Interferon-inducible mechanism of dendritic cell-mediated HIV-1 dissemination is dependent on Siglec-1/CD169

Abstract

Human immunodeficiency virus type 1 (HIV-1) interactions with myeloid dendritic cells (DCs) can result in virus dissemination to CD4⁺ T cells via a trans infection pathway dependent on virion incorporation of the host cell derived glycosphingolipid (GSL), GM3. The mechanism of DC-mediated trans infection is extremely efficacious and can result in infection of multiple CD4⁺ T cells as these cells make exploratory contacts on the DC surface. While it has long been appreciated that activation of DCs with ligands that induce type I IFN signaling pathway dramatically enhances DC-mediated T cell trans infection, the mechanism by which this occurs has remained unclear until now. Here, we demonstrate that the type I IFN-inducible Siglec-1, CD169, is the DC receptor that captures HIV in a GM3-dependent manner. Selective downregulation of CD169 expression, neutralizing CD169 function, or depletion of GSLs from virions, abrogated DC-mediated HIV-1 capture and trans infection, while exogenous expression of CD169 in receptor-naïve cells rescued GSL-dependent capture and trans infection. HIV-1 particles co-localized with CD169 on DC surface immediately following capture and subsequently within non-lysosomal compartments that redistributed to the DC--T cell infectious synapses upon initiation of T cell contact. Together, these findings describe a novel mechanism of pathogen parasitization of host encoded cellular recognition machinery (GM3--CD169 interaction) for DC-dependent HIV dissemination.

Figure 1. Dendritic cell activation by TRIF-dependent TLR ligands or IFNα induces CD169 expression and enhances HIV-1 capture.

A. Capture of VLPs by monocytes, immature DCs or DCs matured with Pam₃Cysk₄, poly(I:C), LPS, IFNα, or TNFα was determined by FACS and is reported as relative capture by cells normalized to that observed with monocytes (mean ± SD). B. Representative expression of DC-SIGN and CD169 on monocytes, immature DCs (day 6 post differentiation), or DCs matured with LPS (last 2 days of differentiation). For each day, cells were gated based on staining with isotype. QFACS measurement of cell surface CD169 (C) and DC-SIGN (D) on immature and LPS-matured DCs. Shown are the average number of ABS per cell (mean ± SD). E. Relative cell surface expression of CD169 on immature DCs or DCs matured with Pam₃Cysk₄, poly(I:C), LPS, IFNα, or TNFα normalized to that observed with monocytes (set as 1), (mean MFI ± SD). The data is from cells derived from 3 independent donors and experiments performed in triplicate.

Figure 2. CD169 mediates HIV-1 capture and trans infection by DCs.

Representative FACS analysis of CD169 (A), DC-SIGN (B), or CD86 (C) expression on DCs transduced with lentivectors expressing scrambled sequence, CD169 or DC-SIGN shRNAs. Relative capture by DC-SIGN or CD169 shRNA expressing DCs to that observed with scrambled shRNA expressing DCs of HIV Gag-eGFP VLPs (D), HIV/Lai-iGFPΔenv (E), and HIV/Lai-iGFP (F) is reported. DC mediated HIV/Lai capture (G) and transfer to autologous CD4⁺ T cells (H) ± isotypes or nAbs against CD169, DC-SIGN, or both. Shown is the average ± SD of cells derived from at least 4 donors (A through F) or one of two experiments done in triplicate (G and H).

Figure 3. Captured HIV-1 particles co-localize with CD169 on DCs.

Mature DCs were either (A) mock infected or exposed to Gag-mCherry VLP for (B) <10 minutes, (C) 120 minutes, or (D) 60 minutes with an additional 60 minute co-culture with autologous CD4⁺ T cells. Representative deconvolved maximum intensity images are shown. Top row = Gag-mCherry (red), second row = CD169 (green), third row = composite images (T cells in blue), bottom row = bright-field. Co-localization is reported as the average Mander's coefficient ± SD. M1 = ratio of Gag-mCherry associated with CD169, M2 = ratio of CD169 that has associated Gag-mCherry.

Figure 4. CD169 is the GSL-dependent HIV-1 attachment factor on inflammatory DCs necessary for virus capture and trans infection.

