Treg-cell marker frequency and density are increased on live BCG-activated CD8⁺ vs. CD4⁺ T cells.

<p>A: BCG induces Treg-cell marker expression on CD4⁺ and CD8⁺ T cells; after live BCG stimulation the percentage of total CD8⁺ T cells expressing CD25, Foxp3, CD39, LAG-3 or CCL4 is significantly higher compared to CD4⁺ T cells, depicted here as frequency of CD8⁺ or CD4⁺ population. Differences in Treg marker expression between heatkilled BCG–activated CD8⁺ vs. CD4⁺ T cells were not significant, except for expression of CCL4; CCL4 expression was also significantly higher on CD8⁺ T cells compared to CD4⁺ T cells in samples not stimulated with BCG (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192.s001" target="_blank">Fig. S1</a>) (<i>*p</i> < 0.05, Wilcoxon signed-ranks test). B: Mean fluorescence intensities (MFIs) of CD25 and CD39 are increased on live BCG-activated CD8⁺ T cells as compared to CD4⁺ T cells. Gating was performed as demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone-0094192-g001" target="_blank">figure 1A</a>. To assess differences in intrinsic intensity of expression on CD4⁺ and CD8⁺ T cells, respectively, MFIs of positive Treg marker populations in samples not stimulated with BCG were compared; this was similar on CD4⁺ and CD8⁺ T cells for MFIs of CD25, Foxp3 and CD39. Data are representative of seven <i>in vitro</i> PPD-responders six days after heatkilled or live BCG stimulation (*<i>p</i> < 0.05; Wilcoxon signed-ranks test).</p

Co-expression of multiple Treg-cell markers enriches for CD8⁺, and not CD4⁺ T cells.

<p>A. The percentage of total CD8⁺ T cells co-expressing CD39, LAG-3, CCL4, CD25 and/or Foxp3 in different combinations is significantly increased, compared to CD4⁺ T cells. Demonstrated is a combined analysis using Boolean gating of cells from ten donors six days after live BCG infection. Gating was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone-0094192-g001" target="_blank">figure 1A</a>. Boxes: 25th to 75th percentiles; line at median; whiskers: minimum to maximum (*<i>p</i> < 0.05, **<i>p</i> < 0.01; Wilcoxon signed-ranks test). B. Combining Treg markers enriches for CD8⁺ T cells as opposed to CD4⁺ T cells. Boolean gating was performed on CD3⁺ T cells of ten donors; CD8 vs. CD4 gating is demonstrated (top) and the CD8⁺ proportion of these gated populations is demonstrated (bottom) for a selection of CD3⁺ Boolean gates. The CD8⁺ proportion increased significantly using a combination of Treg markers as compared to the complete CD3⁺ population. Boxes: minimum to maximum, line at median (Wilcoxon signed-ranks test).</p

Suppressive activity resides predominantly in live BCG-activated CD8⁺ T cells.

<p>Heatkilled BCG-activated and live BCG-activated T cell lines were expanded, and live BCG T-cell lines were enriched for CD4 or CD8 expression using magnetic beads. Suppressive capacity was tested in a co-culture assay by titrating these T cell lines onto a Th1 reporter clone that was stimulated with its cognate peptide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192-Joosten2" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192-Boer1" target="_blank">[31]</a>. Proliferation was measured by (3H)TdR incorporation after three days. Proliferation was divided by Th1 reporter clone proliferation in the absence of Treg cells to obtain relative proliferation as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192-Joosten2" target="_blank">[30]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192-Joosten3" target="_blank">[32]</a>. Dotted lines represent background proliferation of T cells, in the absence of reporter clone peptide, relative to Th1 clone proliferation. A. Suppressive activity was confined to live BCG-activated T cells, and could not be demonstrated for heat-killed BCG-specific T cells. Data are depicted as mean + SE of five different heat-killed BCG-activated T cell lines, and six live BCG-activated T cell lines. B. Suppressive activity resides predominantly in CD8⁺ T cells, and not in CD4⁺ T cells (mean + SE of CD4⁺ and CD8⁺ T cell lines of three donors, tested in at least two independent assays; Wilcoxon signed-ranks test, <i>p</i> < 0.001).</p

Heatkilled vs. live BCG-activated expression of Treg-cell markers on CD4⁺ and CD8⁺ T cells.

