Growth kinetics of OB and OS morphotypes of <i>B</i>. <i>pseudomallei</i> in <i>vitro</i>.

<p>Morphology of OB (WT) and OS (SCV) growth on nutrient agar after (A) 24 hours and (B) 48 hours. (C) Growth curves of OB and OS in LB broth incubated at 37°C for 48 hours. Data are representative of at least 2 independent experiments (A-C).</p

Plasma Th1/Th2/Th17 cytokine ratios.

<p>(A) Th1/Th2 (B) Th1/Th17 (C) Th2/Th17 ratios were measured in uninfected, OB- and OS-infected mice. Box plots show the median value (line), the interquartile range (box), and the minimum and maximum values (whiskers). <i>P</i> values were calculated using Mann-Whitney U test. *<i>P</i><0.025, **<i>P</i><0.005, ***<i>P</i><0.0005 after Bonferroni correction for 2 comparisons.</p

Correlation analysis between Th1/Th2/Th17 cytokines.

<p><i>P</i> values were calculated using Spearman’s Rank Order Coefficient. *<i>P</i><0.008, **<i>P</i><0.002, ***<i>P</i><0.0002 after Bonferroni correction for 6 comparisons.</p

Ten-day survival rate of BALB/c mice infected with WT and SCV <i>B</i>. <i>pseudomallei</i>.

<p>BALB/c mice were infected with different doses of (A) OB and (B) OS, and ten-day survival rate of mice was plotted. (C) Comparison of ten-day survival rate of mice using same log CFU of OB and OS, and <i>P</i> value was calculated using Log-Rank test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Data are representative from 2 independent experiments (A-C, n = 6 per bacterial dose).</p

Levels of plasma Th1/Th2/Th17 cytokines in experimental persistent infection of <i>B</i>. <i>pseudomallei</i>.

<p>(A) IL-2 (B) TNF-α (C) IFN-γ (D) IL-4 and (E) IL-6 (F) IL-10 and (G) IL-17A levels were measured using flow cytometry. Data are pooled from two independent experiments (A–G; n = 6 per group). Box plots show the median value (line), the interquartile range (box), and the minimum and maximum values (whiskers). <i>P</i> values were calculated using Mann-Whitney U test. *<i>P</i><0.025, **<i>P</i><0.005, ***<i>P</i><0.0005 after Bonferroni correction for 2 comparisons.</p

PD-1 and CTLA-4 expressions on FVS 510-/Lymph/CD3+/CD4+ and CD8+ T cells.

<p>(A) Representative histogram of PD-1 and CTLA-4 expressions on CD4+ T cells of uninfected, OB- and OS-infected mice after 60 days of infection. Median fluorescence intensities (MFIs) of PD-1 on (B) CD4+ and (C) CD8+ T cells, and (D) CTLA-4 on CD4+ and (E) CD8+ T cells of uninfected, OB- and OS-infected mice. Mean fluorescence intensity is used in (D) as median shows negative fluorescence values. Logarithm with base 10 is used for (B) and (D) for improved illustration purposes. Data are pooled from two independent experiments (B-E; n = 6 per group). Box plots show the median value (line), the interquartile range (box), and the minimum and maximum values (whiskers). Correlation analyses between CD4+ T-cell frequency and PD-1 expression on (F) CD4+ and CD8+ T cells, and between PD-1 expression on CD4+ and CD8+ T cells. Data are pooled from all uninfected, OB, and OS-infected mice from two independent experiments (F; total n = 18). <i>P</i> values calculated using Mann-Whitney U test (B–E) and Spearman’s Rank Order Coefficient (F). *<i>P</i><0.025, **<i>P</i><0.005, ***<i>P</i><0.0005 after Bonferroni correction for 2 comparisons.</p

Persistent WT and SCV <i>B</i>. <i>pseudomallei</i> infection in BALB/c mice.

