Distinct Phenotype, Longitudinal Changes of Numbers and Cell-Associated Virus in Blood Dendritic Cells in SIV-Infected CD8-Lymphocyte Depleted Macaques

<div><p>Loss of circulating CD123+ plasmacytoid dendritic cells (pDCs) during HIV infection is well established. However, changes of myeloid DCs (mDCs) are ambiguous since they are studied as a homogeneous CD11c+ population despite phenotypic and functional heterogeneity. Heterogeneity of CD11c+ mDCs in primates is poorly described in HIV and SIV infection. Using multiparametric flow cytometry, we monitored longitudinally cell number and cell-associated virus of CD123+ pDCs and non-overlapping subsets of CD1c+ and CD16+ mDCs in SIV-infected CD8-depleted rhesus macaques. The numbers of all three DC subsets were significantly decreased by 8 days post-infection. Whereas CD123+ pDCs were persistently depleted, numbers of CD1c+ and CD16+ mDCs rebounded. Numbers of CD1c+ mDCs significantly increased by 3 weeks post-infection while numbers of CD16+ mDCs remained closer to pre-infection levels. We found similar changes in the numbers of all three DC subsets in CD8 depleted animals as we found in animals that were SIV infected animals that were not CD8 lymphocyte depleted. CD16+ mDCs and CD123+ pDCs but not CD1c+ mDCs were significantly decreased terminally with AIDS. All DC subsets harbored SIV RNA as early as 8 days and then throughout infection. However, SIV DNA was only detected in CD123+ pDCs and only at 40 days post-infection consistent with SIV RNA, at least in mDCs, being surface-bound. Altogether our data demonstrate that SIV infection differently affects CD1c+ and CD16+ mDCs where CD16+ but not CD1c+ mDCs are depleted and might be differentially regulated in terminal AIDS. Finally, our data underline the importance of studying CD1c+ and CD16+ mDCs as discrete populations, and not as total CD11c+ mDCs.</p></div

CD1c+ and CD16+ mDCs, and CD123+ pDCs in whole blood from rhesus macaques.

<p>Upper row: DCs are initially identified based on forward and side scatter (FSC and SSC) profiles of whole blood (<b>A</b>; R1 gate). CD3+ T lymphocytes and CD14+ monocytes are excluded (<b>B</b>; R2 gate) as well as CD20+ B lymphocytes and CD8+ NK cells (<b>B</b>; R3 gate) resulting in the selection of a Lin- population. From this, HLA-DR+ cells are selected (<b>C</b>; R4 gate). <b>D</b>. Within Lin- HLA-DR+ cells, non-overlapping CD1c+ and CD16+ mDCs and CD123+ pDCs are identified. Percentages of positive cells from Lin- HLA-DR+ cells are indicated. <b>E.</b> CD11c is expressed on CD16+ mDCs and at lower levels on CD1c+ mDCs. CD123+ pDCs were negative for CD11c. Grey histograms represent a negative control as CD11c expression by CD3+ T lymphocytes; black histograms show CD11c expression on CD1c+, CD16+ and CD123+ DC. Data are from animal 244–96 analyzed before infection representative of 21 normal rhesus macaques. <b>F</b>. Absolute numbers of DCs per microliter of whole blood determined in normal rhesus macaques. CD16+ mDCs represents the major population of DCs in whole blood of healthy rhesus macaques (left graph and left y-axis). CD1c+ mDCs and CD123+ pDCs are present in lower proportions (right graphs and right y-axis). Data are mean±SD from 21 uninfected animals.</p

Longitudinal assessment of myeloid and plasmacytoid DCs in whole blood of SIV-infected CD8-lymphocyte depleted rhesus macaques.

<p>The absolute cell counts of CD1c+ mDCs (upper row panels), CD16+ mDCs (middle row panels) and CD123+ pDCs (lower row panels) determined over the course of infection are shown. Three independent studies are shown: study I (black symbols and lines; n = 5), study II (grey symbols and lines; n = 4) and study III (black symbols and dotted lines; n = 3). Black arrowheads indicate time of administration of anti-CD8 depleting antibody. <b>A</b>. Absolute DC numbers from individual animals throughout infection. <b>B</b>. Averages of absolute DC numbers throughout infection. <b>C</b>. Individual absolute DC numbers measured in early disease i.e. from pre-infection to 26 dpi including acute (8 dpi) and post-acute phases are shown to emphasize differences between changes in CD1c+ and CD16+ mDC counts. The Kruskal-Wallis test followed by Dunn’s post test was used to determine the significance (asterisks) of differences in absolute numbers during early SIV infection. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001. Box shows symbols for individuals animals.</p

SIV RNA and DNA analysis of FACS-sorted myeloid and plasmacytoid DC subsets from SIV-infected, CD8-lymphocyte depleted rhesus macaques.