Representative FACS analysis of CD169 (A) and DC-SIGN (B) expression on IFN-DCs or IL-4 DCs. The shaded histograms represent isotype controls. Capture of untreated (NT) or GSL-depleted (PDMP) HIV Gag-eGFP VLPs by DCs was determined by FACS (C). Capture of untreated (NT) or GSL-depleted (PDMP) HIV/Lai-Bal (D) or virus capture in the presence of isotype or α-CD169 (10 µg/ml) nAb (E) was determined by measuring cell-associated p24^gag. HIV/Lai-Bal (±PDMP) replication in DCs, CD4⁺ T cells, or DC – T cell co-cultures was determined by measuring p24^gag in supernatants harvested 2 days post infection (F). Average ± SD of one of 3 independent experiments done in triplicate.

Figure 5. Peripheral blood myeloid DCs require CD169 for HIV-1 capture.

CD169 expression was determined for (A) untreated and (B) IFNα matured myeloid DCs by FACS. Shaded histograms represent unstained cells. Capture of HIV Gag-eGFP VLPs (C) and HIV/Lai or HIV/LaiΔenv (D) by untreated (NT) or IFNα-exposed myeloid DCs was determined by FACS and ELISA, respectively. Capture of GSL-depleted (PDMP) or untreated HIV/Lai particles (E) in the presence of isotype or α-CD169 nAb (F) by IFNα exposed myeloid DCs was determined by measuring cell-associated p24^gag. Average ± SD of one of three independent experiments done in triplicate.

Figure 6. Exogenous expression of CD169 rescues GSL-dependent HIV-1 capture and trans infection.

A. CD169 expression on Raji and Raji/CD169 cells was determined by FACS. Shaded histogram represents staining with isotype control. Capture of HIV Gag-eGFP VLP (B) or HIV/Lai and HIV/LaiΔenv (C) by Raji and Raji/CD169 cells was determined by FACS and p24^gag ELISA, respectively. Capture of HEK293T-derived (D) or PBMC-derived (E) HIV/Lai-Bal (±PDMP) by Raji and Raji/CD169 cells was determined by measuring cell-associated p24^gag. F. Raji or Raji/CD169-mediated trans infection of HIV/Lai-Bal-luc (±PDMP) was determined by measuring luciferase activity in Raji/T cell co-cultures 2 days post-infection. Data reported is from one experiment performed in triplicate (mean ± SD) and is representative of 5 independent experiments.

Figure 7. Recognition of α-2,3 sialylated GSL, GM3, by CD169 is essential for HIV-1 capture and trans infection.

A. Capture of fluorescent liposomes by Raji and Raji/CD169 cells was determined by FACS, and is reported as the change in MFI upon challenge with phospholipid-liposome over blank liposomes and is the average MFI ± SD. B. Competitive inhibition of virus capture by Raji/CD169 cells by blank or 1% GM3 containing liposomes was determined by measuring cell-associated p24^gag. C. Expression of human CD169 and mutants in HEK293T cells determined by FACS. Shaded histogram represents staining with isotype control. Capture of HIV/Lai-iGFP (D) and transfer of Lai-Balenv/luc+ (E) by HEK293T cells expressing wildtype-CD169 or mutants (R96A or R116A) were determined by FACS analysis (for %GFP+ cells) and luciferase activity in co-cultures (HEK293T/CD4+ T cells), respectively. The data shown is from one experiment, performed in triplicate (mean ± SD), and is representative of 3 independent experiments.

Figure 8. Retroviral capture and transfer function is conserved between human and mouse CD169.

A. Expression of murine CD169 on HEK293T cells determined by FACS. Capture of untreated (NT) or GSL-depleted HIV Gag-eGFP VLPs (B) or MLV Gag-YFP VLPs (C) by mock transfected or murine CD169 expressing HEK293T cells was determined by FACS. D. Capture of untreated (NT), or GSL-depleted (PDMP) MLV Gag-YFP VLPs, or capture of MLV Gag-YFP VLPs, in the presence of α-CD169 nAb by mDCs was determined by FACS. E. HEK293T/murine CD169 mediated trans infection of Lai-Balenv/luc⁺ to CD4+ T cells (E), or transfer of MLV-E/luc to Rat-2 cells (F) was determined by measuring luciferase activity in HEK293T/CD4⁺ T cell and HEK293T/Rat-2 co-cultures, respectively, 2 days post-infection. G. Transfer of MLV-E/luc by mDCs in the presence or absence of α-CD169 nAb was determined by measuring luciferase activity in mDC/Rat-2 cell co-cultures 2 days post-infection. The data shown is from one experiment, performed in triplicate (mean ± SD), and is representative of 2 independent experiments.