Modulation of PD-1 and GrzB protein co-expression during active TB disease and chemotherapy.

<p>Frozen PBMCs from Pakistan were thawed and PD-1 and GrzB co-expression was investigated by flow cytometry. (A) The proportion of PD-1 cells which are also GzB positive in the CD3⁺CD8⁺ lymphocyte gate, CD3⁺CD8^- lymphocyte gate and CD3^- lymphocyte (NK cell) gate at diagnosis in TB patients (P: n = 18), latently infected contacts (C+: n = 7) and uninfected contacts (C-: n = 4). P values are derived from the Kruskal-Wallis test with Dunn’s multiple testing correction (B) Modulation of PD-1/GzB co-expression with the intensive phase of chemotherapy on CD3⁺CD8⁺ T cells, CD3⁺CD8^- lymphocytes and CD3^- lymphocytes (NK cells), analysed with the Wilcoxon signrank test. The bars show medians and the interquartile range.</p

Study subjects:clinical and demographic information.

<p>Study subjects:clinical and demographic information.</p

PD-1 protein expression during active TB disease and chemotherapy.

<p>Frozen PBMCs from Pakistan were thawed and stained to analyse PD-1 protein expression. (A) PD-1 protein expression on CD3⁺CD8⁺ T cells, CD3⁺CD8^- T cells and CD3^- lymphocytes at diagnosis in 18 TB patients (moderate severity) (P), 7 latently infected contacts (C+) and 5 uninfected contacts (C-). P values are derived from the Kruskal-Wallis test with Dunn’s multiple testing correction. (B) Modulation of PD-1 protein expression on CD3⁺CD8⁺ T cells, CD3⁺CD8^- T cells and CD3^- lymphocytes with the intensive phase of chemotherapy in patients who were sputum smear negative after 2 months of treatment (n = 11), analysed with the Wilcoxon signrank test. The bars show medians and the interquartile range.</p

<i>Ex vivo</i> gene expression of PD-1 and its ligands in active TB disease.

<p>RNA was extracted from whole blood samples added to Tempus tubes and expression investigated by qRT-PCR. (A) PD-1 expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 23), in IGRA+ (C+: n = 11) and in IGRA- contacts (C-: n = 8). (B) PD-L1 expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 24), in latently-infected household contacts (C+: n = 12) and in uninfected contacts (C-: n = 11). (C) PD-L2 expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 24), in latently-infected household contacts (C+: n = 12) and in uninfected contacts (C-: n = 12). The lines in the centre of each group represent medians. (D) ROC comparisons were conducted between TB patients and household contacts (C+ and C- combined) to determine whether PD-1, PD-L1 or PD-L2 expression could distinguish active TB patients from healthy people. PD-1/PD-L1/PD-L2 mRNA expression was determined by qRT-PCR, with results shown normalised against the housekeeping gene Cyclophilin A. P values in A) to C) were derived from Kruskal-Wallis test with Dunn’s multiple testing correction. AUC = Area Under the Curve.</p

<i>Ex vivo</i> gene expression of PD-L1 and PD-L2 in the intensive phase of TB treatment.

<p>RNA was extracted from whole blood added to Tempus tubes and expression investigated by qRT-PCR. (A) PD-1 expression at the beginning and the end of the intensive phase (n = 15 patients). (B) PD-L1 expression at the beginning and the end of the intensive phase (n = 16 patients). (C) PD-L2 expression at the beginning and the end of the intensive phase (n = 15 patients). The lines in the centre of each group represent medians. (D) ROC comparisons were conducted for PD-1, PD-L1 and PD-L2 expression comparising TB diagnosis with month 2 of treatment for the TB patients. PD-L1/PD-L2 mRNA expression was normalised against the housekeeping gene Cyclophilin A.</p

NKG2D <i>ex vivo</i> blood gene expression prior to and during chemotherapy.

<p>RNA was extracted from whole blood Tempus tubes and gene expression investigated by qRT-PCR. (A) NKG2D expression in <i>ex vivo</i> venous blood from untreated active TB cases at diagnosis (n = 26), in latently-infected household contacts (Contacts (+): n = 13) and in uninfected contacts (Contacts (−): n = 11), all recruited in Lahore, Pakistan. The Wilcoxon ranksum test was used to compare the clinical groups. (B) NKG2D expression after the end of the intensive phase in 16 patients who were eventually successfully cured (closed circles) and in 2 patients who subsequently died (open circles). (C) NKG2D expression during the entire course of TB chemotherapy in a different 6 patients, for whom data were available at the end of treatment. The Wilcoxon signrank test was used to analyse the paired data in (B) and (C). The lines in the centre of each group represent medians. NKG2D mRNA expression was determined by qRT-PCR, with results shown normalised against the housekeeping gene Cyclophilin A.</p

NKG2D protein expression during active TB disease and chemotherapy.