<p>A: Gating strategy: cells were gated on single cells, live lymphocytes, CD3⁺ and CD4⁺CD8⁻ vs. CD4⁻CD8⁺. Demonstrated is the synchronized gating on the positive population of interest for CD4⁺CD8⁻ and CD8⁺CD4⁻ T cells; here the CD25-positive population. B: Heatkilled and live BCG activate CD25⁺Foxp3⁺ and LAG-3⁺CD39⁺ T cells. Expression of regulatory T cell markers on CD4⁺ and CD8⁺ T cells of <i>in vitro</i> PPD responders was analysed by flowcytometry six days after heatkilled or live BCG stimulation. For each donor gating was compared to samples not stimulated with BCG (demonstrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094192#pone.0094192.s001" target="_blank">Fig. S1</a>). Data are representative of seven responders.</p

CD8⁺ T-cells from TB patients bind HLA-E/ peptide tetramers and produce Th2 cytokines following peptide stimulation.

<p>PBMCs from patients with pulmonary TB were stained directly <i>ex vivo</i> with HLA-E/ peptide tetramers and analysed by flow cytometry. Data are expressed as the percentage tetramer positive cells within the CD8⁺ population. PBMCs were stimulation with either peptide 62 or peptide 68 for 16 hours in the presence of monensin. Cytokines were stained by intracellular staining followed by flow cytometric analysis, data are expressed as percentage of CD8⁺ T-cells. A. Example flow cytometry results for a representative single TB patient following staining with HLA-E tetramers containing peptide 62 or peptide 68, cells are gated on CD8⁺ T-cells. B. Results of combined TM staining on PBMCs from TB patients for both tetramers containing P62 or P68, data are expressed as percentage of CD8⁺ T-cells. C. Example of intracellular cytokine staining following peptide stimulation for a single representative TB patient, cells are gated on CD8⁺ T-cells. D. Cytokine production by CD8⁺ T-cells following stimulation with peptide 62 (left) and peptide 68 (right). Open circles represent patients with tetramer staining <0.1%, close circles represent patients with tetramer staining >0.1%. Groups were compared using a Mann-Whitney U test and p<0.05 was considered significant.</p

Mtb specific HLA-E restricted T-cell clones produce Th2 cytokines.

<p>A. T-cell clones were cultured and RNA was isolated for gene-expression measurement using dcRT-MLPA, data are normalized to GAPDH as housekeeping gene (left panel); clones were stimulated for 24 hours with αCD3/28 beads before supernatants were collected to determine their maximum cytokine secretion profiles (in pg/ml) using multiplex bead arrays (middle panel); similarly, cells were stimulated for 16 hours with αCD3/28 beads in the presence of brefeldin A followed by intracellular cytokine staining to determine intracellular cytokine levels (right panel). Data are expressed as % of the CD3⁺CD8⁺ T-cell population. Data are coloured according to the amount of the molecules detected, according to the legend in the figure. Nt = not tested. B. HLA-E restricted CD8⁺ T-cell clones were stimulated for 24 hours with αCD3/28 T-cell activator beads and stained intracellular for IL-4 or IL-5 or IL-13 as well as IFN-γ. C. T-cell clones were cultured with peptide pulsed macrophages to assess their specific cytokine production in response to peptide presented by professional antigen presenting cells (top panel), or with BCG infected macrophages to assess their specific cytokine production induced by naturally presented antigen during in vitro mycobacterial infection (bottom panel). Supernatants were collected and cytokine/ chemokine levels were determined using multiplex bead arrays.</p

Peptide specific HLA-E restricted CD8⁺ T-cell clones have an effector memory phenotype.

<p>T cell clones were cultured in the absence of peptide specific stimulation and RNA was isolated from T-cell clones, RNA expression profiles were determined using dcRT-MLPA and data were normalized for GAPDH expression within each sample (A-C). A. RNA expression levels of classical cellular subset and memory markers of T-cell clones; B. RNA expression levels of cytotoxic effector function associated molecules; C. RNA expression levels of lineage associated transcription factors. D. Flow cytometric analysis of T-cell phenotype, T cell clones were directly stained from culture, a representative T-cell clone is shown (D6-2B4). Gating strategy in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004671#ppat.1004671.s001" target="_blank">S1 Fig.</a> E. Flow cytometric analysis of effector molecules, T-cell clones activated with αCD3/28 beads for 24 hours followed by intracellular staining, a representative T-cell clone is shown (D6-2B4). F. Flow cytometric analysis of lineage determining transcription factors, T-cell clones were directly stained from culture using intracellular staining protocols. Dashed lines represent transcription factor staining in PBMCs, grey (D2–1B9) and black are examples of different T-cell clones (D2-4A1, D6-1F11, D2-2A9).</p

HLA-E restricted T-cell clones possess either suppressive or cytolytic activity.

