Superior T cell activation by ESAT-6 as compared with the ESAT-6–CFP-10 complex

Ayman Marei1, Amir Ghaemmaghami1,2, Philip Renshaw3, Martin Wiselka4, Michael Barer1, Mark Carr3 and Loems Ziegler-Heitbrock1

1 Department of Infection, Immunity and Inflammation, Medical Sciences Building, University of Leicester, Leicester LE1 9HN, UK
2 Allergy Research Group, Institute of Infection, Immunity and Inflammation, University of Nottingham, Nottingham, UK
3 Department of Biochemistry, University of Leicester, Leicester LE1 9HN, UK
4 Department of Infectious Diseases, Leicester Royal Infirmary, Leicester, UK

Correspondence to: L. Ziegler-Heitbrock; E-mail: lzh1{at}le.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Using intracellular cytokine staining we show herein that T cells will respond to short-term (6 h) activation with phorbol ester plus ionomycin by production of tumor necrosis factor (TNF), IFN-{gamma} or both. Here CD4 T cells preferentially produce TNF and CD8 cells IFN-{gamma}. The same pattern is seen when T cells are activated with the Mycobacterium tuberculosis protein early secretory antigenic target-6 (ESAT-6). Responses with >0.02% IFN-{gamma}+ CD3 cells were seen in 8 of 10 patients diagnosed with tuberculosis and in 12 of 14 healthy individuals selected for likely exposure to M. tuberculosis. T cell responses to the 1:1 complex of ESAT-6 and culture filtrate protein-10 (CFP-10) were inferior to ESAT-6 alone, and only reached the level of T cell response achieved with CFP-10 alone. Extending the time of incubation to 18 h leads to an increased response to the complex, but it still reached only the level of CFP-10 alone. In vitro digestion with lysosomal enzymes cathepsin L and S at 2000:1 protein to enzyme ratio demonstrates rapid digestion of the individual proteins while the ESAT-6–CFP-10 complex is resistant. The data suggest that the natural complex of ESAT-6–CFP-10 is less amenable to antigen processing leading to a lower T cell response as compared with the individual proteins.

Keywords: antigen presentation, T cells, tuberculosis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
One-third of the world population is infected with Mycobacterium tuberculosis with 10% of the infected developing disease. Diagnosis of the disease can be easy with smear-positive cases but is more difficult in other circumstances such as lymph node-only disease. Skin testing and standard in vitro assays using purified protein derivative (PPD) are confounded by cross-reactivity with the Bacille-Calmette-Guerin (BCG) strain, a strain that in the past was used frequently for vaccination. Here antigens early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) appear to provide improved specificity. These antigens are part of the region of difference 1 (RD1) of the M. tuberculosis genome; a region which is deleted in all BCG substrains but is present in virulent strains of Mycobacterium bovis and M. tuberculosis (1, 2). ESAT-6 has been proposed as a tool for diagnosis of M. tuberculosis infection (3) and has been frequently used in enzyme-linked immunospot (ELISPOT) assays. This assay was shown to be an accurate marker of M. tuberculosis infection and to distinguish infection with M. tuberculosis from the response to BCG vaccination (4). In an elegant analysis of a local outbreak, the ELISPOT assay using ESAT-6 as an antigen was found to be superior to tuberculin (PPD) skin testing in predicting infection with M. tuberculosis (5).

ESAT-6 and CFP-10 may not only be useful diagnostic tools but may also be considered as part of a vaccine, for instance, by co-expressing these proteins in an engineered BCG strain (6). Since these two proteins will form a 1:1 complex when co-expressed (7), the question arises whether this complex will be amenable to appropriate antigen processing and presentation to T cells.

