Fig 2: Uptake of infectious virus into VCCs of human macrophages.NL delta Env virus was pseudotyped with macrophage-tropic BaL Env and added to MDM supernatants at 120 TCID50 per MDM in the presence (B, D) or absence (A, C) of 10µM concentration of the entry inhibitor BMS-626529. Cells were fixed and immunostained for p24 and CD9 (A, B) or p24 and Siglec-1 (C, D) 24 hours following addition of virus.

Fig 3: Model for roles of Siglec-1 and tetherin in capture and internalization of HIV-1 virions to the VCC.(A) Siglec-1 proteins are shown binding to GM3 gangliosides on virion envelope. Tetherin is indicated in the chain on the left, not to scale, illustrating how retention by tetherin followed by capture/internalization by Siglec-1 may occur. Free virions arising from the infected cell or from the surrounding environment are also captured by Siglec-1 (right). (B) Following capture by Siglec-1, virions are internalized and co-transported with Siglec to the VCC. Shown is a single channel leading to the VCC.

Fig 4: Confirmation of novel SSM markers in regional lymph nodes from head and neck cancer patients with multiplex immunohistochemistry. (A) Sequential staining for Hematoxylin (left), CD169, CD16A and CD64 on same lymph node section shows the co-localization of the three markers. Of note, CD169+ macrophages in closer proximity to the sub-capsular sinus were also positive for CD16A and CD64. (B) Sequential staining for Hematoxylin (left), CD169, CD16A and MMP14 on same lymph node section shows the co-localization of the three markers. N = 2; Representative pictures are shown.

Fig 5: Siglec-1 RNAi reduces VCC volumes in HIV-1 NLUdel-infected MDMs.(A) MDMs were infected with VSV-G pseudotyped NLUdel. On the following day, MDMs were transfected with 60 nM scrambled, Siglec-1 or tetherin siRNA. At day 10 post-infection, MDMs were washed, fixed with 4% PFA and immunostained for p24 (green), Siglec-1 (red), tetherin (magenta) and DAPI co-stained. Size bars = 10 µm. (B) P24 VCC volume/cell from 30 NLUdel infected MDMs from each group were quantified using the Volocity 6.3 measurement module. Mean +/- SD shown for each RNAi group. (C) Scatter plot displaying individual p24 VCC volume measurements and mean from (B). (D) p24 release from Siglec-1, tetherin or control siRNA treated and NLUdel-infected MDMs was assessed over 12 days using a p24 ELISA. The efficiency of particle release is plotted as percentage of extracellular p24/total p24 from 3 experiments, with standard deviations indicated. Arrows indicate two timepoints when the media was changed, resulting in a transient drop in the ratio of extracellular p24/total p24.