<p>Bacterial loads in (A) lungs, (B) livers, and (C) spleens harvested from each mouse after ≥60 days of sub-lethal dose of <i>B</i>. <i>pseudomallei</i> infection. Data are pooled from two independent experiments (A-C; n = 6 per group). (D) Liver and (E) splenic abscesses in OS-infected mice. Box plots show the median value (line), the interquartile range (box), and the minimum and maximum values (whisker). <i>P</i> values were calculated using Mann-Whitney U test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. (F) The 397bp band on gel image indicates presence of <i>B</i>. <i>pseudomallei</i> in mice with persistent infection. Lane 1 shows DNA ladder. Lane 2–4 shows presence of OB in lung, liver, and spleen homogenate, respectively. Lane 5–7 shows presence of OS in lung, liver, and spleen homogenate, respectively. Lane 8–9 shows positive and negative control. Picture depicts one representative of all gel images.</p

Expression levels of different markers by CD161⁺⁺CD8⁺ T-cell subsets in the study population.

<p><b>(A)</b> Zebra plots of double-gating strategy (gated on the CD161⁺⁺ CD8⁺ T cells) show staining with 4 different markers (CD103, PD-1, CCR6, CCR5) on representative samples from a HC and a CPTN. HCs showed increased amount of CCR6-expressing MAIT cells and decreased amount of PD-1 expressing MAIT cells compared to CPTNs. <b>(B)</b> HC showed significantly lower expression level of inhibitory receptor, PD-1 while HIV/TB co-infected patients show significantly increased PD-1 expressing MAIT cells. <b>(C)</b> Significantly increased CCR6-expressing MAIT cells were found in HCs compared to HIV and TB infected groups. <b>(D)</b> No significant difference was observed in CCR5 expression levels by MAIT cells among the different study subjects. The significant difference in CCR5 expression between HVTPs and HCs may be due to the limited number of samples. All graphs show median (red bars) and range (blue whiskers); <i>P</i> values are reported for two-sided Mann-Whitney tests with threshold for significance <i>P</i> = 0.025 after Bonferroni correction for 2 comparisons. (Note: TN, treatment naïve; TP, treatment positive; HC, healthy control; CP, HIV/TB co-infection; HV, HIV mono-infection).</p

Demographic, clinical and laboratory characteristics of study participants.

<p>Footnotes: Statistical analyses performed using (A) Fisher’s Exact test and (B) Kruskal-Wallis non-parametric ANOVA test:</p><p>*p<0.05</p><p>**p<0.01</p><p>***p<0.001</p><p>CPTN: HIV/TB co-infection treatment naïve; CPTP: HIV/TB co-infection treatment positive; HVTN: HIV mono-infection treatment naïve; HVTP: HIV mono-infection treatment positive; NA: non- applicable. All values are expressed as mean ±SD.</p><p>Demographic, clinical and laboratory characteristics of study participants.</p

Percentage of CD8⁺ T cells expressing CD161⁺⁺ across different study groups.

<p><b>(A)</b> Scatter plots (gated on the CD3⁺ T-cell population) show co-staining with CD8 and CD161 on representative samples from 5 different clinical groups: CPTNs, CPTPs, HVTNs, HVTPs, and HCs. <b>(B)</b> CD161⁺⁺CD8⁺ T (MAIT cell) frequency in HCs showed significantly increased MAIT cell levels compared to other study groups. <b>(C)</b> CD161⁺CD8⁺ T cell frequency showed no difference across the different study groups. <b>(D-E)</b> CD161⁺⁺CD8⁺ MAIT cell frequency in subjects with HIV mono-infection and HIV/TB co-infection shows no significant correlation with either HIV plasma viral load (copies/mL) or CD4⁺ T-cell counts (cells/mm³). All graphs show median (red bars) and range (blue whiskers); <i>P</i> values are reported for two-sided Mann-Whitney tests with threshold for significance <i>P</i> = 0.025 after Bonferroni correction for 2 comparisons. Correlations between MAIT cell frequency and markers of HIV disease progression were assessed using two-tailed non-parametric Spearman’s rank. (Note: TN, treatment naïve; TP, treatment positive; HC, healthy control; CP, HIV/TB co-infection; HV, HIV mono-infection).</p