In the present study we have analyzed T cell responses to these antigens by analysis of cytokine production. We demonstrate herein that the ESAT-6–CFP-10 complex is inferior to the individual components in triggering human T cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Blood collection and preparation
Heparinized blood samples (10 IU ml–1 final) were collected by venipuncture from 10 patients with tuberculosis (age 54.1 ± 14.0 years, male/female = 3/7). The patient group included five with smear-positive disease and two with miliary tuberculosis. All patients were studied after institution of antibiotic therapy. Eight of the 10 patients gave a positive in vitro response to ESAT-6. Of these eight, two were found to be skin test negative for PPD.

A total of 14 control donors (age 34.9 ± 9.0 years, male/female = 10/4) had been selected for likely exposure to and infection by M. tuberculosis based on origin from countries with high prevalence of M. tuberculosis and on exposure during medical duties. Of the 14 individuals studied nine had exposure to index cases. Twelve of the 14 control donors gave a positive in vitro response to ESAT-6. Of these 12, two were found to be skin test negative for PPD.

The blood samples were washed with an equal volume of RPMI-1640 supplemented with L-glutamine 2 mM, pencillin 100 U ml–1 and streptomycin 100 µg ml–1 (GIBCO Life Technologies Limited, Paisley, UK). Samples were then reconstituted to the original volume with the above medium.

The study protocol was approved by the ethics committee of Leicestershire and Rutland. Written informed consent was obtained from all subjects.

Preparation of ESAT-6 and CFP-10 proteins
The expression of CFP-10 and ESAT-6 in Escherichia coli was investigated using modified methods from a previously published work (7).

Expression and purification of ESAT-6.
Escherichia coli BL21(DE3) transformed with the pET21a-based expression vector for ESAT-6 were grown in LB medium containing 100 µg ml–1 ampicillin. The expression of ESAT-6 was induced in mid-log phase (corresponding to an optical density change at 600 nm of 0.6–0.7) by the addition of isopropyl-1-thio-ß-D-galactopyranoside to 0.45 mM. Cell pellets from 500-ml cultures were re-suspended in 12.5 ml 50 mM Tris, 2 mM EDTA and 0.1% (v/v) Triton X-100 buffer, pH 8.0, and lysed by sonication for 30 s with a 30-s rest period for four cycles. The insoluble fraction of the cell lysate, containing the ESAT-6 as inclusion bodies, was washed three times in a 50 mM Tris, 10 mM EDTA and 0.5% (v/v) Triton X-100 buffer, pH 8.0. Then the ESAT-6 inclusion bodies were solubilized in 6 M guanidine hydrochloride containing 1 mM EDTA and 100 µM phenylmethylsulfonylfluoride (PMSF) to give a final ESAT-6 concentration of 0.5–1.0 mg ml–1. This solution was dialyzed against a 25 mM NaH2PO4, 100 mM NaCl and 1 mM EDTA refolding buffer at pH 6.5 and then into a column running buffer (20 mM Bis–Tris and 1 mM EDTA, pH 6.5). Purification of ESAT-6 was carried out using a 20-ml Q-Sepharose column with a stepwise gradient of increasing NaCl concentration. The ESAT-6, which was eluted at 150 mM NaCl, was dialyzed against a 25 mM NaH2PO4, 150 mM NaCl buffer at pH 6.5 and passed through an Amersham HiLoad 16/60 Superdex 79-pg gel filtration column. The ESAT-6 recovered was judged to be >95% pure by SDS-PAGE (Invitrogen 4–12% Bis–Tris NuPAGE gel system) and electrospray mass spectrometry. A typical yield of purified ESAT-6 was ~35 mg l–1.

