Table 1.
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).
Figure 1.
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.
(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 Figure S1.
Figure 2.
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.
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 [23] and differences between antigen-specific CD4 T cells in Figure B using the student's t-test.
Figure 3.
Comparison of memory maturation and activation profiles of antigen-specific CD4 T cells at baseline between sputum culture (SC) negative and positive individuals.
(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 t-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.
Figure 4.
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.
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 [23] 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.
Figure 5.
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.
(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.
Figure 6.
ROC curve analysis of mycobacteria-specific Ki-67+HLA-DR+ CD4 T cells with time to sputum culture conversion.
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.