Cell sorting, FOXP3 TSDR and gene expression.

<p>A. Flow-cytometry gating strategy used to isolate CD25⁺CD127^low regulatory T cells (Tregs) and CD25⁻CD127⁺ conventional T cells (Tconv) from CD4⁺ T cells (Left Panel). Expansion fold change of Tregs isolated from HIV-1-infected (dark grey) (n = 8 controllers + 13 chronic untreated) and healthy (light grey) (n = 4) individuals during 7 days of cell culture (right Panel). B. Relative mRNA expression in arbitrary units (A.U.) of FOXP3 and IL-10 quantified by real time PCR in expanded Tregs (n = 3 controllers+2 chronic untreated) and Tconvs (n = 2 controllers+2 chronic untreated) isolated from HIV-1 infected individuals after 7 days of culture. C. Frequency of demethylation of the Treg Specific Demethylation (TSDR) region of the FOXP3 gene in expanded Tregs and Tconvs after 7 days of culture as assayed by real time PCR. Empty symbols represent HIV-1 controllers and solid symbols HIV-1 chronic untreated individuals.</p

Phenotyping of expanded Tregs by flow cytometry.

<p>A. Representative examples of gating strategy used for CD25⁺FOXP3⁺ staining by flow-cytometry of <i>ex vivo</i> PBMC (upper panel) isolated from a HIV-1 controller and matched expanded Tregs (lower panel) at day 7 of expansion. B. Expression of different Tregs markers quantified by flow-cytometry of expanded (day 7) and <i>ex vivo</i> unexpanded Tregs and Tconvs. MFI = Mean Fluorescence intensity. Empty symbols represent HIV-1 controllers and solid symbols HIV-1 chronic untreated individuals. C. Representative example of flow-cytometry gating strategy used to phenotype Tregs, Tconvs (n = 3 controllers+9 chronic untreated) and <i>ex vivo</i> CD4 T cells (n = 3 controllers+3 chronic untreated) isolated from HIV-1 positive individuals based on their CD45RA and FOXP3 expression profiles <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086920#pone.0086920-Miyara1" target="_blank">[41]</a>. The left dot plot shows <i>ex vivo</i> CD4⁺ T cells from PBMC, the middle dot plot represents an example of expanded Tregs (black dots) and Tconvs (light grey dots). The right histogram graph quantifies the different Treg subsets in HIV-1 positive individuals. Gate 1 and white columns represent “resting” CD45RA⁺FOXP3^low Tregs, gate 2 and grey columns represent “non-suppressive cytokine-secreting” CD45RA⁻FOXP3^low T cells and gate 3 and black columns represent “activated” CD45RA⁻FOXP3^high Tregs.</p

Suppressive function of expanded Tregs.

<p>A. Suppressive activity of expanded Tregs from HIV-1+ (n = 7 controllers +11 chronic untreated individuals) and healthy controls (n = 4) on activated CD8⁺ T cells (left) and CD4⁺ T cells (right). Columns represent activated T cells (white) co-cultured with autologous expanded Tregs (black) or Tconvs (grey). A suppressive activity of 100% indicates that the proliferation of activated T cells was completely inhibited and a negative suppressive activity signifies that the proliferation of T cells was higher than in the condition “T cells alone”. B. Representative example of a flow-based Treg suppressive assay after 4 days of co-culture. CFSE dilution of activated CD8⁺ T cells (upper panel) and CD4⁺ T cells (lower panel) are represented as histograms. Left columns show CFSE dilution of bead-activated T cells from frozen PBMC, the other columns represent activated T cells co-cultured with autologous expanded Tregs (middle) or Tconvs (right). C. ⁵¹Chromium release assay. Representation of the cytotoxic function (% lysis) of a HIV-1 specific CTL clone (effector) using a HIV-1-peptide-loaded B cell line labeled with [⁵¹Cr] as a target with or without expanded Tregs at a 1 Effector:1 Treg :1 Target ratio. D. Example of gating strategy used to isolate Tregs from the peripheral blood of an HIV-1-infected infant (Left). Numbers of cells counted during the expansion of these Tregs (Middle). Percentage of suppression of the expanded Tregs or expanded Tconvs on activated CD4⁺ T cells when co-cultured with autologous CFSE loaded PBMC at a 1∶1 ratio (Right). E. Example of gating strategy used to isolate Tregs from the colon of an HIV-1-infected individual (Left). The middle panel represents the numbers of cells counted during the expansion of these Tregs (Middle). Percentage of suppression of the expanded Tregs (n = 1 HIV-1-negative sample +4 HIV-1-positive samples) or expanded Tconvs (n = 1 HIV-1-negative sample +4 HIV-1-positive samples) isolated from the GALT on activated CD8⁺ T cells when co-cultured with CFSE loaded PBMC at a 1∶1 ratio (Right).</p

Summary of clinical data of the HIV-1-infected study subjects.

