR) colabel macrophages in the lymph node medulla. Fourth row, Double staining using a commercial anti-DC-SIGN 507 and anti-mannose receptor mAbs.

FIGURE 3

Identical staining by clone 1 anti-DC-SIGN and another established anti-DC-SIGN mAb. Lymph node medulla sections were double stained with our clone 1 and the anti-DC-SIGN mAb MR-1.

FIGURE 4

Immunostaining of the lymph node cortex with anti-DC-SIGN mAbs. First row, The lymph node cortex is shown at ×10 after staining with anti-CD21 (green) to define the B cell follicles (B) and the unstained T cell region (T). Second row, Images at higher magnification within the T cell region reveal that most DCs defined by staining with anti-CD205 are DC-SIGN negative. Third row, The left panel is a double staining with our clone 10 (specific for DC-SIGN and L-SIGN) and CD11c; the middle panel is a double staining with clone 612 (specific for DC SIGN and L-SIGN) and DEC205; the right panel shows a double staining using anti-L-SIGN specific mAb (604) with MR-1.

FIGURE 5

Immunostaining of the lymph node cortex. Higher magnification of the cortex where rare fields can be found in which red DC-SIGN-positive cells colabel with CD11c and anti-CD205.

FIGURE 6

HIV-1 binding to DC-SIGN-expressing cells. Cells were incubated with anti-DC-SIGN mAbs or isotype control at 20 μg/ml for 20 min at room temperature and then infected with HIV-1 for 2 h at 37°C. Cells were washed and lysed in 0.5% Triton. The amount of p24 in the cell lysates was quantified by ELISA.

FIGURE 7

Effect of anti-DC-SIGN mAbs on HIV-1 transmission to T blasts. A, The indicated cells (left panel, immature DCs; right panel, Raji-DC-SIGN) were incubated with anti-DC-SIGN mAbs as described in Fig. 6, and then infected with HIV-1 BaL in the continued presence of mAb. The cells were washed four times, and 5 × 10⁴ infected cells were cocultured with 10⁵ T blasts. After 5 days, p24 secreted in the culture supernatants was quantified by ELISA. Each symbol corresponds to an individual experiment. B, DC-SIGN-expressing cells were transduced with siRNA lentiviral vector or empty vector at an MOI of 20, sorted for low DC-SIGN expression, and infected with HIV-1 before addition to T cells. The resulting cells were cocultured and assayed as described above.

FIGURE 8

DC-SIGN expression is induced by IL-4 and is not required for HIV-1 transmission by blood myeloid DCs. A, CD14⁺ cells and BDCA1⁺ cells were cultured for 48 h with IL-4 (10 ng/ml), GM-CSF (100 IU/ml), or IL-4 and GM-CSF. Cells were stained with anti-DC-SIGN and analyzed by FACS. B, Myeloid DCs were cultured for 48 h with or without IL-4 (10 ng/ml), the latter to induce DC-SIGN expression. Part of the cultures were incubated for 20 min with 20 μg/ml anti-DC-SIGN mAb and AZN-D1 and then infected with BaL for 2 h and washed. Infected DCs (5 × 10⁴) were cultured with 10⁵ T blasts. Supernatants were collected at different time points and analyzed for p24 secretion by ELISA. C, Raji transfectants were studied in parallel.

FIGURE 9

Inability of DC-SIGN to contribute to DC stimulation of the MLR. A, Immature DCs were pretreated for 20 min at room temperature with 20 μg/ml anti-DC-SIGN mAbs. DCs were cultured with allogeneic CFSE-labeled CD3 T cells at a ratio of 1:60. After 5 days, the presence of an MLR was documented by CFSE dilution. B, Immature DCs were transduced with siRNA DC-SIGN lentiviral vector or empty vector at an MOI of 20 and after 48 h were stained for DC-SIGN. The siRNA DC-SIGN (thick black line), empty vector (gray line), and isotype control (thin black line) are indicated. C, The low DC-SIGN-expressing cells were sorted and separated on a FACS. D, DC-SIGN^low sorted immature DCs were used to stimulate an MLR as described in A, and additional anti-DC-SIGN mAbs were tested for inhibition of the MLR.