HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells

Abstract

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1. HIV-1 gp120 Induces IL-10 Secretion from MDDCs in a Donor- and Concentration-Dependent Manner

(A) MDDCs from different human donors were cultured in GM-CSF + IL-4 for 6 d and then incubated for 24 h (n = 71 donors) with or without JR-FL gp120 (3 μg/ml) before measurement of IL-10 production by ELISA. The fold-increases in IL-10 production after gp120 treatment compared with untreated cells are depicted on the y-axis. Another set of MDDC from each donor was stimulated for 24 h with TNIL + LPS (±CD40L), instead of gp120. The corresponding fold-increase in IL-10 secretion compared with untreated cells is also plotted. (B) MDDCs from 11 pedigreed donors were cultured and stimulated with gp120 or TNIL + LPS, as in (A), twice within a 3-mo interval. IL-10 production was measured 24 h after stimulation. Black bars represent the first assay on each donor, white bars the second. (C) The secretion of IL-10 from iMDDCs after 24 h is depicted as a function of the gp120 concentration. The data points show mean values ± SD (the error bars lie within the symbols) of duplicate ELISA determinations. Each curve represents data derived from a single donor, the same symbol representing the same donor in each panel. Left: Three different donors, JR-FL gp120. Middle: Four different donors, LAI gp120. Right: Three different donors, KNH1144 gp120 (the square symbol is overlaid by the circle symbol). (D) iMDDCs from five donors were treated with JR-FL gp120 (3 μg/ml), LAI gp120 (10 μg/ml), KNH1144 gp120 (10 μg/ml), or TNIL + LPS before measurement of IL-10 production at 24 h.

Figure 2. The Induction of IL-10 Secretion by gp120 Is Mannose-Dependent

(A) The bars represent IL-10 production from MDDCs on day 6 after 24 h (black bars) of treatment with JR-FL gp120 (3 μg/ml). The reagents listed on the horizontal axis were incubated with gp120 or iMDDCs for 1 h prior to addition of gp120 to the cells (see Materials and Methods for the inhibitor concentrations tested). The bars represent the mean value ± SEM for data derived from five different, gp120-responsive donors. The left and right panels show data derived from different experiments. The various reagents were also tested in the absence of gp120 and found not to stimulate IL-10 production (2+-chelator EGTA and the anti-DC-SIGN mAb, AZN-D1 (M-gp120 black bar; D-gp120 white bar). (C) Left panel: A reducing SDS-PAGE gel shows the reduction in JR-FL gp120 m.wt. caused by treatment with α-(1,2,3,6) mannosidase. Right panel: western blotting with anti-gp120 serum ARP3119 confirms the m.wt reduction, and blotting with mAb 2G12 shows that its mannose-dependent epitope has been removed from gp120. (M = m.wt markers; enzyme only = no gp120 present). (D) The experimental design was the same as in (A). The gp120 proteins (or influenza HA or TNIL + LPS + CD40L) tested are listed on the x-axis. The bars represent the mean values ± SEM for data derived from five different donors (black bars, IL-10 production after 24 h; white bars, after 48 h).

Figure 3. Involvement of the ERK1/2 and p38 MAP Kinase Signaling Pathways in the Induction of IL-10 and IL-12p70 by gp120 and TNIL + LPS

(A) Day-6 MDDCs were incubated with or without JR-FL M-gp120 or D-gp120 (3 μg/ml), or with TNIL + LPS, for 10 min before pERK1/2 (upper panel) and p-p38 levels (lower panel) were measured by ELISA (black bars). The white bars show the effects of adding the pERK1/2 inhibitor UO126 (5 μM) or the p38 inhibitor SB 203580 (10 μM) 1 h before the gp120s or TNIL + LPS. The bars represent the mean values ± SEM for data derived from four different donors. (B) The experimental design was based on that used for (A), except that the iMDDCs were incubated with or without UO126 or SB 203580 for 1 h prior to the addition of M-gp120 or TNIL + LPS + CD40L and continued incubation for 24 h. IL-10 (upper panel) or IL-12p70 (lower panel) production was measured by ELISA after 24 h. The bars represent the mean values ± SEM for data derived from five different donors, which were not the same as the ones used in (A).

Figure 4. Gp120 Impairs iMDDC Maturation via Interaction with an MCLR(s)

The maturation status of MDDCs was evaluated after treatment for 48 h (days 6–8) with TNIL + LPS + CD40L ± 3 μg/ml of JR-FL M-gp120 or D-gp120. The cell surface expression of CD80, CD83, CD86, DC-SIGN, and MR on CD11c+ cells was measured by flow cytometry as described in Supporting Information. (A) The histograms show expression of the surface markers on MDDCs from one donor whose expression marker response to gp120 was of average magnitude. The grey shaded profiles depict the use of isotype control mAbs, the other profiles were derived using the various specific test mAbs. The black curves represent control MDDCs; red curves, MDDCs treated with TNIL + LPS + CD40L; blue curves, TNIL + LPS + CD40L + D-gp120; green curves, TNIL + LPS + CD40L + M-gp120. (B) The average fold-changes (for 14 or 15 donors) in MFI values for MDDC cell-surface marker expression are depicted. The background MFIs obtained with the respective isotype controls were subtracted from MFIs for all conditions. Then the ratios of MFI for presence of gp120 over the MFI for absence of gp120 were calculated. For each marker, the MFI value derived from MDDC matured with TNIL + LPS + CD40L alone is thus defined as 1.0 (log ratio = 0). The means of the 10-logarithms of the ratios for all donors were calculated. The mean log MFI ratio ± SD for cells also treated with either M-gp120 (black bars) or D-gp120 (white bars) are plotted relative to this baseline value corresponding to TNIL + LPS + CD40L alone.

Figure 5. Treatment with gp120 Inhibits MDDC-Induced T Cell Proliferation

(A) Day-6 MDDCs were incubated for 48 h with or without TNIL + LPS and/or JR-FL M-gp120, D-gp120, or influenza HA as specified on the x-axis. The MDDCs were then co-cultured for 5 d with CFSE-labeled CD4+ T cells before determination of the extent of the allogeneic mixed T lymphocyte reaction on day 13. Relative CD4+ T cell proliferation was calculated by first subtracting the background value for CFSE-negative cells obtained using unstimulated iMDDCs (9.95% ± 0.70%, n = 15) from the value obtained when the MDDCs were stimulated with TNIL + LPS (46.4% ± 1.54%, n = 15). The net value was defined as 100% and used for normalization. The bars represent the mean values ± SD for data derived from 15 donors (except for influenza HA; ten donors) tested in 15 independent experiments. The superantigen SEB served as a positive control for CD4+ T cell stimulation in the absence of any MDDCs; CFSE dilution in response to SEB was 130% ± 2.9% (n = 15) of that seen with TNIL + LPS (unpublished data). (B) The extent of T cell proliferation in co-cultures (as in A) containing iMDDCs exposed to M-gp120 + TNIL + LPS + CD40L is plotted as a function of the IL-10 response of the iMDDCs to M-gp120 after 24 h. There was no correlation for iMDDCs from 15 donors (r2 = 0.0008). (C) In a subset of the experiments shown in (A), extracellular cytokine levels were measured at the end of the MDDC-T cell co-culture (i.e., on day 13). The bars represent the mean values ± SEM from five different donors. Upper panel, IL-10; lower panel, IL-12p70.