Fig 2: CD169, as an HIV-1 receptor, mostly impacts transfer from mature MDDCs rather than immature MDDCs. (A) Flow cytometry plots showing CD86, DC-SIGN, and CD169 expression levels on MDDCs (pre-gated on SSC FSC, living cells, CD3− cells and singlets). Panels show the expression of these proteins in iMDDCs (left panel) and MDDCs with LPS pretreatment at 100 ng/ml for 48 or 24 h before co-culture (middle and right panels, respectively). (B) Percentage of variation of HIV-1 transfer when using iMDDCs or LPS-treated MDDCs (100 ng/ml LPS for different lengths of time) incubated with a blocking antibody against CD169 as compared to an isotype control for each condition. Results are displayed for 4 different blood donors with the mean ± SD of technical triplicates. (C) Percent of variation in HIV-1 transfer to assess the impact of blocking CD169 and TSPAN7 knockdown as compared to scramble shRNA on MDDCs matured with LPS for 48 h treated by an isotype control. Mean ± SD of seven different blood donors in 4 experiments. (B,C) NS, not significant. **p < 0.01; ***p < 0.001. (D) Confocal microscopy images of iMDDCs (left panel) and mature MDDCs (mMDDCs) right panel, to assess the degree of colocalization between CD169 (magenta) and incoming X4–HIV-1–Gag-iGFP (green). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue). Four hundred nanometers of Z-stacks were taken 40 h after the start of the co-culture with CD4+ T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.

Fig 3: Validation of proteomic data. (A) Proteomic quantitation of a subset of proteins stimulated by IFNα2a in primary CD14+ monocytes. Each point represents a single donor. Data is a subset of that shown in Figure 3A , and Table S1 . (B) Flow cytometry-based validation of IFNα2a-stimulated change in BST2 expression at the surface of primary CD14+ monocytes (top) and CD4+ T cells (bottom). Similar data from an additional donor is shown in Figure S7A . (C) Validation by flow cytometry of a selection of proteins stimulated by IFNα2a in primary CD14+ monocytes. Early activation antigen CD69, CD38, CD40, sialoadhesin SIGLEC1 and programmed cell death 1 ligand 1 (CD274) were quantified in CD14+ monocytes from one donor, while SLAMF7 was quantified in cells donated by a second individual. Red (unstimulated) and blue (stimulated with IFNα2a) coloring as in Figure 5B . For SLAMF7 staining, the gray line represents a control sample stained only with secondary antibody (anti-mouse-AF647). For all other samples, the gray line represents unstained samples as directly conjugated antibodies were employed. Similar data from two additional donors is shown in Figures S7B , (C). (D) Proteomic quantitation of cell surface ECE1 in different cell types. Full data is given in Table S1 . (E) ECE1 protein is upregulated in the whole cell protein lysate from IFNα2a-stimulated primary CD4+ T cells. Data is from a single donor. (F) qPCR confirming IFNα2a-stimulated upregulation of ECE1 in primary CD4+ T cells, at the level of mRNA (Data is from a single donor, with three technical replicates). (G) Immunoblot confirming IFNα2a-stimulated upregulation of ECE1 in whole cell protein lysates from two cultured T cell lines Jurkats and SUPT1s (n=1). (H) Immunoblot after immunoprecipitation (IP) of biotinylated cell surface proteins confirming IFNα2a-stimulated upregulation of ECE1 at the cell surface of Jurkat T cells. ‘Whole cell input’ represents protein lysates prior to PM enrichment, and ‘PM IP’ represents PM-enriched samples. For the quantitation shown in the bar chart below the immunoblot, signals from the ECE1 bands following immunoprecipitation were normalized according to the relative amounts of PM protein loaded on both lanes of the gel. This was measured using a total protein stain and quantified using Image Studio Lite (n=1). (I) Proteomic quantitation of cell surface TMEM123, demonstrating upregulation by IFNα2a in all cell types. Full data is given in Table S1 . (J) TMEM123 upregulation by IFNα2a was validated in THP-1s by qPCR (three technical replicates).

Fig 4: Role of TSPAN7 and actin nucleation in HIV transfer from mature MDDCs to T cells. (A) Percentage change of HIV-1 transfer following inhibition of actin nucleation by CK-666 (100 μM), compared to DMSO-treated cells; in immature MDDCs (iMDDCs) and MDDCs matured by 100 ng/ml LPS, 48 h before transfer experiment (mMDDCs). Mean ± SD of triplicates for four different healthy blood donors are represented. (B) Percentage change of X4–HIV-1–GFP transfer following TSPAN7 knockdown compared to scramble shRNA-expressing cells, in iMDDCs and MDDCs matured by 100 ng/ml LPS, 48 h before transfer experiments. Mean ± SD of six different healthy blood donors. (C) Percentage change of X4–HIV-1–GFP transfer according to kinetic of maturation of MDDCs [LPS added 48 or 24 h before co-culture or at the time of co-culture (time 0)], compared to iMDDCs. Mean ± SD of six different healthy blood donors. (A–C) NS, not significant. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Confocal microscopy images of MDDCs at different stages of maturation: iMDDCs (left panel); mMDDCs stimulated by LPS (100 ng/ml) 48 h before (middle panel) or at the same time of start of co-culture (right panel). Actin filaments and nuclei were stained with phalloidin (red) and DAPI (blue), respectively and Siglec-1/CD169 was detected using an anti-CD169 antibody conjugated to APC (magenta). Incoming X4–HIV-1–Gag-iGFP particles are seen in green based on (GFP presence). Four hundred nanometers of Z-stacks were taken 20 h after the start of the co-culture with CD4+ T cells and X4–HIV-1–Gag-iGFP. The pictures presented here are from a representative donor from four unrelated blood donors.