Expression and purification of CFP-10.
Escherichia coli cells transformed with the pET28a-based expression vector for CFP-10 were grown in Luria-Bertani medium containing 40 µg ml–1 kanamycin and were harvested 4 h after induction by isopropyl-1-thio-ß-D-galactopyranoside in mid-log phase. The cell pellets were lysed with Bugbuster HT (Novagen) to which was added EDTA to 0.5 mM and PMSF to 100 µM to inhibit protease activity. The soluble fraction containing CFP-10 was then dialyzed into a 20 mM Tris and 1 mM EDTA column running buffer at pH 8.0. Initial purification of CFP-10 was carried out on a 20-ml Q-Sepharose column pre-equilibrated with the pH 8.0 Tris buffer. The column was washed with a stepwise gradient of increasing NaCl concentration and CFP-10 eluted in the 75 mM NaCl wash. Fractions containing CFP-10 were pooled, dialyzed against a 20 mM piperazine, 1 mM EDTA buffer, pH 5.8, and applied to a pre-equilibrated 20-ml Q-Sepharose column. CFP-10 was eluted from this column in a 50 mM NaCl wash and was dialyzed against a 25 mM NaH2PO4, 150 mM NaCl buffer, pH 6.5, and passed through an Amersham HiLoad 16/60 Superdex 79-pg gel filtration column. The CFP-10 recovered was judged to be >95% pure by SDS-PAGE and electrospray mass spectrometry, as described above for ESAT-6. A typical yield of purified CFP-10 was ~15 mg l–1.

Generation of 1:1 complex of ESAT-6–CFP-10.
The 1:1 ESAT-6–CFP-10 complex was produced by mixing equimolar solutions of CFP-10 and ESAT-6 in 25 mM NaH2PO4, 100 mM NaCl and 0.02% (w/v) NaN3, pH 6.5, at room temperature. The 1:1 complex structure was confirmed as detailed previously (7).

Cytokine flow cytometry assay
A cytokine flow cytometry (CFC) assay was used to detect intracellular IFN-{gamma} and tumor necrosis factor (TNF) in CD3+, CD4+ and CD8+ T lymphocytes. A total of 1 ml of whole blood was stimulated with antigen at a final concentration of 5 µg ml–1 for ESAT-6 and CFP-10 individually and with 10 µg ml–1 for the ESAT-6–CFP-10 complex. For each sample, a positive control blood sample was stimulated with phorbol myristate acetate (catalog no. P-1585, Sigma, Munich, Germany) (20 ng ml–1 final concentration) and ionomycin (catalog no. I-0634, Sigma) (1 µM final concentration). The samples were incubated at 37°C in a humidified atmosphere of 5% CO2 for 6 and 18 h.

Brefeldin A (10 µg ml–1 final, B-7651, Sigma), a fungal metabolite that interferes with vesicular transport from the rough endoplasmic reticulum to the Golgi complex, was added at 2 h into the 6-h incubation or at 6 h into the 18-h incubation. After the incubation, 14 ml of lysis solution (4.15 g NH3Cl, 0.5 g KHCO3 and 26 µl 0.5 M EDTA in 500 ml) was added to lyse the erythrocytes and the mixture incubated for 5 min at room temperature. The cells were centrifuged for 5 min at 400 x g and washed in 1 ml PBS/2% FCS. The pellets were then re-suspended in 100 µl PBS/2% FCS.

Lymphocytes were defined by cell-surface staining for CD3 (FITC-conjugate antibody; no. 92-0001; BD Biosciences PharMingen), CD4 (TRI-color conjugate antibody; no. MHCD0406; Caltag Laboratories, Burlingame, CA, USA) and CD8 (TRI-color conjugate antibody; no. MHCD0806; Caltag Laboratories). Monocytes were identified using a CD14 PC5 antibody (no. IM 2640; Beckman Coulter). Antibodies were added to cell suspensions and incubated for 20 min on ice. The cells were washed in 1 ml PBS/2% FCS and centrifuged for 5 min at 400 x g. The pellets were re-suspended and fixed overnight in 500 µl of 0.5% formaldehyde in PBS. After the overnight incubation, the cells were washed twice in 1 ml PBS/2% FCS and centrifuged for 5 min at 400 x g. The pellets were re-suspended and washed twice in 1 x perm wash buffer (no. 554723; BD Biosciences PharMingen). All the wash steps were done at 4°C. The pellets were re-suspended in 100 µl of 1 x perm wash and were stained with anti-TNF–allophycocyanin (catalog no. 554514, Beckman Coulter) and anti-IFN-{gamma}–PE (catalog no. IM 2717, BD Biosciences PharMingen) at 10 ng µl–1 final concentration or with the respective isotype controls. The pellets were incubated 30 min on ice and washed twice with PBS/2% FCS. The cells were re-suspended in 400 µl of PBS for acquisition and analysis by flow cytometry.

