p24 Gag_209–218/HLA-Cw*0102 complexes on T2 cells lead to functional inhibition of primary KIR2DL2(+) NK cells.

<p>(<b>A</b>) Representative histograms of degranulation of KIR2DL2(−) (left panel) and KIR2DL2(+) (right panel) NK cells after co-incubation with peptide-pulsed T2 cells (tinted and black) or without target cells (clear). NK cell degranulation was measured flow cytometrically by surface expression of CD107a. (<b>B</b>) Different levels of NK cell degranulation between KIR2DL2(−) (dark grey) and KIR2DL2(+) (light grey) NK cells after co-incubation with peptide-pulsed T2 cells. Each column represents mean±SEM percentage of CD107a(+) NK cells of 5 of different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column.</p

HIV-1 p24 peptide-dependent HLA-Cw*0102 stabilization on T2 cells.

<p>(<b>A</b>) Representative histograms of HLA-Cw*0102 stabilization on T2 cells as determined by flow cytometry. Histograms display HLA-Cw*0102 expression on T2 cells in the presence of control peptides VAP-FA and VAP-DA (black) at a concentration of 40 µg/ml as compared to unloaded T2 cells (grey tinted) and isotype control (clear). (<b>B</b>) HLA-Cw*0102 expression on HIV-1 p24 peptide-pulsed T2 cells. HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 5 independent experiments for each HIV-1 p24 OLP. A total of 59 HIV-1 p24 OLPs were analyzed, which previously showed strongest HLA-A/B/C stabilization as determined by an HLA-A/B/C antibody. Of those, 11 peptides with the highest HLA-Cw*0102 expression (>five-fold) were selected for subsequent KIR-Fc binding assays.</p

Ability of p24 Gag_209–218 peptide variants for HLA-Cw*0102 stabilization and KIR2DL2-Fc binding.

<p>(<b>A</b>) Differential expression of HLA-Cw*0102 on T2 cells after co-incubation with peptide variants of HIV-1 p24 Gag_209–218-L (AAEWDRLHPV). Peptide variants differed in length [9 or 10 amino acids (aa)] as well as in sequence (substitution of Leucine in position 7 with various amino acids). HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 4 independent experiments for each peptide variant. (<b>B</b>) Relative binding of KIR2DL2-Fc to T2 cells after co-incubation with selected HIV-1 p24 Gag_209–218variants. KIR2DL2-Fc binding is illustrated as RFI as compared to VAP-DA-pulsed T2 cells. Each column represents mean±SEM RFI of 3 independent experiments for each 10 aa variant. (<b>C</b>) Different levels of NK cell degranulation after co-incubation with T2 cells in the presence of different p24 Gag_209–218-L variants. Each column represents mean±SEM percentage of CD107a(+) NK cells of 4 different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column. * indicates statistical significance as defined <i>p</i><0.05.</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

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

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

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

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