HIV-1 co-receptors, CCR5 and CXCR4, expression profiles and <i>ex vivo</i> R5 HIV-1 infectivity of CD4⁺ T Cells in hNOJ mice.

<p>(A) Changes in the percentage of CCR5⁺ (left) and CXCR4⁺ (right) cells within the peripheral blood CD4⁺ T cell population isolated from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 18 and <i>n</i> = 6, respectively). Data are expressed as the mean ± SD. (B) Percentage of CCR5⁺ and CXCR4⁺ cells within the naïve, CM, EM_early, and EM_int/late subsets of peripheral blood CD4⁺ T cells isolated from hNOJ mice at 16 wk post-transplantation (<i>n</i> = 18 and <i>n</i> = 6, respectively) and from humans (<i>n</i> = 5). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^***<i>P</i><0.001) were determined by two-way factorial ANOVA with the Bonferroni multiple comparison test. (C, D, E) Fusion assay using R5 HIV-1 and CD4⁺ T cells. Splenic CD4⁺ T cells from hNOJ mice at ≥17 wk post-transplantation (<i>n</i> = 4; three hNOJ (IR+) mice and one hNOJ (IR−) mouse) or peripheral blood CD4⁺ T cells from humans (<i>n</i> = 5) were infected <i>ex vivo</i> with HIV-1_{NL-AD8-D-BlaM-Vpr}. (C) Naïve, CM, and EM subsets of CD4⁺ T cells (gated on CD3⁺CD4⁺CD8⁻) were defined as CD45RA⁺CCR7⁺, CD45RA⁻CCR7⁺, and CD45RA⁻CCR7⁻, respectively, by flow cytometry. (D) Percentage of R5 HIV-1 fusion cells within the total CD4⁺ T cell population and within the naïve, CM, and EM subsets in hNOJ mice and humans. Individual data points are plotted. The black lines represent the mean. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01) were determined by the unpaired <i>t</i> test. (E) Relative ratio of R5 HIV-1 fusion among the naïve, CM, and EM subsets from hNOJ mice and humans. The level of R5 HIV-1 fusion in each of the CD4⁺ T cell subsets relative to that in the corresponding CM subset. Data are expressed as the mean ± SD. Significant differences (^***<i>P</i><0.001) were determined by Tukey’s multiple comparison test.</p

<i>In vivo</i> R5 HIV-1 infection in hNOJ mice.

<p>hNOJ mice were challenged intravenously with HIV-1_NL-AD8-D and divided into two groups: naïve-rich hNOJ (IR+) mice at 10 wk post-transplantation (<i>n</i> = 7) and memory-rich hNOJ (IR−) mice at ≥12 wk post-transplantation (<i>n</i> = 8), based on the percentage of each individual CD4⁺ T cell subsets at pre-challenge. (A) Weekly analysis of the plasma viral load. Individual hNOJ (IR−) mice are denoted by different colors in this and in the following figures. (B) The plasma viral load at 1 wk post-challenge. Data are plotted individually along with the mean (black lines). Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01) between hNOJ (IR+) mice (<i>n</i> = 7) and hNOJ (IR−) mice in which the plasma viral load was detectable (>5000 VL, <i>n</i> = 6) or all hNOJ (IR−) mice (<i>n</i> = 8) were determined by the Mann-Whitney U test. (C) The absolute number of CD4⁺ T cells in the peripheral blood at pre-challenge [hNOJ (IR+) mice; <i>n</i> = 7 and hNOJ (IR−) mice; <i>n</i> = 8]. Each CD4⁺ T cell subset (Naïve, CM, and EM) was defined as outlined in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053495#pone-0053495-g006" target="_blank">Figure 6</a>. Data are plotted individually along with the mean (black lines). Significant differences (^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by the Mann-Whitney U test. (D) The peak plasma viral load during 5 wk post-challenge [hNOJ (IR+) mice; <i>n</i> = 6 and hNOJ (IR−) mice; <i>n</i> = 7]. Data are plotted individually along with the mean (black lines). Significant differences (^**<i>P</i><0.01) were determined by the Mann-Whitney U test.</p

The number of mice used in the present study.

<p>The number of mice used in the present study.</p

Possible occurrence of HSP of CD4⁺ T Cells in hNOJ mice.

<p>(A) Association between the percentage of hCD45⁺ cells within the PBMC population and that of CD34⁺ cells in the BM cells from hNOJ (IR+) and hNOJ (IR−) mice at ≥16 wk post-transplantation (<i>n</i> = 12 and <i>n</i> = 8, respectively). Spearman’s rank correlation coefficient was used for statistical analysis. (B) Percentage of Ki-67⁺ cells among naïve, CM, EM_early, and EM_int/late subsets of splenic CD4⁺ T cells from hNOJ (IR+) and hNOJ (IR−) mice at ≥16 wk post-transplantation (<i>n</i> = 6 and <i>n</i> = 6, respectively) and from human PBMCs (<i>n</i> = 10). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by Tukey’s multiple comparison test. (C and D) <i>Ex vivo</i> IFN-γ production by CD4⁺ T cells after stimulation with PMA/ionomycin. CD4⁺ T cells were prepared from the spleens of hNOJ (IR+) and hNOJ (IR−) mice at ≥16 wk post-transplantation or from human PBMCs. (C) Representative flow cytometry profiles showing the proportion of IFN-γ⁺ cells within each of the CD4⁺ T cell subsets from a hNOJ (IR+) mouse at 16 wk post-transplantation. (D) Cumulative data showing the percentage of IFN-γ⁺ cells within each of the CD4⁺ T cell subsets from hNOJ (IR+) and hNOJ (IR−) mice and humans (<i>n</i> = 3, <i>n</i> = 3, and <i>n</i> = 4, respectively). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by Tukey’s multiple comparison test.</p

Development of human hematopoietic cells in hNOJ mice.

