Baseline characteristics of included and non-included TB-patients with or without HIV ^a and healthy controls ^b.

<p>^a Total number of recruited subjects with active TB in the cohort was 1116</p><p>^b Total number of recruited healthy controls in the cohort was 298</p><p>c All patients with CD4<500 cells/mm³ were selected for inclusion. For patients with CD4≥500cells/mm³ a consecutive number of patients were selected for inclusion.</p><p>^d Patients could have a simultaneous diagnosis of extrapulmonary and pulmonary TB (included HIV+ patients, n = 3, included HIV-negative patients, n = 2).</p><p>^e BMI is the calculated Body mass index (weight/height²).</p><p>^f MUAC is the mid upper arm circumference measured by a measuring tape.</p><p>Baseline characteristics of included and non-included TB-patients with or without HIV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144292#t001fn001" target="_blank">^a</a> and healthy controls <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144292#t001fn002" target="_blank">^b</a>.</p

Neopterin and CRPlevels in different CD4 cell count strata in 365 TB patients with and without HIV and 31 controls.

<p>^a Neopterin levels (nmol/l), median (IQR) and number of study subject (n =)</p><p>^b CRP levels (μg/ml), Median (IQR) and number of study subject (n =)</p><p>Neopterin and CRPlevels in different CD4 cell count strata in 365 TB patients with and without HIV and 31 controls.</p

Baseline neopterin in relation to evolution of CD4 cell count at completion of ATT subdivided into two groups; No increase (CD4 cell count ≤50 cells/mm³ or unchanged CD4 cell count, ± 50 cells/mm³ after 6 months ATT), or increase (CD4 ≥50 cells/mm³ after 6 months ATT).

<p>Graph <b>A</b> displays <b>HIV-</b>patients (n = 85 of whom 58 had an increase in CD4 cell count). Baseline median CD4 cell count for those with increasing CD4 cell count was 405 cells/mm³ and 496 cells/mm³ for those without increasing CD4 cell count (n = 27). Graph <b>B</b> displays <b>HIV+</b> patients who did not start ART (n = 43 of whom 22 had an increase in CD4 cell count). Baseline median CD4 cell count for those with increasing CD4 cell count was 255 cells/mm³ and 411 cells/mm³ for those without increasing CD4 cell count (n = 21).</p

ROC curves showing AUC for neopterin and CRP for prediction of CD4 cell strata using three different cut-off levels: <500 cells/mm³, <350 cells/mm³, <100 cells/mm³.

<p>A: CD4 cell count <500 cells/mm³. Includes all participants. B: CD4 cell count <350 cells/mm³. Includes HIV+ patients. C: CD4 cell count <100 cells/mm³. Includes HIV+ patients.</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

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

Performance of QuantiFERON-TB Gold Plus for detection of latent tuberculosis infection in pregnant women living in a tuberculosis- and HIV-endemic setting

<div><p>We evaluated the performance of QuantiFERON-TB Gold Plus (QFT-Plus), which includes two <i>Mycobacterium tuberculosis</i> antigen formulations (TB1 and TB2), for detection of latent tuberculosis infection during pregnancy. Eight-hundred-twenty-nine Ethiopian pregnant women (5.9% HIV-positive) were tested with QFT-Plus, with bacteriological sputum analysis performed for women with clinically suspected tuberculosis and HIV-positive women irrespective of clinical presentation. QFT-Plus read-out was categorized according to the conventional cut-off (0.35 IU/ml) for both antigen formulations. In addition, we analysed the distribution of QFT-Plus results within a borderline zone (0.20–0.70 IU/ml), and interferon-γ response in relation to HIV infection and gestational age. Two-hundred-seventy-seven women (33%) were QFT-Plus-positive (HIV-positive 16/49 [33%]; HIV-negative 261/780 [33%]). There was a strong agreement between the two antigen formulations (κ = 0.92), with discordant results in 29 cases (3.5%). Whereas discordant QFT-Plus results were rare in pregnancy, several results with both TB1 and TB2 within the borderline range were observed (11/49 [22%] vs. 43/780 [5.5%] in HIV-positive and HIV-negative women, respectively; p<0.0001). HIV-positive women had lower absolute interferon-γ levels (TB1: 0.47 vs. 2.16 IU/ml; p<0.001, TB2: 0.49 vs. 2.24 IU/ml, p<0.001, considering results ≥0.20 IU/ml) compared to HIV-negative women. QFT-Plus-positive women who submitted samples at later stages of pregnancy had lower mitogen- (p<0.001) but higher TB-antigen-specific (p = 0.031 for TB1, p = 0.061 for TB2) interferon-γ response. Considering their lower capacity to produce TB-specific interferon-γ, a lower cut-off level for defining QFT-Plus-positivity may be considered in HIV-positive pregnant women.</p></div