Co-expression of inhibitory receptors on CD8⁺ and CD4⁺ T cells in chronically HIV-infected BLT mice.

<p><b>A</b>) Representative flow cytometry data of peripheral blood from an HIV-infected BLT mouse at 13 weeks post infection. Co-expression of CD244, CD160, and LAG-3 with PD-1 was determined on human CD8⁺ and CD4⁺ cells. <b>B</b>) Percentages of PD-1 expressing CD8⁺ and CD4⁺ T cells co-expressing CD244, CD160, and LAG-3 at 13 weeks and 26 weeks post infection. Horizontal lines within data points depict mean values. *<i>P</i> = 0.036, Mann-Whitney test.</p

Effects of anti-PD-1 mAb or control treatment on HIV viral loads in chronically infected BLT mice.

<p>BLT mice infected with HIV-1 for 13 weeks were injected intraperitoneally with anti-PD-1 mAb, control mAb, or no Ab on days 0, 3, 7 and 10 (200 µg/dose, arrow). Peripheral blood was collected at multiple time points and HIV-1 plasma viral load was measured by quantitative RT-PCR. Graph represents mean viral load of Control (n = 10, control mAb or no Ab), anti-PD-1 mAb-treated PD1-LO mice (n = 3), and anti-PD-1 mAb-treated PD1-HI mice (n = 5). *<i>P</i><0.05, Mann-Whitney test.</p

PD-1 expression on T cells in HIV-infected BLT mice.

<p>Peripheral blood was obtained at various time points after HIV infection. <b>A</b>) Percentages of human CD8⁺ and CD4⁺ T cells expressing PD-1. Horizontal lines within data points depict mean value. Uninfected controls were from peripheral blood samples obtained at time points when littermates were infected for 9-13 weeks. (HIV infected: n = 9 mice at wk 3, n = 18 mice at other time points; Uninfected: n = 7 mice). *<i>P</i> = 0.01, Wilcoxon matched pairs test; **<i>P</i> = 0.001, Mann-Whitney test. <b>B</b>) Representative flow cytometry data of PD1 expression on CD8⁺ or CD4⁺ T cells at 13 weeks post infection. PD1-HI representative is mouse #4 and PD1-LO is mouse #1 depicted in the next panel. <b>C</b>) Percentages of CD8+ and CD4+ T cells expressing PD-1 in PD1-LO (defined as having <30% PD-1⁺CD8⁺ cells) and PD1-HI (>30% PD-1⁺CD8⁺ cells).</p

Schematic of BLT humanized mouse generation and timeline of HIV-1 infection and anti-PD-1 mAb treatment.

<p>Schematic of BLT humanized mouse generation and timeline of HIV-1 infection and anti-PD-1 mAb treatment.</p

Colocalization of PD-L1 on DCs to CD80 expressed by HSV-CD80 and its effect on virus replication.

<p><u>A) FACS analyses of infected WT DCs</u>. Subconfluent monolayers of DCs isolated from WT C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-HSV-1 gC, and anti-PD-L1 antibodies and three-color FACS analysis was performed. CD11c⁺ gated cells were analyzed for expression of HSV-1 gC and PD-L1. Experiments were repeated twice; <u>B) FACS analyses of DCs from knockout mice.</u> DCs from C57BL/6 WT, C57BL/6-PD-L1^-/- and C57BL/6-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV-CD80, parental virus, or mock infected. At 24 hr PI, cells were harvested and reacted with anti-CD11c, anti-CD80, and anti-PD-L1 antibodies and three-color FACS analysis was performed. CD11c⁺ gated cells were analyzed for expression of CD80 and PD-L1. Experiments were repeated twice; <u>C) Immunostaining of DCs from knockout mice</u>. DCs from WT BALB/c, BALB/c-PD-L1^-/- and BALB/c-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV-CD80 or parental virus. At 24 hr PI, cells were reacted with anti-HSV-1 gC, anti-CD80, and anti-PD-L1 antibodies. DAPI is shown as a nuclear counter-stain; <u>D) Replication of HSV-CD80 in DCs isolated from knockout mice</u>. Subconfluent monolayers of DCs isolated from WT BALB/c, BALB/c-PD-L1^-/- and BALB/c-PD-L-2^-/- mice were infected with 1 PFU/cell of HSV- CD80 or parental virus as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. Virus yield was determined at the indicated times by standard plaque assays. Each point represents the mean ± SEM (n = 6) from two separate experiments; and <u>E) Replication of HSV-CD80 in WT C57BL/6 DCs in presence of blocking antibody</u>. Subconfluent monolayers of DCs were incubated with 10F.2H11 antibody, irrelevant antibody, or no antibody and infected with 1 PFU/cell of HSV-CD80 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. Virus yield was determined at the indicated times by standard plaque assays. Each point represents the mean ± SEM (n = 6).</p

Construction and structure of the HSV-CD80 recombinant virus.

