Neutralizing antibodies inhibit HIV-1 infection of plasmacytoid dendritic cells by an FcγRIIa independent mechanism and do not diminish cytokines production

Abstract

Plasmacytoid dendritic cells (pDC) expressing FcγRIIa are antigen-presenting cells able to link innate and adaptive immunity and producing various cytokines and chemokines. Although highly restricted, they are able to replicate HIV-1. We determined the activity of anti-HIV-1 neutralizing antibodies (NAb) and non-neutralizing inhibitory antibodies (NNIAb) on the infection of primary pDC by HIV-1 primary isolates and analyzed cytokines and chemokines production. Neutralization assay was performed with primary pDC in the presence of serial antibodies (Ab) concentrations. In parallel, we measured the release of cytokines and chemokines by ELISA and CBA Flex assay. We found that NAb, but not NNIAb, inhibit HIV-1 replication in pDC. This inhibitory activity was lower than that detected for myeloid dendritic cells (mDC) infection and independent of FcγRIIa expressed on pDC. Despite the complete protection, IFN-α production was detected in the supernatant of pDC treated with NAb VRC01, 4E10, PGT121, 10-1074, 10E8, or polyclonal IgG44 but not with NAb b12. Production of MIP-1α, MIP-1β, IL-6, and TNF-α by pDC was also maintained in the presence of 4E10, b12 and VRC01. These findings suggest that pDC can be protected from HIV-1 infection by both NAb and IFN-α release triggered by the innate immune response during infection.

Figure 1. Primary pDC express FcγRII and are productively infected by HIV-1.

(A) The percentage of FcγRII-positive cells was determined in living CD123-positive pDC; isotype controls are in grey and FcγRII staining is the black line. (B) Dot plot representation of pDC analysis by flow cytometry. Events were sorted using forward and side scatter (B, left), doublet events were excluded and only living cells, selected by Live/Dead exclusion, were included in the analysis (B, right). (C) Productive infection was detected by intracellular immunolabeling of the p24 HIV antigen in living CD123-positive pDC (in blue). Dot plots show non-infected controls (Mock, left), HIV-1_BaL replication (middle, in pink), and negative controls for HIV-1_BaL replication with reverse transcriptase inhibitor AZT (5 µM) (right). (D) Viability of primary pDC during neutralization assay: primary pDC were stained with Live/Dead to ensure viability of the cells. Means of the percentage of alive CD123-positive cells is represented for non-infected control (Mock), HIV-1_BaL infected cells (HIV), infected cells treated with the reverse transcriptase inhibitor AZT (5 µM), and cells were infected in the presence of various concentrations of anti-HIV Abs. (E) Maturation of primary pDC during neutralization assay: maturation of primary pDC was analyzed by CD83 labeling. Mean of the CD83 MFI (mean of fluorescence intensity) measured in each experiment is represented for pDC treated under the same conditions described in (D). One-way ANOVA showed no significant difference between these conditions. Data are means ± standard deviation (SD) of independent experiments performed with primary pDC from at least three different healthy donors. Groups were compared by one-way ANOVA (Kruskal-Wallis test), with P < .05 considered significant.

Figure 2. Antibody-mediated inhibition of pDC infection.

(A) The percentage of infection of pDC by HIV-1_BaL in the presence of various concentrations of NAb VRC01 and 2F5 compared to control without NAb is represented. The IC₉₀ (dotted line) corresponds to the concentration of Ab needed to inhibit 90% of the infection. (B) The IC₉₀ (µg/ml) measured with NAb 2F5 and VRC01 are represented. Data are the means ± SD of independent experiments performed with cells from 2F5 (n = 7 donors) and VRC01 (n = 6 donors) for (A) and (B). The percentage of infection of pDC by HIV-1_BaL in the presence of the most recent broadly anti-gp120 NAb PGT121 (C), 10-1074 (D) and anti-gp41 NAb 10E8 (E) compared to control without Ab are represented. Results of one representative experiment. (F) The percentage of infection of pDC by HIV-1_BaL in the presence of anti-gp41 Ab 4B3, 2F5 and 4E10 compared to control without Ab is represented. Ab were tested together on pDC from two different healthy donors and data represent the means ± SD of two independent experiments.

Figure 3. Role of FcγRII in the inhibition of infection of primary pDC by HIV-1.

(A) Data represent the percentage of pDC infected by HIV-1_BaL in the presence of various concentrations of NAb b12 or LALA (µg/ml) compared to the control condition without NAb. Data are representative of three independent experiments with primary pDC from three different healthy donors. (B) Detection of FcγRII on pDC (black line) or after 30 minutes of incubation with anti-FcγRII Ab (10 µg/ml) (grey line, arrowhead); isotype control in grey. (C) pDC were pre-incubated with or without anti-FcγRII Ab (10 µg/ml) for 30 minutes at 37°C before being added to the HIV-1_BaL/VRC01 mix. Percentage of infected cells in the presence of NAb VRC01 compared to the control of infected cells in the absence of NAb is shown. Data are means ± SD of two independent experiments performed with primary pDC purified from blood of two healthy donors.

Figure 4. Production of IFN-α by primary pDC during neutralization assay.

The supernatants of pDC infected in the presence or absence of different Ab were collected at 48 h post-infection. Histograms represent the concentration of IFN-α detected in the supernatants (black bars, left axis) of pDC incubated alone (Mock) or with HIV-1_BaL (HIV) or with HIV-1_BaL in the presence of NAb b12 or LALA (A), VRC01 (B), 4E10 (C), IgG purified from serum of HIV-seronegative (IgG Neg) and HIV-1-posive (IgG44) individuals (D), and the most recent broadly NAb PGT121, 10-1074 and 10E8 (E). In parallel, the percentage of infected pDC in the presence of NAb compared to the control condition without NAb is shown (triangle, right axis). Data are means ± SD for IFN-α (ng/ml), representative of three independent experiments performed with primary pDC from three healthy blood donors.

Figure 5. Kinetics of IFN-α production by primary pDC during neutralization assay.

Supernatants of the neutralization assays performed with pDC were collected 6, 12, 24 and 48 h and screened by ELISA for IFN-α. The concentration of IFN-α was measured in the supernatants of pDC uninfected (Mock) or incubated with HIV-1_BaL (HIV) or incubated with HIV-1_BaL in the presence of two concentrations of NAb b12 and LALA (A), 4E10 (B), or VRC01 (C). Data are representative of three independent experiments performed with primary pDC from three healthy blood donors.

Figure 6. Production of MIP1-α, MIP1-β, IL-6 and TNFα by primary pDC during neutralization assay.

Supernatants of pDC were collected after 48 h of infection and screened by CBA Flex to measure MIP1-α, MIP1-β, IL-6 and TNFα. The concentration of MIP1-α, MIP1-β, IL-6 and TNFα in the supernatants from pDC incubated alone (Mock) or with HIV-1_BaL (HIV) or with HIV-1_BaL in the presence of NAb 4E10, b12 or VRC01 is shown. Data are representative of three independent experiments performed with primary pDC from three healthy blood donors.

Figure 7. Kinetics of MIP1-α, MIP1-β, IL-6 and TNFα production by primary pDC during neutralization assay.

The concentration of MIP1-α (A), MIP1-β (B), IL-6 (C) and TNFα (D) were determined by CBA Flex in the supernatants of pDC collected at different time (6, 12, 24 and 48 h) following incubation without virus (Mock) or with HIV-1_BaL (HIV) or with HIV-1_BaL in the presence of two concentrations of NAb 4E10 or VRC01. Data are representative of three independent experiments performed with primary pDC from three healthy blood donors.