<p>A. T-cell clones were expanded, after which they were added in different ratios to an unrelated reporter Th1 cell clone (Rp15 1-1; Mtb hsp65 p3–13 specific, HLA-DR3 restricted) in the presence of irradiated HLA-DR3 expressing PBMCs as antigen presenting cells together with the cognate peptide recognized by Rp15-1-1 (closed bars). After 3 days of co-culture the proliferative response of the Th1 clone was determined by 3H-TdR incorporation. There was no proliferation in the absence of the cognate p3–13 peptide stimulating the Th1 clone (open bars). Data are expressed in counts per minute (CPM), averaged for triplicate wells (+/- standard deviation). B. T-cell clones were titrated onto ⁵¹Cr labelled adherent HLA-A2 negative monocytes that were infected with live BCG, and the release of ⁵¹Cr was determined after 5 hours. Data are expressed as percentage specific lysis. Black bars represent BCG infected monocytes, open bars represent uninfected control monocytes. A ratio of 10:1 (T-cells: monocytes) is shown here. T-cell clones from donor 2 (peptide 62 specific) and from donor 6 (peptide 68 specific) specifically lysed BCG infected target cells. Data represent the average +/- standard deviation of triplicate wells. C. Combined analyses of suppressive and cytolytic activity for all clones tested. The percentage of suppression was calculated by dividing deltaCPM (CPM in presence of p3–13 to activate Rp15 1-1 proliferation minus CPM in absence of p3–13) of 5x10e4 T-cell clones by the deltaCPM of the Th1 clone in the absence of HLA-E restricted T-cell clones (left panel) as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004671#ppat.1004671.ref058" target="_blank">58</a>]. Similarly, the percentage specific lysis was plotted in the middle panel. The percentage of specific Mtb killing for each individual clone is plotted in the right panel. The percentage of Mtb killing was calculated after subtraction of the average experimental variation within each experiment and was tested in 3–4 different macrophage donors for each T-cell clone. Nt = not tested. D. T-cell clones were added to Mtb (H37Rv) infected HLA-A2 negative macrophages for 24 hours in a ratio of 5:1 (T-cells: monocytes), subsequently macrophages were lysed and plated for assessment of colony forming units (CFU). CFU were counted and are expressed as CFU/ml lysate. Data represent the average +/- standard deviation of duplicate wells. E. The percentage of intracellular Mtb growth inhibition was calculated by dividing CFU outgrowth from infected macrophages with and without the addition of T-cells for each individual clone. All clones were tested in duplicate in at least 3 independent experiments, using independent macrophage donors. The percentage Mtb growth inhibition was expressed as average of these experiments. The percentage of Mtb growth inhibition was plotted against the percentage of CD8⁺ T-cells expressing perforin (left), perforin and granulysin (middle) and perforin and granzyme B (right), as assessed by flow cytometry. Linear regression analysis was performed to obtain an R² value.</p

Phenotype of human CD8⁺ T-cell clones reactive with Mtb peptides presented by HLA-E.

<p>Phenotype of CD8⁺ T-cell clones was determined using flow cytometry for all 16 individual clones, and results were scored for all clones tested for the specific marker (combination) according to the percentage of positive cells as indicated in the last column, the cut-off was set based on population characteristics and staining patterns on PBMC. Individual data (% of cells expressing particular markers) for all clones can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004671#ppat.1004671.s003" target="_blank">S1 Table</a>.</p><p>Phenotype of human CD8⁺ T-cell clones reactive with Mtb peptides presented by HLA-E.</p

HLA-E restricted Mtb specific T-cell clones utilize IL-4 to provide B-cell help.

<p>T cell clones were co-cultured with primary CD19⁺ B-cells in a 1:1 ratio for 48 hours, subsequently B-cell activation was determined by flow cytometry and by measurement of IL-6 in supernatants. A. Flow cytometric analysis of B-cells only (top row), or B-cells co-cultured with 2 independent T-cell clones and stained for CD80, CD86, and CD25. Cells are gated on CD3⁻CD19⁺ cells. B. B-cell activation induced by HLA-E restricted Mtb specific T-cell clones as indicated by expression of CD80, CD86, and CD25. C. B-cell activation induced by panel of unrelated, (CD4⁺) control T-cell clones and by recombinant cytokines. B-cell activation is assessed by flow cytometry. D. IL-6 production in supernatants of co-cultures of B-cells with HLA-E restricted Mtb specific CD8⁺ T-cells, B-cell activators (CpG, αIgG/M), recombinant cytokines and unrelated control T-cell clones. Data are expressed as pg/ml in supernatant. E. Co-culture of B-cells with HLA-E restricted Mtb specific T-cell clones in the presence of blocking antibodies against IL-4, IL-5 or IL-13, supernatants were collected and IL-6 measured by ELISA. Data are expressed as percentage inhibition of IL-6 production in supernatants of specific antibody blocking compared to the isotype control.</p