Siglec-1 is up-regulated in highly <i>trans</i>-infecting LPS mDCs.

<p>(A) (Left) Comparative HIV-1 capture of LPS and ITIP mDCs: cells were cultured with HIV-1, washed, and lysed to measure viral p24^Gag antigen by ELISA. (Right) Comparative transmission of captured HIV-1 from LPS and ITIP mDCs to a reporter CD4⁺ cell line. Graphs show mean values and standard error of the means (SEMs) from two independent experiments including cells from six donors. (B) Plot of <i>SIGLEC</i> genes (in open circles), <i>CD86</i> and <i>DC-SIGN</i> (in grey circles) computing the fold change in LPS mDCs compared to ITIP mDCs, and the average gene expression across all samples. Circle size is inversely proportional to adjusted <i>p</i> values. Highlighted in red are statistically differentially expressed genes. Analysis was performed with DCs from four donors matured in parallel with the different stimuli. (C) Relative quantification of <i>SIGLEC1</i> mRNA expression levels in distinct DCs analyzed by qRT-PCR. Measurements were normalized using the endogenous control housekeeping gene <i>Beta Glucuronidase</i>. Data show means and SEMs of samples from six donors. (D) Cell surface expression of Siglec-1 in distinct DCs analyzed by FACS with mAb 7–239-PE. (Left graph) Geometric mean fluorescence intensity (MFI) of Siglec-1. (Right graph) Percentage of Siglec-1 positive cells. Data show mean values and SEM from two experiments, including cells from six donors. (Histograms) Representative profiles of Siglec-1 staining in distinct DCs derived from one donor.</p

Siglec-1 expressed in LPS mDCs capture distinct ganglioside containing vesicles, such as HIV-1 viral-like particles, liposomes, and exosomes.

<p>(A) Relative capture of VLP_HIV-Gag-eGFP by LPS mDCs that had been pre-incubated with 10 µg/ml of the indicated mAbs or 500 µg/ml of mannan before VLP exposure for 30 min at 37°C. Values are normalized to the level of VLP capture by mock-treated LPS mDCs (set at 100%). Data show mean values and SEMs from three experiments including cells from nine donors. (B) Relative capture of GM1 containing LUV_HIV-tRed by LPS mDCs as described in (A). Data show mean values and SEMs from two experiments including cells from six donors. (C) Relative capture of Exosomes_DiI by LPS mDCs that had been pre-incubated with 10 µg/ml of the indicated mAbs before exosome exposure for 4 h at 37°C. Values are normalized to the level of exosome capture by isotype-treated LPS mDCs (set at 100%). Data show mean values and SEMs from two experiments including cells from five donors. (D) Capture of VLP_HIV-Gag-eGFP by LPS mDCs that had been pre-incubated with decreasing concentrations of α-Siglec-1 mAb 7D2 before VLP exposure for 30 min at 37°C. Titration of α-Siglec-1 mAb 7–239 is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s001" target="_blank">Figure S1</a>. Data show mean values and SEMs from three experiments including cells from six donors. (E) Capture of VLP_HIV-Gag-eGFP by LPS mDCs that had been pre-incubated with or without 2 µg/ml of α-Siglec-1 mAb 7D2 previously treated or not with at least a 100-fold molar excess of the indicated human recombinant proteins. Of note, Siglec-14 shares 100% of amino acid homology with Siglec-5 in the V-set domain. Data show mean values and SEMs from three experiments including cells from nine donors. (F) Kinetics of VLP_HIV-Gag-eGFP capture by iDCs (left graph) and LPS mDCs (right graph) compared to the expression of Siglec-1 over time, assessed after LPS addition to mDCs. Cells were pulsed for 1 h at 37°C with VLP_HIV-Gag-eGFP and labeled for Siglec-1 and HLA-DR in parallel at the indicated time points. For comparative purposes, the maximum geometric MFI values obtained by FACS for each donor were set at 100%. Data show mean values and SEMs including cells from three donors. (G) Positive correlation (ρ = 0.9695) between the geometric MFI of captured VLPs and the mean number of Siglec-1 Ab Binding Sites per cell in different DC subtypes (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s002" target="_blank">Figure S2</a> to compare VLP capture capacity among LPS mDCs derived from the same donor). Data show values from three experiments including cells from nine donors.</p

Siglec-1 captures HIV-1 and traffics with the virus to the same sac-like compartment.

<p>(A) Comparative capture of HIV-1 by distinct DCs that had been pre-incubated with 10 µg/ml of the indicated mAbs or 500 µg/ml of mannan for 30 min before viral exposure. Cells were cultured with HIV-1 in the presence of the indicated reagents, washed, and lysed to measure p24^Gag by ELISA. Viral binding at 4°C in LPS mDCs is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s003" target="_blank">Figure S3</a>. Data show mean values and SEMs from two experiments including cells from six donors. (B) Comparative capture of HIV-1 by distinct DCs first exposed to the virus and then treated with the indicated reagents for 30 min before washing. Cells were lysed and assessed by p24^Gag ELISA. Data show mean values and SEMs from two experiments including cells from six donors. (C) Comparative capture of HIV-1 by distinct blood myeloid DCs that had been pre-incubated with 10 µg/ml of the indicated mAbs for 30 min before viral exposure as in panel A. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s004" target="_blank">Figure S4</a> depicts Siglec-1 surface expression levels of blood myeloid cells. Data show mean values and SEMs from two experiments including cells from six donors. (D) Confocal microscopy analysis of LPS mDCs pulsed for 4 h with GM1-containing LUV_HIV-tRed, VLP_{HIV-Gag-Cherry} or HIV-1_Cherry, fixed, permeabilized, and then stained for Siglec-1 with mAb 7–239-Alexa 488. (Inset) Merge of the bright field and maximun fluorescence intensity (scale bar: 5 µm). (3D images) Isosurface representation of DAPI stained nucleus and maximum fluorescence intensity of the sac-like compartment where particles and Siglec-1 accumulate are shown in a 3D volumetric x-y-z data field. (Bar graphs) Quantification of the percentage of GM1-containing LUV_HIV-tRed, VLP_{HIV-Gag-Cherry} or HIV-1_Cherry co-localizing with Siglec-1-Alexa 488 7–239 and vice versa, obtained analyzing at least 50 compartments from LPS mDCs of two donors. The mean and standard deviation of the thresholded correlation coefficient of Pearson (obtained considering all the images) were 0.77±0.07, indicating co-localization. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s010" target="_blank">Movies S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s011" target="_blank">S2</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s012" target="_blank">S3</a> or <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001448#pbio.1001448.s005" target="_blank">Figure S5</a> to observe the compartment in relation to the plasma membrane or the cytoplasm of the cells. (E) Confocal microscopy analysis showing the sac-like compartment pattern of Siglec-1 in LPS mDCs after internalization of the α-Siglec-1 mAb 7D2. Cells were labeled with the mAb for 30 min at 16°C, revealed with an Alexa 488 secondary Ab, shifted to 37°C for 4 h, and analyzed. (3D image) 3D reconstruction (representative of 69% of the analyzed DCs) was done as in (D). (Inset) Merge of the bright field and maximun fluorescence intensity (scale bar: 5 µm).</p