Assay characteristics of the chromium release assay vs. BLI assay.

<p>The spontaneous death, maximal killing, range, and the signal to noise (max:min) ratios measured by ⁵¹Cr release and BLI are shown. Results are presented as mean ± SD of three independent experiments.</p><p>* p<0.01 by Student t test.</p

Measurement of chromium release and luciferase activity in cells lysed by water or by 1% NP40.

<p>Three luciferase-transduced human cell lines (K562, UCI191 and U266) and 3 mouse cell lines (P815, YAC1, and A20) were labeled with radioactive chromium for 4 hours. The cells were lysed in water or 1% NP40 for 4 hours. (A) Chromium release in the supernatant of cells lysed in water or in 1% NP40 was determined. (B) Luciferase activity was detected by BLI in cells lysed in water or in 1% NP40 was determined. The results are represented as mean ± SD of n = 3–4 independent experiments. * p<0.001 by Wilcoxon Rank test.</p

Comparison of cytotoxicity obtained at 4 hours by the chromium release and BLI method at higher E:T ratios.

<p>Luciferase-transduced the YAC1 cell line was co-cultured with mouse effector cells for 4 hours at various E:T ratios ranging from 100∶1 to 20∶1. The % specific lysis obtained by the chromium release assay (closed circles) or the BLI assay (open circles) is plotted against multiple E:T ratios. Results are represented as mean ± SD of triplicate values. One representative of 2 independent experiments is shown. The p value obtained from the statistical analysis performed permutated two-way ANOVA is shown.</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

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

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

Comparison of cytotoxicity obtained at 2 hours by the chromium release method and BLI method.

<p>Luciferase-transduced human and mouse cell lines were co-cultured with human or mouse effector cells for 2 hours at various E:T ratios. The % specific lysis of the human cell lines, (A) K562, (B) U266, and (C) UCI101 obtained by the chromium release assay (closed circles) or the BLI assay (open circles) is plotted against multiple E:T ratios. The % specific lysis of the human cell lines, (D) P815, (E) YAC1, (F) EL-4, and (G) A20 obtained by the chromium release assay (closed circles) or the BLI assay (open circles) is plotted against multiple E:T ratios. Results are represented as mean ± SD of n = 3 independent experiments. The p values obtained from the statistical analysis performed by permutated two-way ANOVA are shown in each graph.</p

Comparison of cytotoxicity obtained at 4 hours by the chromium release and BLI method.

<p>Luciferase-transduced human and mouse cell lines were co-cultured with human or mouse effector cells for 4 hours at various E: T ratios. The % specific lysis of the human cell lines, (A) K562, (B) U266, and (C) UCI101 obtained by the chromium release assay (closed circles) or the BLI assay (open circles) is plotted against multiple E:T ratios. The % specific lysis of the murine cell lines, (D) P815, (E) YAC1, (F) EL-4, and (G) A20 obtained by the chromium release assay (closed circles) or the BLI assay (open circles) is plotted against multiple E:T ratios. Results are represented as mean ± SD of n = 3 independent experiments. The p values obtained from the statistical analysis performed by permutated two-way ANOVA are shown in each graph.</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