Immune activation and up-regulation of endogenous <i>A3H</i> expression in the human CD4⁺ T cells of infected humanized mice.

<p>(<b>A, B</b>) RNA-seq of the splenic human MNCs of infected humanized mice. (<b>A</b>) RNA-seq. Results of differentially expressed gene analysis from the splenic human MNCs of HIV-1-infected (n = 4) and mock-infected (n = 4) humanized mice are shown as a heatmap. (<b>B</b>) GSEA analysis. The procedure is described in Material and Method, and the top 50 annotations are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s018" target="_blank">S8 Table</a>. The vertical broken line indicates <i>Q</i> value = 0.05. (<b>C, D</b>) <i>A3H</i> expression in the human CD4⁺ T cells of infected mice. (<b>C</b>) Flow cytometry. Human MNCs were isolated from the spleen of HIV-1-infected (n = 6) and mock-infected (n = 6) humanized mice and analyzed the proportion of CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ cells) and activated CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁺ cells) by flow cytometry. Representative dot plots (left), the percentage of CD3⁺ CD8⁻ cells in CD45⁺ cells (middle) and the percentage of CD25⁺ cells in CD3⁺ CD8⁻ cells (right) are respectively shown. *<i>P</i> < 0.05 versus mock-infected mice by Mann-Whitney U test. In panel <b>C</b>, the numbers on each dot plot indicates the percentage of gated cells. (<b>D</b>) Real-time RT-PCR of <i>A3H</i>. Activated CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁺ cells) and non-activated CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁻ cells) of mock-infected mice (n = 6 each) and CD25⁻ CD4⁺ T cells (CD45⁺ CD3⁺ CD8⁻ CD25⁻ cells) of HIV-1-infected mice (n = 6) were sorted using FACSJazz (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006348#ppat.1006348.s007" target="_blank">S7 Fig</a>). The mRNA expression level of <i>A3H</i> in each population was analyzed by real-time RT-PCR as described in Materials and Methods. The value of CD25⁻ CD4⁺ T cells of mock-infected mice is set as 1. *<i>P</i> < 0.05 versus CD25⁻ CD4⁺ T cells of mock-infected mice by Mann-Whitney U test. NS, no statistic difference. Note that CD25⁺ CD4⁺ T cells of infected mice were not available because this fraction was severely depleted (panel <b>C</b>).