Selective Transmission of R5 HIV-1 over X4 HIV-1 at the Dendritic Cell–T Cell Infectious Synapse Is Determined by the T Cell Activation State

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against HIV. On the other hand, due to the susceptibility of DCs to HIV infection, virus replication is strongly enhanced in DC–T cell interaction via an immunological synapse formed during the antigen presentation process. When HIV-1 is isolated from individuals newly infected with the mixture of R5 and X4 variants, R5 is predominant, irrespective of the route of infection. Because the early massive HIV-1 replication occurs in activated T cells and such T-cell activation is induced by antigen presentation, we postulated that the selective expansion of R5 may largely occur at the level of DC–T cell interaction. Thus, the immunological synapse serves as an infectious synapse through which the virus can be disseminated in vivo. We used fluorescent recombinant X4 and R5 HIV-1 consisting of a common HIV-1 genome structure with distinct envelopes, which allowed us to discriminate the HIV-1 transmitted from DCs infected with the two virus mixtures to antigen-specific CD4+ T cells by flow cytometry. We clearly show that the selective expansion of R5 over X4 HIV-1 did occur, which was determined at an early entry step by the activation status of the CD4+ T cells receiving virus from DCs, but not by virus entry efficiency or productivity in DCs. Our results imply a promising strategy for the efficient control of HIV infection

Expansion of activated memory CD4+ T cells affects infectivity of CCR5-tropic HIV-1 in humanized NOD/SCID/JAK3null mice.

Humanized mice reconstituted with human hematopoietic cells have been developed as an experimental animal model for human immunodeficiency virus type 1 (HIV-1) infection. Myeloablative irradiation is usually performed to augment the engraftment of donor hematopoietic stem cells (HSCs) in recipient mice; however, some mouse strains are susceptible to irradiation, making longitudinal analysis difficult. We previously attempted to construct humanized NOD/SCID/JAK3(null) (hNOJ) mice, which were not irradiated prior to human HSC transplantation. We found that, over time, many of the reconstituted CD4(+) T cells expanded with an activated effector memory phenotype. Therefore, the present study used hNOJ mice that were irradiated (hNOJ (IR+)) or not (hNOJ (IR-)) prior to human HSC transplantation to examine whether the development and cellularity of the reconstituted CD4(+) T cells were influenced by the degree of chimerism, and whether they affected HIV-1 infectivity. Indeed, hNOJ (IR+) mice showed a greater degree of chimerism than hNOJ (IR-) mice. However, the conversion of CD4(+) T cells to an activated effector memory phenotype, with a high percentage of cells showing Ki-67 expression, occurred in both hNOJ (IR+) and hNOJ (IR-) mice, probably as a result of lymphopenia-induced homeostatic expansion. Furthermore, when hNOJ (IR+) and hNOJ (IR-) mice, which were selected as naïve- and memory CD4(+) T cell subset-rich groups, respectively, were infected with CCR5-tropic HIV-1 in vivo, virus replication (as assessed by the plasma viral load) was delayed; however, the titer subsequently reached a 1-log higher level in memory-rich hNOJ (IR-) mice than in naïve-rich hNOJ (IR+) mice, indicating that virus infectivity in hNOJ mice was affected by the different status of the reconstituted CD4(+) T cells. Therefore, the hNOJ mouse model should be used selectively, i.e., according to the specific experimental objectives, to gain an appropriate understanding of HIV-1 infection/pathogenesis

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

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

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