Ccr5 apc

Cervical cells were obtained from Digene cytobrushes, processed within 4 hours, and stained immediately [11 (link)]. The antibodies included: APC-H7-CD3, BV605-CCR6, and APC-CCR5 (all from BD Biosciences, San Jose, CA); BV711-CD8, PE-CCR10, and Alexa-700-human leukocyte antigen (HLA) DR isotope (Biolegend, San Diego, CA); PE-Cy5.5-CD4 (Invitrogen, Carlsbad, CA); and PE-Cy7-cluster of differentiation 38 (CD38) (eBioscience, San Diego, CA). Staining for LIVE-DEAD cells, Pac-blue-CD14, and Pac-blue-CD19 was performed to exclude dead cells, monocytes, and B-cells, respectively (all from Invitrogen). Cells were acquired using the FORTESSA (BD Immunocytometry Systems, San Jose, CA). FlowJo v9.9.3 (FlowJo, LLC, Ashland, OR) was used for the data analysis. The gating strategy is shown in Supplementary Figure 1.

Cervical cells were obtained from Digene cytobrushes, processed within 4 h, and stained immediately to measure T cell frequencies and activation by flow cytometry, as previously described (21 (link)). The gating strategy is shown in Fig. S1 in the supplemental material. The following panel of antibodies was included: APC-H7-CD3, BV605-CCR6, and APC-CCR5 (BD Biosciences); BV711-CD8, PE-CCR10, and Alexa-700-human leukocyte antigen (HLA) DR isotope (BioLegend); PE-Cy5.5-CD4 (Invitrogen); and PE-Cy7-cluster of differentiation 38 (CD38) (eBioscience, Inc.). LIVE-DEAD cell, Pacific-blue-CD14, and Pacific-blue-CD19 (Invitrogen) staining were performed to exclude dead cells, monocytes, and B-cells, respectively. Cells were acquired using an LSRFFortessa (BD Immunocytometry Systems). FlowJo v9.9.3 (FlowJo, LLC) was used for the data analysis.

Macaque PBMCs (2 × 10⁶) isolated from peripheral blood were stained to determine T cell, B cell, and monocyte populations. PBMCs were washed with Roswell Park Memorial Institute Media (RPMI)-1640 + 10% FBS and resuspended in 100 μL of PBS. The cells were stained with antibodies (1 μg) for 30 min at room temperature with the following antibody panel (panel 1; Supplementary Figure S1): AmCyan-Live/Dead (Invitrogen, Grand Island, NY, USA); Qdot 655-CD8; PerCP-CD3 (BD Biosciences, San Jose, CA, USA); PE-CD45; Pacific Blue-CD14; APC-Cy7-CD4; PE-Cy7-CD16 (Biolegend, San Diego, CA, USA); ECD-CD20 (Beckman-Coulter, Brea, CA, USA) FITC-CD38 (StemCell Technologies, Vancouver, BC, Canada); APC-CD66 (Miltenyi Biotech, Auburn, CA, USA). PBMCs were stained for 30 min at room temperature to assess SIV co-receptor expression with the following panel (panel 2; Supplementary Figure S2): AmCyan-Live/Dead (Invitrogen, Grand Island, NY, USA); Qdot 655-CD8; PerCP-CD3 (BD Biosciences, San Jose, CA, USA); APC-Cy7-CD4 (Biolegend, San Diego, CA, USA); AF488-CXCR4 (R&D Systems); APC-CCR5 (BD Biosciences). Cells were washed with 2 mL of PBS, re-suspended in 300 μL of PBS + 1% paraformaldehyde (PFA), and acquired on an LSRII (BD Biosciences). Data were analyzed using FACS DIVA and FlowJo (TreeStar, Ashland, OR, USA) software.