Figure 3.

DCs from cervical mucosa mediate viral uptake via Siglec-1 and are detected in vivo. (A) Cervical mononuclear cells isolated from the ectocervix and endocervix of benign hysterectomies were pulsed with VLPs for 18 h at 37°C, extensively washed, labeled with the indicated mAbs and assessed by FACS. Colored gates and arrows indicate populations analyzed, and corresponding matching colors are used to identify bar graphs showing frequencies of those populations. Representative dot plot and frequency of HLA-DR⁺ and Siglec-1⁺ cells on hematopoietic cervical cells. (B) Representative dot plot and frequency of cells capturing HIV-1_Gag−eGFP VLPs among the myeloid HLA-DR⁺ fraction. Smaller dot plot in between depicts the control without VLPs. (C) Representative dot plot showing reduced expression of Siglec-1 in the myeloid HLA-DR⁺ cells not capturing HIV-1_Gag−eGFP VLPs. (D) Representative dot plot of Siglec-1⁺ cells among the cells capturing HIV-1_Gag−eGFP VLPs. Bar graphs show mean values and SEM from the ectocervix and endocervix of 4 to 5 donors. (E) Images of Siglec-1⁺ cervical cells pulsed and labeled as in (A). Cells were acquired by Amnis-imaging FACS, and showed green fluorescent HIV-1_Gag−eGFP VLPs accumulation within a sac-like virus-containing compartment enriched in Siglec-1 (labeled in red). (F) Paraffin-embedded cervical tissue from one viremic HIV-infected woman stained for HIV-1 p24 antigen (labeled in red), Siglec-1 (in green), and nucleus (in blue). Scale bar 50 μm. (Inset panels) zoom in of squared region with distinct fluorescences (scale bar 20 μm). (G) 3D volumetric x-y-z data fields reconstruction of Siglec-1⁺ cells from four distinct areas of the cervical tissue of the viremic HIV-infected woman. Opacity representation of DAPI stained nuclei and fluorescence of the sac-like virus-containing compartment (VCC; white arrows). Right bottom image displays a characteristic cell pattern with p24⁺ dots reflecting viral production (Infection).