Flow cytometric analysis was performed with a FACSCalibur flow cytometer (BD Biosciences) and Cell Quest software. At least 250 000 T cell events were acquired for each sample. This was done by first gating on lymphocytes defined by the light scatter signals and followed by gating on CD14-negative cells in order to completely exclude monocytes that may generate background signals. Then we gated on the T cells (CD3, CD4 or CD8) to generate the TNF/IFN-{gamma} histograms.

The percentages of CD3+, CD4+ and CD8+ cells which are INF-{gamma}+, TNF+ or IFN-{gamma}+/TNF+ were calculated by setting quadrant markers for the bivariate dot plot. Background signals for unstimulated cells were subtracted from signals for ESAT-6 and CFP-10 proteins to give specific T cell responses. A percentage of 0.02% reflecting at least 20 events when analyzing 100 000 T cells was considered positive. Student's t-test (paired samples) was used for statistical analysis.

Digestion of ESAT-6 and CFP-10 with cathepsin L and S
Samples of purified CFP-10 and ESAT-6 were dialyzed overnight against either 20 mM CH3COONa, 100 mM NaCl, 2 mM EDTA, 2 mM dithiothreitol (DTT), pH 5.5 (reaction buffer A), or 25 mM NA2HPO4, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, pH 6.5 (reaction buffer B). Individual samples of CFP-10 and ESAT-6 were prepared to give 50 µg protein in 100-µl final volumes of reaction buffers A and B. The ESAT-6–CFP-10 complex was formed by mixing equal quantities of the individual proteins which were incubated at room temperature for 30 min. Spectroscopic analysis was used to determine complex formation as described previously (7). The complex was then added to the above reaction buffers to give 50 µg complex in 100-µl final volumes. Human, recombinant cathepsin L [15 U mg–1, Calbiochem, via Merck Biosciences Ltd (Nottingham)] was added to samples in reaction buffer A at ratios of 200:1, 2000:1 and 20 000:1 (protein to enzyme by mass) and were incubated at 30°C with 20-µl samples taken at time 0, 15 and 30 min and activity halted. Similarly, human recombinant cathepsin S (30 U mg–1, Calbiochem) was added to samples in reaction buffer B at ratios of 200:1, 2000:1 and 20 000:1 (protein to enzyme by mass) and were incubated at 37°C with 20-µl samples taken at time 0, 15 and 30 min and activity halted. Time point samples were run on 4–12% acrylamide gradient, pre-cast NuPAGE® Bis–Tris gels (Invitrogen) and gels were stained for 20–30 min with Coomassie Brilliant Blue [2.5 g l–1 in 45% (v/v) methanol and 10% (v/v) acetic acid] and then destained in a 10% (v/v) methanol and 5% (v/v) acetic acid solution.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Since both TNF and IFN-{gamma} are important cytokines in the control of M. tuberculosis, we studied the expression of these mediators in blood T lymphocytes. We initially looked at T cells activated for 6 h after polyclonal activation with a combination of 12-O-tetradecanoylphorbol 13-acetate (TPA) and ionomycin in the presence of the protein transport blocker Brefeldin A.

When looking at CD3+ cells in a two-color plot for the two cytokines, we can detect a major population that expresses TNF only, a population that expresses both cytokines and a small fraction producing IFN-{gamma} only (Fig. 1A). The signals detected are specific as shown by the isotype controls for IFN-{gamma} (Fig. 1B) and for TNF (Fig. 1C). In an average of five donors CD3+ cells after this type of activation responded by producing either TNF only (12.9 ± 1.7%), TNF and IFN-{gamma} (12.0 ± 7.5%) or IFN-{gamma} only (5.0 ± 2.5%) (Table 1).



