IL-7 induces CD95 upregulation on B cells via IFN-γ released from T cells.

<p><b>A.</b> Purified T cells were cultured in the presence or absence of 25 ng/ml IL-7 and IFN-γ concentrations were measured at the indicated time points using ELISA. Mean values and standard deviations are calculated from the results of 7 independent experiments. <b>B.</b> IFN-γ expression was analyzed by flow cytometry in naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+), effector memory (CD45RA-CCR7−) and TEMRA (CD45RA+CCR7−) T cell subsets cultured in the presence (black line) or absence (grey line) of 25 ng/ml IL-7 for five days. Dashed lines represent isotype control staining. Data are representative of 3 independent experiments. <b>C.</b> IFN-γ mRNA levels were measured in IL-7 treated or untreated T cells by real time PCR. <b>D.</b> IFN-γ concentrations measured in the IL-7 treated or non-treated T cell supernatants are correlated with the ability of the same supernatants to induce CD95 expression on B cells (using the samples described in panel A). Black dots represent IL-7 treated, white dots represent non treated T cell supernatants collected at day 2, 5, 8 or 11. <b>E.</b> CD95 expression is shown on freshly isolated B cells (dashed line) or following a 12 h (grey lines) or 48 h (black lines) treatment with IL-7 treated T cell supernatants or 20 ng/ml recombinant human IFN-γ. <b>F.</b> STAT-1 phosphorylation is measured using flow cytometry in sorted B cells treated with supernatants of IL-7 treated T cells in combination with 10 mg/ml neutralizing anti-IFN-γ or isotype control antibodies. Data are representative of 3 experiments. <b>G.</b> B lymphocytes were cultured for five days with IL-7 treated or non treated T cell supernatants or were left untreated in the presence of 10 mg/ml neutralizing anti-IFN-γ or isotype control Abs. Thereafter the expression of CD95 was analyzed by flow cytometry. Data are representative of 3 different experiments.</p

T cells treated with IL-7 induce STAT1 activation in B cells.

<p><b>A.</b> B cells were purified from peripheral blood and were treated with supernatants of T cells cultured with or without IL-7 for 30 minutes at 37°C. In order to identify signaling pathways specifically induced by the IL-7 treated T cell supernatants we lysed the B cells and used a protein array to detect a wide range of intracellular phophorylation events (see table). Individual molecules are plotted in duplicates. <b>B.</b> STAT1 phosphorylation was studied in sorted B cells treated with supernatants of IL-7 treated or non treated T cells using flow cytometry. <b>C.</b> CD95 expression on B cells cultured in the presence of the STAT1 inhibitor fluodarabine or DMSO control in the presence of supernatants of IL-7 treated or untreated T cells. Data are representative of 3 independent experiments.</p

IL-7 presenting stroma cells increase IL-7 induced IFN-γ production in T cells.

<p><b>A.</b> IL-7Rα expression and surface IL-7 binding was measured on the HS27 BM stromal cells as well as on CD14+ peripheral blood monocytes and CD3+ T cells using flow cytometry. <b>B.</b> The HS27 cell line (10000 or 1000 cells/well) or human monocytes (30000cells/well) were cultured in 96-well plates, pretreated with different concentrations of IL-7 for 2 hours at room temperature and thereafter purified peripheral blood T lymphocytes were added to the cultures for 3 days. IFN-γ production was measured after 3 days of culture using ELISA. Representative data of 3 independent experiments are shown.</p

Induction of CD95 upregulation on B cells by IL-7 treated T cells.

<p><b>A.</b> Representative histogram of CD95 expression among T and B lymphocytes from untreated (dashed line) or IL-7 treated (solid line) PBMCs cultured for 5 days. Grey filled histograms correspond to isotype control stainings. <b>B.</b> Peripheral B cell subsets were distinguished by measuring CD10, CD21 and CD27 expression. A representative CD95 staining on B cell subsets is shown after culturing PBMC for 5 days in presence (solid line) or absence (dashed line) of IL-7. Data are representative of 3 independent experiments. <b>C.</b> Cell surface levels of CD95 on different B cell subsets are shown measured after culturing PBMCs for 5 days in the presence of 25 or 2.5 ng/ml IL-7, or without the cytokine. D. Analysis of CD95 expression on freshly purified B cells or following a 3 day culture with and without IL-7. Alternatively, purified B cells were co-cultured with T cells using 1∶1 cell ratio allowing direct cell contact or separating the cells using a trans-well system. B cells were also plated in the presence of IL-7 treated or non treated T cell supernatants collected after culturing T cell for 5 days in the presence or absence of 25 ng/ml IL-7. Data are representative of 5 experiments.</p

Enhanced CD95 mediated apoptosis of T and B cells upon IL-7 treatment.

