DataSheet_1_Deciphering decidual leukocyte traffic with serial intravascular staining.docx

The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, the degree, timing, and subset-specific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques (Macaca fascicularis) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy.</p

Pegivirus avoids immune recognition but does not attenuate acute-phase disease in a macaque model of HIV infection

<div><p>Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis–as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses–suggesting that HPgV’s protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses–an understudied group of viruses with a high prevalence in the global human population–and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.</p></div

SIV pathogenesis in SIV-only vs. SIV+SPgV infected macaques.

<p>(<b>A</b>) Peripheral CD4+ T cell counts were obtained by multiplying absolute lymphocyte counts by the percentage of lymphocytes that were CD3+ CD4+ CD20- CD8- (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006692#ppat.1006692.g003" target="_blank">Fig 3</a> for gating strategy details). (<b>B</b>) Gut CD4+ T cells were stained within sections of colonic tissues via IHC with an anti-CD4 antibody and manually quantified. Significant differences between the SIV-only and SPgV+SIV groups were analyzed using a two-tailed unpaired t-test (solid line) with error bars representing SEM. Significant changes in all animals over the course of acute SIV infection were quantified using a two-tailed paired t-test (dashed line). (<b>C</b>) A representative set of colonic tissue from Cy0883 (SIV+SPgV) and Cy0887 (SIV-only) are shown pre and post SIV infection at 400x for comparison. Arrows highlight representative cells with membranous CD4 staining.</p

SPgV and SIV viral loads in infected macaques.

<p>Titers for each virus were measured from plasma using highly sensitive virus-specific quantitative RT-PCR assays. (<b>A</b>) SPgV titers in the four macaques infected with SPgV+SIV. (<b>B</b>,<b>C</b>,<b>D</b>) SIV titers in four macaques infected with SPgV+SIV (green) and four macaques infected with SIV-only (black). <i>P</i> values reflect a two-tailed unpaired t-test and error bars represent SEM. The symbols used for each animal in this figure are consistent throughout the manuscript.</p

Activation of immune tissues in SIV-only vs. SIV+SPgV infected macaques.

<p>Proliferating cells were stained within sections of lymph nodes (<b>A</b>) and colon (<b>B</b>) via IHC with an anti-Ki67 antibody and manually quantified. Significant changes over time were quantified using a two-tailed paired t-test (dashed line). A representative set of lymph nodes from Cy0883 (SIV+SPgV) and Cy0881 (SIV-only) is shown at 400X pre and post SIV infection for comparison in (<b>A</b>). A representative set of colon tissues from Cy0886 (SIV+SPgV) and Cy0887 (SIV-only) is shown pre and post SIV infection at 400x for comparison in (<b>B</b>).</p

SPgV co-infection does not alter recognition of MHC class I restricted SIV epitopes by CD8+ T cells.

<p>Lymph nodes were collected from macaques at 125/126 days post SIV infection and cells were stained for analysis with MHC class I tetramers folded with SIV peptides that are immunodominant on the M3/M4 MHC background. (<b>A</b>) Flow cytometry gating strategy used for defining tetramer-positive CD8+ T cells. (<b>B</b>) Percentage of CD8+ T cells that were positive for each tetramer. <i>P</i> values represent a two-tailed unpaired t-test with error bars reflecting SEM.</p

Peripheral immune activation in SIV-only vs. SIV+SPgV infected macaques.

<p>(<b>A</b>) Flow cytometry gating strategy used for defining immune cell subsets. Fresh whole blood was used for staining and flow cytometry at each time point. (<b>B-D</b>) Activation of immune cell subsets. <i>P</i> values represent a two-tailed unpaired t-test with error bars reflecting SEM. Note: Cy0886 did not exhibit a distinct peak or nadir of CD69+ Ki67+ expression in the CD3+ CD8+ T cell population, and so is not included in these analyses.</p

Immune activation following SPgV vs. SIV infection.

<p>(<b>A-C</b>) Fresh whole blood was used for analysis by flow cytometry at each time point. <i>P</i> values are from a two-tailed paired t-test comparing the average immune activation pre-any-virus-infection to the average of all post-SPgV or post-SIV data points within the first 26 days of infection for each virus for which flow cytometry data was available. (<b>D</b>) Proliferating cells were stained within sections of lymph nodes via IHC with an anti-Ki67 antibody and manually quantified (SPgV: day -8 vs day 24; SIV: day -34 vs day 25). Significant changes over time were quantified using a two-tailed paired t-test. (<b>E</b>) Representative set of lymph node tissue from Cy0885 is shown at 400x.</p

Molecularly barcoded Zika virus libraries to probe <i>in vivo</i> evolutionary dynamics

<div><p>Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a “synthetic swarm” whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects <i>in vivo</i>, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time <i>in vivo</i> to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.</p></div

Vector competence of <i>Aedes aegypti</i> following peroral exposure to ZIKV-BC-1.0-infected pregnant macaque 4 days post inoculation.

<p>Vector competence of <i>Aedes aegypti</i> following peroral exposure to ZIKV-BC-1.0-infected pregnant macaque 4 days post inoculation.</p