<p>Summary of clinical data of the HIV-1-infected study subjects.</p

The TCR repertoire is not altered after <i>in vitro</i> expansion of Tregs.

<p>A. Degree of expansion of the TCRß repertoire (i.e. number of TCRs in a sample that belongs to an individual clone and expressed as percentage of total reads) from 2×10⁴<i>ex vivo</i> sorted unexpanded (light grey) and 2×10⁴<i>in vitro</i> expanded (Day 14; dark grey) Tregs isolated from the same original PBMC specimen. B. Distribution of variable-gene (Vß-gene) variants from 2×10⁴<i>ex vivo</i> sorted unexpanded (light grey) and 2×10⁴<i>in vitro</i> expanded (Day 14; dark grey) Treg TCR-β clones isolated from the same PBMC specimen. C. Distribution of joining-gene (Jß-gene) variants from 2×10⁴<i>ex vivo</i> sorted unexpanded (light grey) and 2×10⁴<i>in vitro</i> expanded (Day 14; dark grey) Treg TCR-β clones isolated from the same PBMC specimen.</p

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Delivery to human immune cells.

<p><b>A)</b> Human T cells and MDDCs were tested for delivery of cascade blue labeled 3kDa dextran, fluorescein labeled 70kDa dextran, and APC labeled IgG1. The representative histograms for a 30–4 (T cells) and 10–7 (MDDCs) device (left) and replicates across device designs (right) are displayed. <b>B)</b> SiRNA mediated knockdown of CD4 and DC-SIGN protein levels in CD4⁺ T cells and MDDCs respectively. <b>C)</b> Knockdown of CD4 expression in human regulatory T cells in response to treatment by a 30–4 device. Dead cells were excluded for delivery or knockdown analysis. <b>D)</b> Comparison of device performance in T cells to nucleofection by Amaxa. Protein expression 72hrs after siRNA delivery and cell viability after treatment are shown. <b>E)</b> Intracellular staining for the p24 antigen was used as an indicator of HIV infection level in treated human CD4⁺ T cells 24hrs after infection. In these studies, vif and/or gag, siRNA was delivered 24hrs prior to infection while CD4 siRNA was delivered 48hrs prior to infection. <b>F)</b> Median fluorescence intensity of the p24 antigen stain across repeats (min. N = 4) of the experimental conditions. Data are represented as mean + 1 standard error.</p

Delivery methodology and performance in mouse cells.

<p><b>A)</b> Illustration of device design and delivery mechanism. <b>B)</b> Illustration of the system setup and delivery procedure. <b>C)</b> Representative histograms of T cells, B cells and myeloid cells (CD11b⁺) treated by the CellSqueeze device to deliver APC-labeled IgG1. <b>D)</b> Delivery efficiency of Cascade blue-labeled 3 kDa dextran, fluorescein-labeled 70 kDa dextran, and APC-labeled IgG1. All results were measured by flow cytometry within an hour of treatment. Dead cells were excluded by propidium iodide staining. Viability is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118803#pone.0118803.s002" target="_blank">S2 Fig</a></b>. Data in <b>D)</b> (mean ± SD) are from 3 independent experiments. Untreated cells were not put through the device or exposed to the biomolecules. The ‘no device’ samples were incubated with the biomolecules, but were not treated by the device. This control is meant to account for surface binding, endocytosis and other background effects.</p

Relationship between plasma IP-10 levels and cell-associated DNA levels during primary HIV-1 infection.

<p><b>A.</b> IP-10 plasma concentrations stratified on cell-associated DNA levels during PHI (M0). <b>B.</b> Correlation between IP-10 concentrations and cell-associated DNA levels during PHI (M0). M0 = primary HIV-1 infection and time of inclusion (Fiebig stage III/IV).</p

CXCL10/IP-10 dynamics in blood before HIV-1 infection and during the early infection, and its impact on the CD4 T-cells count.

<p>IP-10 levels were determined in sera from HIV+ individuals enrolled in the Amsterdam Cohort Studies on HIV/AIDS. <b>A.</b> Plasma IP-10 levels. The numbers of patients per group are indicated. <b>B.</b> Longitudinal follow-up of the same 16 individuals. <b>C.</b> Positive correlation between IP-10 concentrations and viremia at PHI. <b>D.</b> Positive correlation between IP-10 concentrations and viremia at M3 post SC. <b>E.</b> Impact of pre-infection blood IP-10 concentrations on the CD4 T-cells count after seroconversion. <b>F.</b> Impact of M3 blood IP-10 concentrations on the CD4 T-cells count after seroconversion. <b>E</b> and <b>F</b>: Kaplan Meier survival analysis. Pre-inf. = Pre-infection (median 11 months before seroconversion), PHI = Primary HIV-1 infection, M3 and M6 = 3 and 6 months post seroconversion. * p<0.05, ** p<0.001, **** p<0.00001</p