Clonal Analysis of the T-Cell Response to <i>In Vivo</i> Expressed <i>Mycobacterium tuberculosis</i> Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method

<div><p>Tuberculosis (TB), caused by <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), remains a leading cause of death worldwide. A better understanding of the role of CD4⁺ and CD8⁺ T cells, which are both important to TB protection, is essential to unravel the mechanisms of protection and to identify the key antigens seen by these T cells. We have recently identified a set of <i>in vivo</i> expressed <i>Mtb</i> genes (IVE-TB) which is expressed during <i>in vivo</i> pulmonary infection in mice, and shown that their encoded antigens are potently recognized by polyclonal T cells from tuberculin skin test-positive, <i>in vitro</i> ESAT-6/CFP10-responsive individuals. Here we have cloned T cells specific for one of these newly identified <i>in vivo</i> expressed <i>Mtb</i> (IVE-TB) antigens, Rv2034. T cells were enriched based on the expression of CD154 (CD40L), which represents a new method for selecting antigen-specific (low frequency) T cells independent of their specific function. An Rv2034-specific CD4⁺ T-cell clone expressed the Th1 markers T-bet, IFN-γ, TNF-α, IL-2 and the cytotoxicity related markers granzyme B and CD107a as measured by flow cytometry. The clone specifically recognized Rv2034 protein, Rv2034 peptide p81–100 and <i>Mtb</i> lysate. Remarkably, while the recognition of the dominant p81–100 epitope was HLA-DR restricted, the T-cell clone also recognized a neighboring epitope (p88–107) in an HLA-DR- as well as HLA-DQ1-restricted fashion. Importantly, the T-cell clone was able to inhibit <i>Mtb</i> outgrowth from infected monocytes significantly. The characterization of the polyfunctional and <i>Mtb</i> inhibitory T-cell response to IVE-TB Rv2034 at the clonal level provides detailed further insights into the potential of IVE-TB antigens as new vaccine candidate antigens in TB. Our new approach allowed the identification of T-cell subsets that likely play a significant role in controlling <i>Mtb</i> infection, and can be applied to the analysis of T-cell responses in patient populations.</p></div

PBMC recognition of TB10.4 and IVE-TB antigen Rv2034.

<p>PBMC from a PPD⁺ donor were stimulated with different stimuli for 6 days and IFN-γ production (pg/ml) was determined in the supernatants. Both TB10.4 protein (10 µg/ml) (A) and Rv2034 protein (10 µg/ml) (B) were analyzed as well as control mitogen PHA and <i>Mtb</i> derived PPD (A and B). Medium values (unstimulated PBMC) were subtracted. IFN-γ concentrations were determined from triplicate-pooled supernatant. A cut-off value was set arbitrarily at 100 pg/ml.</p

Analysis of TB10.4 clonal cultures.

a<p>Single live CD14-CD19-CD3+ cells (population>100 cells).</p><p>Non-responding clonal cultures included CD4 (n = 9), CD8 (n = 2) and DN (n = 2) cells.</p><p>DN = Double negative.</p><p>+ = >1% positive cells.</p><p>+/− = <1% positive cells.</p

Rv2034 responsive CD4⁺ T-cell clone phenotype.

<p>The shown CD4⁺ T-cell clone that had been expanded was restimulated with the Rv2034 peptide pool and analyzed for the expression of CD154 expression, IFN-γ, TNF-α and IL-2 (black dots). Data is representative of over three independent experiments. CD154 and Th1 cytokine expression of non-activated T cells is indicated in grey dots. Dot blots show single live CD14⁻CD19⁻CD3⁺CD4⁺ T cells. The frequency of all CD3⁺CD4⁺ T cell subsets identified upon stimulation are indicated in the corners of each plot.</p

Identification of immunogenic epitope(s) of Rv2034 recognized by CD4 T-cell clone.

<p>To identify the immunogenic epitope(s) in Rv2034, the CD4⁺ T-cell clone was stimulated with all individual Rv2034 20-mer peptides with 10 aa overlap; Rv2034 recombinant protein; Rv2034 peptide pool; control ESAT-6/CFP10 fusion protein; an Ag85B/ESAT-6/Rv2034 trimeric fusion protein; and negative and positive control conditions. Autologous irradiated PBMC were used as APCs. Both IFN-γ (open bars) and T-cell proliferation (black bars) were determined. CPM bars represent median ranging the highest and lowest value (n = 3) (A). To determine the Rv2034 p81–100 specific response by flow cytometry, the CD4⁺ T-cell clone was stimulated with Rv2034 p81–100 (B), Rv2034 protein (C) and Rv2034 p11–30 (D) using autologous irradiated PBMC, in the presence of BFA. Intracellular CD154 and Th1 cytokine expression was determined. Data is representative of three independent experiments. Flow cytometry plots show single live CD14⁻CD19⁻CD3⁺CD4⁺ T cells, the frequency of all subsets of CD3⁺CD4⁺ T cells are indicated in the corners of each plot.</p

CD4⁺ T-cell clone has <i>Mtb</i> inhibitory properties.