<p>CD1c+ mDCs, CD16+ mDCs and CD123+ pDCs from were sorted by flow cytometry before infection (Pre) and at days 8, 21 and 40 post-infection (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119764#pone.0119764.s002" target="_blank">S2 Fig</a> for gating strategy and post-sort purity analysis). CD4+ T lymphocytes (CD4+ T Ly) from 3 animals were sorted as positive controls for SIV infection. Symbols represent individual animals. Sorted cells were analyzed by PCR for SIV gag. Symbols indicate RNA (A) or DNA (B) copy numbers in samples of sorted DC populations and CD4+ T lymphocytes. Samples that gave negative PCR reaction (limit of the PCR assay: 30 total copies per cell sample) are represented as symbols located below threshold detection. All values have been normalized per 10⁵ diploid genome cell equivalents.</p

Longitudinal analyssis of the percentage change in absolute numbers of DCs in SIV infected CD8-lymphocyte depleted rhesus macaques.

<p>Percent changes in absolute numbers of CD1c+ mDCs (<b>A</b>), CD16+ mDCs (<b>B</b>) and CD123+ pDCs (<b>C</b>) were calculated at different time points post-infection by comparison to pre-infection (baseline: horizontal dashed line “0”). Negative percentages indicate cell loss while positive percentages represent a gain in circulating DCs. Percent changes measured at necropsy time (†) are shown as black symbols. Symbols represent individual animals and horizontal bars represent medians. The Kruskal-Wallis test followed by Dunn’s post test was used to determine the significance (asterisks) of differences in percent change absolute numbers during SIV infection. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Unlike CD16+ mDCs and CD123+ pDCs, numbers of CD1c+ mDCs with AIDS are not significantly decreased compared to pre-infection.

<p>Absolute numbers of CD1c+ mDCs (<b>A</b>), CD16+ mDCs (<b>B</b>) and CD123+ pDCs (<b>C</b>) were measured before infection (Pre, white symbols) and at necropsy (†, black symbols) with AIDS are shown. The Wilcoxon rank sum test was used to perform pair-wise analysis for the absolute numbers of DC before infection and at necropsy with AIDS. P values and asterisks indicative of significant differences are shown; n.s., not significant.</p

Modulation of absolute counts and percent changes of DCs in SIV-infected rhesus macaques with no CD8 depletion.

<p><b>A.</b> Absolute numbers of CD1c+ mDCs CD16+ mDCs and CD123+ pDCs were measured before infection (Pre) and after SIV infection of rhesus macaques without depletion of CD8+ cells. Each symbol represents an individual animal. <b>B.</b> Percent changes in absolute numbers of CD1c+ mDCs, CD16+ mDCs and CD123+ pDCs were calculated at different time points post-infection by comparison to pre-infection (baseline: horizontal dashed line “0”). Negative percentages indicate cell loss while positive percentages represent a gain in circulating DCs. Symbols represent individual animals and horizontal bars represent medians. The Friedman test followed by Dunn’s multiple comparison post test was used to determine significant differences in absolute number and percent change during SIV infection. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

Longitudinal flow-cytometry analysis of BLyS/BAFF expression by blood myeloid dendritic cells (mDC), monocytes, CD4⁺ T-cells and granulocytes.

<p>Relative percentages (left panels) and geometric mean fluorescence intensity (GeoMFI) (right panels) of BLyS/BAFF expression on (A) CD16⁺CD1c^- mDC, (B) CD16^-CD1c⁺ mDC, (C) CD14⁺CD16⁺ monocytes, (D) CD14⁺CD16^- monocytes, (E) CD4⁺ T-cells and (F) granulocytes obtained from the blood of 5 SIV-infected rhesus macaques. Values were compared to that obtained at pre-infection using the paired Student's <i>t</i> test (*) or between time points with repeated-measures ANOVA test followed by Tukey’s post-test (#). Significances levels are shown as *,# p < 0.05, **,## p < 0.001 and ***,###p < 0.0001. dpi, days post-infection.</p

Longitudinal monitoring of plasma SIV viral loads.

<p>Viral loads (log copies/ml) of 5 SIV-infected macaques were quantified by quantitative RT-PCR and compared to values obtained at pre-infection stage using the paired Student's t test. No significant differences were observed between animals. dpi, days post-infection.</p

Longitudinal flow-cytometry analysis of blood myeloid dendritic cells (mDC), monocytes, CD4⁺ T-cells and granulocytes.

<p>Relative percentages (left panels) and absolute numbers (right panels) of (A) CD16⁺CD1c^- mDC, (B) CD16^-CD1c⁺ mDC, (C) CD14⁺CD16⁺ monocytes, (D) CD14⁺CD16^- monocytes, (E) CD4⁺ T-cells and (F) granulocytes obtained from the blood of 5 SIV-infected rhesus macaques. Values were compared to that obtained at pre-infection using the paired Student's <i>t</i> test (*) or between time points with repeated-measures ANOVA test followed by Tukey’s post-test (#). Significances levels are shown as *,# p < 0.05, **,## p < 0.001 and ***,###. dpi, days post-infection.</p