<p>Frozen PBMCs were thawed and stained to analyse NKG2D protein expression. (A) NKG2D protein expression on CD3⁺CD8⁺ T cells and CD3^- lymphocytes at diagnosis in 21 TB patients (P), 8 latently infected contacts (C+) and 6 uninfected contacts (C-). Data were analysed using the Wilcoxon rank sum test. (B) Modulation of NKG2D protein expression on CD3⁺CD8⁺ T cells and CD3^- lymphocytes with the intensive phase of chemotherapy in successfully cured patients (n = 13). The graphs are box and whisker plots, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070063#pone-0070063-g003" target="_blank">Figure 3</a>, and analysed using the Wilcoxon signed rank test. (C) NKG2D protein expression in 2 patients who subsequently died.</p

NKG2D gene expression at diagnosis and during chemotherapy in <i>in vitro</i> stimulated RNA samples.

<p>PBMC were isolated and stimulated <i>in vitro</i> with <i>M. bovis</i> BCG (MOI 1∶1) or anti-CD3 mAb for 16 hours and NKG2D mRNA expression analysed by qRT-PCR. (A) NKG2D mRNA expression at TB diagnosis in 26 untreated TB patients (P), 13 latently infected contacts (C+) and 11 uninfected contacts (C-). (B) Modulation of NKG2D mRNA expression during the intensive phase of treatment in 16 successfully cured TB patients. (C) NKG2D mRNA expression following stimulation in 2 patients who subsequently died. (D) NKG2D mRNA expression in a separate group of 6 successfully cured patients throughout the full treatment course. The line in the centre of the box and whisker plots represents the median whereas the top and bottom lines represent the 75^th and 25^th quartile respectively, and whiskers represent minimum and maximum data points. The Wilcoxon ranksum test (A.) and the Wilcoxon signrank test (B) and (D) were used for statistical analyses of unpaired and paired data respectively.</p

Regulation of NKG2D and DAP10 mRNA following mycobacterial stimulation <i>in vitro</i>.

<p>CD4⁺ and CD8⁺ T cells were isolated from ten healthy BCG-vaccinated donors after 7 days of PBMC stimulation with live <i>M. tuberculosis</i> H37Rv, on ten separate occasions. NKG2D (A) and DAP10 (B) mRNA expression levels in each T cell subset were determined by qRT-PCR. Data are normalised against the HPRT housekeeping gene, with the mean of duplicate technical replicates shown. (C) Diluted whole blood cultures from five healthy donors were incubated on five separate occasions with live <i>M. bovis</i> BCG in the absence or presence of IL-2 for the time indicated. NKG2D mRNA expression was determined for each sample in duplicate by qRT-PCR and is shown normalised against HPRT and normalised against the unstimulated control at each time point, with mean and standard error of the mean for the five donors shown. *P<0.05 compared to the medium control cultures. (D) Diluted whole blood cultures from nine BCG-vaccinated donors were incubated with live <i>M. bovis</i> BCG or live <i>M. tuberculosis</i> for six days in three separate experiments. NKG2D mRNA expression is shown normalised against HPRT mRNA expression, with the mean and standard error of the mean of the nine donors shown. Statistical comparisons were performed using the Wilcoxon signed rank test for paired data.</p

Modulation of NKG2D/GrzB protein co-expression during active TB disease and chemotherapy.

<p>Frozen PBMCs were thawed and NKG2D/GrzB co-expression was investigated by flow cytometry. (A) Percentage of NKG2D GrzB double-positive cells in the CD3+CD8+ lymphocyte gate or CD3- lymphocyte (NK cell) gate at diagnosis in TB patients (P: n = 18), latently infected contacts (C+: n = 7) and uninfected contacts (C-: n = 4). Modulation of NKG2D/GzB co-expression with the intensive phase of chemotherapy on CD3⁺CD8⁺ T cells (B) or CD3- lymphocytes (NK cells) (C) in patients who were successfully cured (n = 8) or who subsequently died (n = 2). The graphs are box and whisker plots, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070063#pone-0070063-g003" target="_blank">Figure 3</a>. The Wilcoxon ranksum test (A.) and the Wilcoxon signrank test (B) and (C) were used for statistical analyses of unpaired and paired data respectively.</p