<p>(A) Changes in the percentage of human CD45⁺ (hCD45⁺) cells within the PBMC population from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 22 and <i>n</i> = 13, respectively). Data are expressed as the mean ± SD. Significant differences (^**<i>P</i><0.01) were determined by the Mann-Whitney U test. (B) Percentage of human CD34⁺ cells within the BM cells isolated from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 5 and 6, respectively) at 8 wk post-transplantation. Data are expressed as the mean ± SD. Significant differences (^**<i>P</i><0.01) were determined by the Mann-Whitney U test. (C) Association between the percentage of hCD45⁺ cells within the PBMC population and that of CD34⁺ cells within the BM cells of hNOJ (IR+) and hNOJ (IR−) mice at 8 wk post-transplantation (11 plots from five hNOJ (IR+) and six hNOJ (IR−) mice). Spearman’s rank correlation coefficient was used for statistical analysis. (D, E) Changes in the percentage of human CD19⁺ B cells, CD14⁺ monocytes, CD4⁺ T cells (CD3⁺CD4⁺CD8⁻ cells), and CD8⁺ T cells (CD3⁺CD4⁻CD8⁺ cells) within the peripheral blood hCD45⁺ cell population (D) or total PBMC populstion (E) from hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 22 and <i>n</i> = 13, respectively). Data are expressed as the mean ± SD. Significant differences (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001) were determined by the Mann-Whitney U test.</p

Influence of irradiation on the survival and growth of hNOJ mice.

<p>Newborn NOJ mice (1−2 days after birth) were irradiated (1 Gy) or not before transplantation with CD34⁺CD133⁺ HSCs isolated from human cord blood. (A) Survival curves for hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 16 and <i>n</i> = 28, respectively). Significant differences (^***<i>P</i><0.001) were determined by the log-rank test. (B) Changes in the body weight of hNOJ (IR+) and hNOJ (IR−) mice (<i>n</i> = 7 and <i>n</i> = 10, respectively). Data are expressed as the mean ± SD. Significant differences (^***<i>P</i><0.001) were determined by the unpaired <i>t</i> test.</p

FACS-based analysis of dual infection in primary CD4⁺ T cells.

<p>(A) FACS analysis of primary CD4⁺ T cells (1) unstimulated, (2) stimulated with 5 µg/ml anti-CD3 and 10 µg/ml anti-CD28 for 24 h (strong activation), (3) stimulated with the same concentrations of anti-CD3 and anti-CD28 for 2 h (medium activation), or (4) stimulated with 10-fold lower concentrations of anti-CD3 and anti-CD28 for 2 h (weak activation). Cells were then infected with equal amounts of X4 and R5 HIV-1 and analyzed at 5 dpi. (B) Quantitative PCR analysis of CD4⁺T cells separately infected with either R5 or X4 HIV-1. R-U5 and U5-Gag was analyzed by qPCR in two donors. The amount of HIV-1–specific DNA per cell was normalized to β-globin gene expression. The data represents the average ±SD of three independent experiments. **, <i>P</i><0.005; ***, <i>P</i><0.0005.</p

Replication of recombinant HIV-1 encoding EGFP or DsRed.

<p>(A) Concentration of X4 HIV-1 (HIV_NL-432), X4 HIV-1 expressing EGFP (HIV_NL-E), X4 HIV-1 expressing DsRed (HIV_NL-D), or R5HIV-1 expressing DsRed (HIV_NLAD8-D) in PHA-stimulated PBMCs of two donors. Virus production was monitored by in-house p24 antigen ELISA. (B) FACS analysis of HIV-1-infected T cells expressing EGFP or DsRed at 7 dpi.</p

Infectivity of HIV-1 in MDDCs.

<p>(A) Surface expression of CCR5 and CXCR4 in MDDCs. MDDCs were stained with anti-CCR5 (left) and anti-CXCR4 mAb (right), or with isotype control mAbs (dotted line). The reproducible representative of the FACS profiles of several individuals is depicted. (B) Quantitative PCR analysis of R5 (HIV_NLAD8-D) and X4 (HIV_NL-E) HIV-1-infected MDDCs. Data represent the average ±SD of three independent experiments.</p

Genomic structure and co-receptor usage of recombinant HIV-1 encoding EGFP or DsRed.

<p>(A) The structure of provirus DNA encoding <i>EGFP</i> or <i>DsRed</i> designated as pNL-E and pNL-D for X4 HIV-1 and pNLAD8-D for R5 HIV-1. EGFP or DsRed is not expressed as a fusion protein of Env because of one base insertion after the Env stop codon. Nef is also independently expressed under the control of IRES. To confirm the coreceptor usage of these fluorescent HIV-1, 1G5 (B) cells, 1G5/CCR5 (C) cells were infected with HIV-1_NL432 (parent strain), HIV-1_NL-E, HIV-1_NL-D, or HIV-1_NLAD8-D. After 48 h post-infection, cell lysates were prepared and the Luc assay was performed. The data represents the averages ±SD of three independent experiments.</p