A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR + Ki67 + on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67 + HLA-DR + Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67 + HLA-DR + T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ + IL2 + TNFα + CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67 + HLA-DR + ) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+ Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+ HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+ HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+ IL2+ TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+ HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment

Microbiological characteristics of patients enrolled into the study showing pre-admission and on-admission (visit 0) AFB smear, time between smears and time to first sputum culture negative, culture conversion and which antigen specificities were detectable in the ICS assay (% responders).

%<p>Y =  yes; N =  no.</p>§<p>MDR Classification: 2, New MDR after TB treatment failure; 4, MDR after TB default; 6, Primary MDR; 9, MDR 2nd episode.</p>1<p>Acid Fast Bacilli (intensity score: +).</p>2<p>Mitogen (QuantiFERON-TB Gold).</p><p>*culture conversion calculated as the interval between the date of admission/start of MDR regimen to date of the first of 2 consecutive negative cultures at least 30 days apart.</p>#<p>based on first negative sputum culture, no follow up sputum culture results recorded.</p>a<p>RIF/INH/EMB/PZA (Reg 1).</p>b<p>Reg 1 plus Streptomycin.</p>c<p>Reg 1 plus Ofloxacin/Ethionamide/Streptomycin.</p>d<p>PAS/Terizodone/High dose INH/Kanamycin/Clarithromycin.</p

Comparison of memory maturation and activation profiles of antigen-specific CD4 T cells at baseline between sputum culture (SC) negative and positive individuals.

<p>(A) Representative overlay flow cytometry dot/density plots of TB-, PPD- and mitogen (mito)- specific CD4 T cell subsets (red) onto total CD4 sub-population (grey) for an individual with positive SC (top panel) and negative SC (bottom panel). Different CD4 subsets are shown as Naïve, Early Differentiated memory (ED), Late Differentiated memory (LD) and Terminally Differentiated (TD). The numbers in each quadrant represent the proportion of antigen-specific cell within each subset (red dots). (B) Proportion of naïve, ED, LD and TD subsets in Mtb (4 SC negative and 8 SC positive), PPD (6 SC negative and 12 SC positive) and mitogen (6 SC negative and 12 SC positive) responsive CD4 T cells. The open symbols represent patients who were SC negative and solid symbols represent patients who were SC positive. The horizontal line corresponds to the median. Non-Parametric Mann-Whitney <i>t</i>-test was used for statistical comparisons. (C) Representative flow cytometry dot-plots of the level of expression of Ki-67 and HLA-DR within PPD-specific CD4 T cell in a SC negative and a SC positive representative patient. The numbers represent the proportion of cells co-expressing Ki-67 and HLA-DR. (D) Proportion of activated cells (co-expressing Ki-67 and HLA-DR) in Mtb (4 SC negative and 8 SC positive), PPD (6 SC negative and 12 SC positive) and mitogen (6 SC negative and 12 SC positive) responsive CD4 T cells. Non-Parametric Mann-Whitney t-test was used for statistical comparisons.</p

ROC curve analysis of mycobacteria-specific Ki-67⁺HLA-DR⁺ CD4 T cells with time to sputum culture conversion.

<p>Receiver Operating Characteristic curve for the proportion of mycobacteria-specific Ki-67⁺HLA-DR⁺ CD4 T cells at baseline with the time to sputum clearance. The area under the curve (AUC), p-value and 95% confidence interval (c.i.) are shown on the graph. The dotted line represents an AUC of 0.5, which would depict a random test.</p

Comparison of the polyfunctional profiles of Mtb-, PPD- and Mito-specific CD4 T cells at baseline between patients who were sputum culture (SC) negative or positive.

<p>Pie and Bar charts showing the proportion (A) and relative frequency (B) of each cytokine combination after Mtb, PPD and mitogen (Mito) stimulation. The colors of the pies correspond to the permutation color grid at the foot of 2B, where this corresponds to the proportional frequency of cytokine-expressing combinations. Twelve of 18 patients responded in the ICS assay to the Mtb antigen cocktail: 4 SC negative and 8 SC positive. All 18 patients responded to PPD and Mito stimulations: 6 SC negative and 12 SC positive for each ICS stimulus. Statistical analysis of T cell multi-cytokine expression differences between groups, represented in the pie charts in Figure A, was made using a non-parametric partial permutation method within SPICE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102178#pone.0102178-Roederer1" target="_blank">[23]</a> and differences between antigen-specific CD4 T cells in Figure B using the student's t-test.</p

Evolution of the polyfunctional profile of Mtb- and PPD-specific CD4 T cells over time of TB chemotherapy in individuals with positive sputum culture (SC) at baseline.

<p>Pie and Bar charts showing changes in the proportions and relative frequency of multi-cytokine combinations after Mtb (A) and PPD (B) stimulation over time from baseline (BL, pre-treatment), 2, 4 and 6 months (M) of treatment. The color codes in the pies correspond to the permutation color blocks at the foot of each bar chart. Eight of the patients who were SC positive at baseline and responded to the Mtb antigen cocktail are shown in A. Twelve of the patients who were SC positive and responded to PPD stimulations and are shown in B. Statistical analysis of the changes in CD4+ T cell multi-cytokine proportions over time, represented in the pie charts, was made using the non-parametric partial permutation method within SPICE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102178#pone.0102178-Roederer1" target="_blank">[23]</a> and differences between the relative frequency of antigen-specific CD4+ T cells over time, represented by the solid colored symbols in the bar chart, was assessed using the Wilcoxon T-test.</p

Comparison of the frequencies of Mtb, PPD and mitogen responsive CD4 T cells at baseline between patients who were sputum culture (SC) negative or positive.

<p>(A) The magnitude of antigen-specific CD4 T cells expressing any of the 3 cytokines measured (i.e. IFNγ, IL2 and TNFα) and expressed as a % of total CD4 population. Open symbols represent SC negative patients (n = 6) and solid symbols represent SC positive patients (n = 12). The horizontal line shows the median. Non-Parametric Mann-Whitney t-test was used for statistical comparisons. (B) Representative flow cytometry dot plots showing the level of expression of IFNγ, IL2 and TNFα after no stimulation (NS), Mtb peptides, PPD and mitogen (MITO) in the whole blood ICS assay. The numbers in the quadrant represent the frequency of cells expressing each cytokine. The gating strategy is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102178#pone.0102178.s001" target="_blank">Figure S1</a>.</p

Changes in the proportion of antigen-specific CD4 T cells co-expressing HLA-DR and Ki67 over time of chemotherapy in individuals with positive SC at baseline.

<p>(A) Representative flow cytometry dot-plots showing the level of Ki-67 and HLA-DR co-expression within PPD-specific CD4 T cells at baseline (BL), 2, 4 and 6 months (M) of treatment. The numbers in the quadrants represent the proportion of antigen-specific CD4 T cells co-expressing Ki-67 and HLA-DR. (B) Proportion of activated (Ki-67⁺HLA-DR⁺) cells within Mtb, PPD and mitogen responsive CD4 T cells over time (months). The statistical differences were assessed using Wilcoxon matched pairs test.</p