FIG 4 .

CCR2, JAM-A, and ALCAM are therapeutic targets to block and reduce preferential transmigration of HIV⁺ CD14⁺ CD16⁺ monocytes. (A to H) HIV-infected (both HIV⁺ and HIV^exp) mature CD14⁺ CD16⁺ monocytes (1.5 × 10⁵) were added to the BBB model in the presence or absence of 100 nM CVC or 20 μg/ml anti-JAM-A or anti-ALCAM antibody or an IgG1 isotype control antibody. Cells were allowed to transmigrate in response to 200 ng/ml CCL2 for 16 h. A subset of CD14⁺ CD16⁺ monocytes was stained with CD14 and CD16 for analysis by flow cytometry pretransmigration, and DNA was collected from 10⁶ CD14⁺ CD16⁺ monocytes to quantify the number of HIV Gag copies pretransmigration. Transmigrated cells were collected from the bottom of 4 quadruplicate wells for flow cytometric analysis and transmigrated cells from 10 wells for qPCR HIV DNA analysis. Data are represented relative to baseline (media). (A) Schematic representation of expected transmigration results. The graph indicates the expectation of transmigration analyzed by flow cytometry, determining the number of HIV-infected (HIV⁺ and HIV^exp) CD14⁺ CD16⁺ monocytes that transmigrated across the BBB. (B, left panel) Schematic representation of the expected results by qPCR HIV DNA assay if there is no preferential blocking of HIV⁺ CD14⁺ CD16⁺ monocytes by CVC or the anti-JAM-A or anti-ALCAM blocking antibodies. If there is no preferential blocking, the ratio of HIV⁺ and HIV^exp cells would be the same posttransmigration as pretransmigration, and all posttransmigration conditions would have the same EF. (B, right panel) Schematic representation of the expected results by qPCR HIV DNA assay if there is preferential blocking of HIV⁺ CD14⁺ CD16⁺ monocytes by CVC or the anti-JAM-A or anti-ALCAM blocking antibodies. Preferential blocking of HIV⁺ CD14⁺ CD16⁺ monocytes would mean there would be fewer or no HIV⁺ cells in the posttransmigration population of the blocking conditions, and the EF would be reduced. (C, E, and G) Quantification by flow cytometry of the number of CD14⁺ CD16⁺ monocytes that transmigrated across the BBB in the presence or absence of (C) CVC (n = 8), (E) anti-JAM-A or IgG1 isotype control antibody (n = 7), and (G) anti-ALCAM or IgG1 isotype control antibody (n = 9). (D, F, and H) qPCR HIV DNA analysis quantified the number of HIV Gag copies per 10⁶ CD14⁺ CD16⁺ monocytes pretransmigration (pre-trans) and posttransmigration. An EF was calculated for BBB transmigration assays with (D) CVC (n = 8), (F) anti-JAM-A antibody or IgG1 isotype control (n = 7), and (H) anti-ALCAM antibody or IgG1 isotype control (n = 9). Data are represented as mean ± SEM. Significance was determined by Wilcoxon’s signed-rank test. *, P < 0.05; **, P < 0.01; NS, not significant.