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Fig. 1. Effect of polyclonal stimulation by TPA plus ionomycin on cytokine expression by T cells. Whole blood from a healthy control donor was stimulated with TPA/ionomycin for 6 h in the presence of Brefeldin A. Cells were then cell-surface stained for CD3, CD4 or CD8 and intracellular cytokine was determined with anti-TNF and anti-IFN-{gamma} antibodies. (A) CD3 cells and anti-IFN-{gamma}/anti-TNF. (B) CD3 cells and isotype for the anti-IFN-{gamma}/anti-TNF antibodies. (C) CD3 cells and anti-IFN-{gamma} and isotype for the anti-TNF antibody. (D) CD4 cells and anti-IFN-{gamma}/anti-TNF. (E) CD8 cells and anti-IFN-{gamma}/anti-TNF. The values within or next to the quadrants give the percentage of reactive cells. One of three to five experiments as summarized in Table 1.

 

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Table 1. Cytokine expression by T cells after stimulation with TPA/ionomycina

 
We then asked whether the cytokines could be assigned to different T cell sub-populations. When looking at CD8+ T cells, we observed an exclusive production of IFN-{gamma} after polyclonal activation by TPA/ionomycin (Fig. 1E). In contrast, CD4+ T cells predominantly expressed TNF only and some TNF plus IFN-{gamma} (Fig. 1D). This pattern was consistent in three donors as summarized in Table 1. The results demonstrate a differential expression of the two cytokines in CD4+ helper cells and CD8+ cells after direct ex vivo stimulation of whole blood.

We then studied the response of T cells to the M. tuberculosis protein ESAT-6 in whole-blood cultures. For this, heparinized blood samples were incubated with the ESAT-6 protein at 5 µg ml–1 for a total of 6 h. In order to allow for antigen processing and presentation on the cell surface of antigen-presenting cell (APC), the protein transport blocker Brefeldin A was only added for the final 4 h. As shown in Fig. 2(A), an ESAT-6-specific response can be readily induced in CD3+ T cells. Similar to the response to TPA/ionomycin, ESAT-6 also induces T cells that produced TNF only, IFN-{gamma} only or both cytokines. When looking at CD4+ and CD8+ sub-populations, we could demonstrate a clear production of IFN-{gamma} in both subsets (Fig. 2B and C). TNF production was, however, low in CD8 cells (Fig. 2C), while CD4 cells clearly produce TNF. The average values are given in Fig. 3.



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Fig. 2. Cytokine response of T cells to ESAT-6. Whole blood was stimulated for 6 h with ESAT-6 with addition of Brefeldin A for the final 4 h. Cells were then cell-surface stained for CD3, CD4 or CD8 and intracellular cytokine was determined with anti-TNF and anti-IFN-{gamma} antibodies. (A) CD3 cells and anti-IFN-{gamma}/anti-TNF. (B) CD4 cells and anti-IFN-{gamma}/anti-TNF. (C) CD8 cells and anti-IFN-{gamma}/anti-TNF. The values within the quadrants give the percentage of reactive cells. One representative experiment with cells from control donor C14. Results from three to five experiments are summarized in Fig. 3.

 


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Fig. 3. Cytokine response of T cells to ESAT-6. Whole blood was stimulated and processed as given in Fig. 2. Given is the average ± SD for n = 5 for (A) (donors C10, C11, C12, C14 and P12), n = 3 for (B) (donors C10, C11 and C12) and n = 3 for (C) (donors C10, C11 and C12). These data summarize experiments exemplified in Fig. 2.

 
These data confirm that CD4 cells are the main producers of TNF. Also, T cells producing both TNF and IFN-{gamma} were rare after ESAT-6 stimulation (8% of all reactive cells, see Fig. 3) compared with the polyclonal activation (40% of all reactive cells, see Table 1). This suggests that T cells responsive to ESAT-6 have a biased cytokine pattern and tend to produce either TNF or IFN-{gamma} but not both.

