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

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

GPR15 is mostly present on central memory CD4⁺Tcells in HIV-1 infected individuals and uninfected controls.

<p>PBMCs were isolated from whole blood by Lymphoprep gradient centrifugation and stained for CD3, CD4, CCR7, CD45RA and GPR15. (A) Cells were gated for lymphocytes, CD3⁺, CD4⁺ and CCR7⁺CD45RA⁻ (CM: central memory), CCR7⁻CD45RA⁻ (EM: effector memory) or CCR7⁺CD45RA⁺ (N: naïve) (A) and GPR15 expression on the subsets was analyzed via FACS (B). The GPR15 expression on CD4⁺ T cell subpopulations was analyzed in eight uninfected controls and eleven HIV-1 infected patients (C) as indicated in (A, B). GPR15 expression is shown as the percent of the analysed subpopulation which expresses the co-receptor (C). Blood samples taken two month later from the two high GPR15 expressing HIV-1 infected patients and two controls (shown in C) were stained for GPR15, CD4 and CD8 (D) or CD4 and other co-receptors like CXCR4, CCR5 and CXCR6 (E). The co-receptor expressions are shown as a percent of CD4⁺ and CD8⁺ T cells expressing GPR15 (D) or of CD4⁺ T cells expressing CXCR4, CCR5 or CXCR6 (E). Statistical analysis was done using Wilcoxon signed-rank test with GraphPad Prism.</p

TLR3 stimulation up-regulates GPR15 also on CD8⁺ T cells and CD19⁺ B cells.

<p>The GPR15 expression was studied on whole PBMCs (A,B) or separated T and B cells (C) using additional anti-CD8 (A) or CD19 (B) antibodies. GPR15 is shown as a percent of CD8⁺ T or CD19⁺ B cells.</p

HIV-1 infection increases GPR15 expression on infected cells.

<p>The PM1 T cell line was infected with three different primary HIV-1 isolates the multitropic isolates 25 and 4052 and the R5-tropic 2195 for 3 days and afterwards stained for GPR15 surface expression and intracellular p24. The percent of uninfected (p24−) or infected (p24+)cells expressing GPR15 is shown.</p

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

GPR15 is strongly up-regulated on gut homing CD4⁺Tcells and is highly expressed on colon CD4⁺Tcells.

<p>TLR3 stimulation up-regulates GPR15 on gut homing (α4β7-integrin⁺) (A, C) and on CD4⁺ T cells homing to lymph nodes (CD62L⁺) (B, D). The different symbols in the Figures C and D specify different donors. Before TLR3 stimulation both subsets express GPR15 to a similar level (E). The different symbols describe individual donors (E). PBMCs were isolated from whole blood by Lymphoprep gradient centrifugation and co-stained for CD4, GPR15 and CD62L or β7-integrin. The cells were gated on lymphocytes, CD4⁺ as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1 A</a>. The graphs show GPR15 expression as a percent of CD62L⁺ CD4⁺ T cells or β7⁺ CD4⁺ T cells expressing the co-receptor. The experiments were done at least two times including two donors each time. Statistical analysis was done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g003" target="_blank">Figure 3</a> using paired t-test. Human colon intraepithelial mononuclear cells (IEMC) and lamina propria mononuclear cells (LPMC) express GPR15 on high level. IEMC and LPMC were isolated following the described protocol and co-stained for CD45, CD3, CD4 and GPR15. Cells were gated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1A</a> with the accretion that CD45 were gated out to exclude epithelial cell contamination (F). Colon biopsies of HIV-1 infected and uninfected individuals were immunofluorescently stained for GPR15, CD4 and cell nuclei using DAPI. Slides were analysed by confocal microscopy. Three biopsies per patient and 15–20 images per biopsy were acquired at 63×. Cells were enumerated using ImageJ cell counting software for % of CD4⁺ cell expressing GPR15 (G).</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

TLR3 stimulation increases GPR15 on the surface of CD4⁺ T cells.

<p>PBMCs were incubated with different ligands for TLRs and GPR15 expression on CD4⁺ T cells was analyzed. Cells were gated for lymphocytes, CD3⁺, CD4⁺ as already shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1A</a>. The bar graph is a summary of four donors which were analysed in two independent experiments (A). Upon TLR3 triggering, GPR15 is mostly up-regulated on central memory T cells (B). To exclude cell-cell interaction effects T cells were further separated using negative selection with magnetic beads and stimulated with TLR3 ligand polyIC (C). Pre-treatment of T cells with TLR3 signalling inhibitor PepinhTRIF abrogates the increase of GPR15 on the T cell surface (D). To test if TLR3 stimulation can up-regulate other co-receptors it was also stained for CXCR6 (E), CCR5 (F) and CXCR4 (G). GPR15 expression is shown as a percent of the gated CD4⁺ T cell subpopulations. Statistical analysis was done with GraphPad Prism using paired t-test.</p