<p>Kinetics of CD95 expression (left panels) and CD95 mediated apoptosis sensitivity (right panels) of CD3+ T cells (<b>A</b>) and CD19+ B cells (<b>B</b>) are shown, measured in PBMC cultures treated with 25 ng/ml IL-7, with supernatants of IL-7 treated or non treated T cell cultures or left untreated. Apoptosis was triggered using recombinant CD95L added for 24 hours to cultures. Data are representative of 3 independent experiments.</p

The effect of γ-chain cytokines on IFN-γ production by T cells.

<p><b>A.</b> IFN-γ levels were measured in supernatants of T cells cultured in the presence of IL-2, IL-4, IL-7, IL-15 and IL-21 for five days. <b>B.</b> The same supernatants were tested for the ability to trigger CD95 upregulation on B lymphocytes. Representative data out of 3 independent experiments are shown. <b>C.</b> Purified B cells were cultured in the presence of IL-2 or IL-15 treated T cell supernatants for 3 days and then CD95 expression was analyzed by flow cytometry. IFN-γ was neutralized in the supernatants using 10 µg/ml anti-IFN-γ antibodies (black lines). Histograms with grey line represent samples containing isotype control antibody, the grey filled histograms represent samples without antibodies added and the dotted histograms represent stainings with isotype control antibody.</p

HIV-specific responses are significantly upregulated after the vaccination with LIPO-5-DC vaccine.

<p>Each patient is designated with its individual color/symbol code (refer to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004752#ppat.1004752.s008" target="_blank">S1 Table</a>). Symbols are attributed based on maximum viral load rebound after treatment interruption as indicated in the figure. (A) Representative plots for a single patient prior (wk -4) and after (wk 16) the vaccination. Cells were stimulated with gag p24 pool, LIPO-5 vaccine or CMV lysate as a control, stained and analyzed by flow cytometry 44 hours later. Plots show viable CD4⁺ T cells. (B) Graphs show the percentages of antigen-specific cells (CD134⁺CD25⁺) among CD4⁺ T cells after the stimulation with the indicated antigens (n = 14). (C) IFN-γ, TNF-α and IL-2 production among LIPO-5- specific cells (CD134⁺CD25⁺) (n = 14). (D) Graph shows the correlation between the relative increase in LIPO-5-specific response (response after the vaccination-response before vaccination) and maximal viral load rebound after HAART interruption (n = 14). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.</p

Patients’ characteristics.

<p>Patients’ characteristics.</p

Overall strength of the HIV-specific responses is inversely correlated with maximum viral load after ATI.

<p>(A) Graph showing the responses for all individual patients to indicated antigens. (B) Correlation between the relative increase in the strength of the response (sum of the strengths of the response post vaccination–sum of the strengths of the response prior to vaccination) and maximal viral load rebound after HAART interruption (n = 9). Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.</p

HIV-specific Tregs/Teffs ratio is affected by vaccination.

<p>(A) Gag p24-, LIPO-5- and CMV-Tregs responses prior and after vaccination (n = 14). (B) Pie chart showing LIPO-5-specific Tregs among LIPO-5-specific (CD134⁺CD25⁺) cells prior and after vaccination (n = 14). (C) Representative profiles after in vitro stimulation with LIPO-5 showing good and poor responder to vaccination (based on a viral load rebound). (D) Correlation between IFN-γ-producing and Tregs cells among LIPO-5-specific cells after in vitro stimulation. Data were analyzed by Wilcoxon matched-pairs signed rank test. *p < 0.05; **p < 0.01; ***p < 0.001. Spearman coefficient is indicated (r) as well as p value.</p