<p>(A) The top schematic diagram shows the HSV-1 McKrae genome in the prototypic orientation. TR_L and IR_L represent the terminal and internal (or inverted) long repeats, respectively, and TR_S and IR_S represent the terminal and internal (or inverted) short repeats, respectively. U_L and U_S represent the long and short unique regions, respectively. The solid rectangle represents the very stable 2 kb LAT. (B) dLAT2903 (parental virus for HSV-CD80) has a deletion from LAT nucleotides −161 to +1667 in both copies of LAT and makes no LAT RNA. (C) HSV-CD80 was constructed from dLAT2903 by homologous recombination between dLAT2903 DNA and a plasmid containing the complete LAT promoter and the entire structural CD80 gene (including its 3′ poly(A) signal) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>.</p

Replication of HSV-CD80 recombinant virus in BM-derived DCs.

<p>Subconfluent monolayers of DCs isolated from C57BL/6, 129SVE, and BALB/c mice were infected with 10 PFU/cell of HSV-CD80 or parental virus as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. In some experiments DCs from C57BL/6 mice were infected with 1 PFU/cell of each virus, while DCs from 129SVE were infected with 10 PFU/cell of wt HSV-1 strain McKrae. Virus yield was determined at the indicated times PI by standard plaque assays. Panels: A) DCs from C57BL/6 mice were infected at 1 PFU/cell of HSV-CD80 or parental virus; B) DCs from C57BL/6 mice were infected at 10 PFU/cell of HSV-CD80 or parental virus; C) DCs from 129SVE mice were infected at 10 PFU/cell of HSV-CD80, parental, and wt McKrae viruses; and D) DCs from BALB/c mice were infected at 10 PFU/cell of HSV-CD80 or parental virus. Each point represents the mean ± SEM (n = 6) from two separate experiments.</p

Replication of HSV-CD80 recombinant virus in RS cells.

<p>Subconfluent RS cell monolayers in triplicate from two separate experiments were infected with 1/cell of HSV-CD80 or parental dLAT2903 virus as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. Total virus was harvested at the indicated times PI by two cycles of freeze-thawing. The amount of virus at each time point was determined by standard plaque assays on RS cells. Each point represents the mean + SEM (n = 6).</p

Level of HSV-1 immediate early, early, and CD80 in HSV-CD80 infected DCs.

<p>Subconfluent monolayers of DCs from C57BL/6 mice were infected with 1 PFU/cell of HSV-CD80 or parental virus as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#pone-0087617-g001" target="_blank">Fig. 1</a>. For ICP-0 and TK measurements, cells were isolated 4, 8, and 12 hr PI, while for CD80 measurements cells were harvested 2, 4, 8, 12, 24, and 48 hr PI. Total RNA was isolated and TaqMan RT-PCR was performed using ICP0-, TK-, and CD80-specific primers as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087617#s4" target="_blank">Materials and Methods</a>. ICP0 and TK mRNA levels were normalized in comparison to each transcript at 0 hr PI, while CD80 mRNA level was normalized to the level of CD80 in mock infected DCs. GAPDH was used as internal control. Each point represents the mean ± SEM (n = 6) from two separate experiments. Panels: A) ICP-0; B) TK, and C) CD80.</p

CD160 is expressed on CD8 and CD4 T cells.

<p>Naïve or stimulated (ConA) splenocytes from wildtype C57BL/6 (WT), CD4^−/−, CD8^−/− and CD28^−/− mice were stained with anti-CD160. Mean fluorescence intensity of CD160 on naïve CD8⁺ and CD4⁺ T cells or CD8⁺ and CD4⁺ T cells expressing an effector/memory phenotype (CD44^highCD62L^low) was analyzed. <b>Upper Panel</b>: Representative dot plots of CD160 expressing cells (shaded histograms) in WT mice. <b>Lower Panel</b>: The histograms demonstrate the MFI of CD160⁺ cells of the overall CD4⁺ or CD8⁺ T cell population as the mean ± SEM of 3–5 independent experiments.</p