<p>Autologous PBMC were loaded with <i>Mtb</i> (10 MOI) and the Rv2034 T-cell clone was added at an effector/target (E/T) ratio of 20∶1 and 50∶1 as indicated on the <i>x</i>-axis. CFU were determined after o/n incubation. Eight replicates from two independent experiments were included for 50∶1 and only target cells condition, four replicates were included for 20∶1 condition (A). The HA-specific clone was included as a negative control (n = 4) (B). The horizontal line indicates the median value and the outer boundaries of the box represents the 25^th and 75^th percentiles. The whiskers indicate the highest and lowest values. A Mann-Whitney <i>U</i> test was performed to analyze the difference between CFU. A <i>p</i>-value≤0.05 was considered significant and indicated with an asterisk.</p

Restriction of Rv2034 p81–100 and p88–107 presentation by HLA-DR and -DQ molecules.

<p>To determine the HLA-DR and -DQ restriction of Rv2034 p81–100 responding CD4⁺ T-cell clone, T cells were incubated with Rv2034 p81–100 (A), Rv2034 protein (B) or Ag85B/ESAT-6/Rv2034 fusion protein (C), in the presence of irradiated PBMC with (mis)matched HLA-DR and/or DQ molecules. Both IFN-γ production (white bars) and proliferation (CPM) (black bars) was determined. CPM bars represent median ranging the highest and lowest value (n = 3). Matching HLA alleles are indicated in bold on the <i>x</i>-axis. HLA-DR and HLA-DQ molecules of APCs pulsed with either Rv2034 protein, Rv2034 p81–100, Rv2034 p88–107 (5 µg/ml) or control conditions, were blocked using monoclonal antibodies directed against HLA-DR or -DQ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099203#pone.0099203-Ottenhoff3" target="_blank">[21]</a>, and T-cell proliferation was analyzed (CPM) (D). Data is representative for three independent experiments.</p

Kinetics of IP-10 production in WBA.

<p>(<b>A</b>): IP-10 concentrations produced in stimulated whole blood cultures of leprosy patients (upper panel; LP; n = 10: 5 BL (Ethiopia); 2 BT (Ethiopia); 3 BT (The Netherlands) and healthy endemic controls (lower panel; EC, n = 8) in response to <i>M. leprae</i> WCS (left panel; 10 µg/ml), <i>M. leprae</i> unique protein ML2478 (middle panel; 10 µg/ml) and PHA (right panel; 1 µg/ml). IP-10 concentrations were determined by ELISA after 1 h, 4 h, 6 h and 24 h antigen stimulation. Values on the y-axis are concentrations corrected for background values. (<b>B</b>): Comparison of IP-10 concentrations determined by ELISA after 6 h stimulation with ML2478 (10 µg/ml) of whole blood samples.</p

Correlation between ELISAs and UCP-LFAs.

<p>Levels of IP-10 (<b>A</b>) and IL-10 (<b>B</b>) in 24 h whole blood samples of 77 <i>M. leprae</i> (antigen), LPS and PHA stimulated WBA samples of Dutch healthy controls were simultaneously determined by ELISAs and wet-format UCP-LFAs. <b>Left panels</b>: results for ELISAs are indicated in pg/ml (ELISA) or as the ratio of the relative fluorescence units (RFUs) measured at Test and Flow-Control lines (UCP-LFA). <i>R²</i> equals the square of the Pearson correlation coefficient. <b>Right panels</b>: Spearman ranking.</p

Combined cytokine profiles in response to <i>M. leprae</i>.

<p>Production of IFN-γ (<b>A</b>), IP-10 (<b>B</b>) and IL-10 (<b>C</b>) determined by ELISA, in response to medium (-), PHA, <i>M. leprae</i> WCS or the <i>M. leprae</i>-unique protein ML2478 in 24 h WBA for Ethiopian leprosy patients (n = 11: 2 BT (○) and 9 BL (•), and healthy endemic controls (EC; n = 12; □). For comparison between BT and BL, significant differences were found for <i>M. leprae</i> WCS (Mlep) induced IFN-γ responses (p = 0.036) and ML2478 induced IL-10 responses (p = 0.035). (<b>D</b>): IP-10/IL-10 ratios are depicted for unstimulated samples after 24 h {LP (•) and EC (□)} or after 1 h WBA {LP (▵) and EC (▾)}. (<b>E</b>): Anti-PGL-I antibodies for BL (○) and BT (•) patients were detected by ELISA using natural disaccharide of PGL-I linked to HSA <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002845#pntd.0002845-Cho2" target="_blank">[31]</a> (ND-O-HSA). Optical density (OD₄₅₀) readings were performed using 1∶800 serum dilutions. Median values per group are indicated by horizontal lines. The cut-off for positivity is indicated by the dashed horizontal line.</p