Responses to ESAT-6 with at least 0.02% IFN-{gamma}-positive cells were seen in 8 of 10 patients with tuberculosis and in 12 of 14 donors that had been selected for likely exposure to and infection by M. tuberculosis. Among the responders to ESAT-6, we did not detect a dependence on PPD status. ESAT-6+PPD+ control donors showed an average of 0.15 ± 0.15% IFN-{gamma}-positive CD3+ cells and for the ESAT-6+PPD– control donors the respective value 0.32 ± 0.18%. Also, among ESAT-6+PPD+ patients showed an average of 0.18 ± 0.14% IFN-{gamma}-positive CD3+ cells and for the ESAT-6+PPD– patients the respective value was 0.17 ± 0.21%.

Mycobacterium tuberculosis co-expresses ESAT-6 and CFP-10, both of which are encoded by the RD1 in the mycobacterial genome. Since these two proteins form a stable 1:1 complex, we next studied the T cell IFN-{gamma} production induced by this complex as compared with the two antigens alone. As shown in Fig. 4(A), the response to CFP-10 after 6 h stimulation was clearly lower compared with ESAT-6. Unexpectedly, the complex of the two antigens was not the sum of the two responses but was as low as CFP-10. These findings would suggest that presentation of the complexed antigens is less efficient compared with the individual antigens, possibly because of a higher resistance of the complex to digestion within the APCs.



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Fig. 4. Effect of stimulation by ESAT-6, CFP-10 and ESAT-6–CFP-10 complex on T cell responses. (A) Whole blood was stimulated for a total of 6 h with ESAT-6 (E6) at 5 µg ml–1, CFP-10 (CFP) at 5 µg ml–1 or ESAT-6–CFP-10 complex at 10 µg ml–1. Brefeldin A was added for the final 4 h. Cells were then cell-surface stained for CD3 followed by intracellular staining for IFN-{gamma}. The response is expressed as percentage of the result obtained with ESAT-6 alone set as 100%. The average response for ESAT-6 alone was 0.8 ± 1.1% (n = 5, donors C1, C2, P1, P3 and P6). *P < 0.001 compared with ESAT-6 alone. (B) Whole blood was stimulated for a total of 18 h with Brefeldin A added after the first 6 h. The response is expressed as percentage of the result obtained with ESAT-6 alone set as 100%. The average response for ESAT-6 alone was 0.29 ± 0.15% (n = 3, donors C11, C13 and C14). (C) Whole blood was stimulated as under (A) and cells were then stained for intracellular TNF. The average response for ESAT-6 alone was 0.09 ± 0.05% (n = 4, donors C10, C11, C12 and C14).

 
We therefore extended the time allowed for processing to 6 h before Brefeldin A was added, followed by overnight culture. With this approach, the fraction of T cells responding to CFP-10 alone reached the level of ESAT-6 alone (Fig. 4B). However, the complex of the two antigens failed to give a response equal to the sum of the responses from the two individual antigens. This suggests a persistent resistance to antigen processing of the ESAT-6–CFP-10 complex.

Also, when TNF production, which by and large reflects activity of CD4 T cells, was analyzed, we noted the same pattern of responses, in that T cell activation by the complex only reached the level of CFP-10 alone (Fig. 4C).

The inferior T cell response to the ESAT-6–CFP-10 complex compared with the individual components may be due to a higher resistance to digestion during antigen processing. We therefore tested the complex and the individual components in in vitro digestion experiments using cathepsin L and S. As shown in Fig. 5, cathepsin L completely digested CFP-10 at a 1:2000 dilution after 30 min (upper left panel, lane 7). Under the same conditions ESAT-6 alone was almost completely digested (middle left panel, lane 7), while the ESAT-6–CFP-10 complex was resistant (lower left panel, lane 7). When studying digestion by cathepsin S, a similar pattern was observed (see lane 6 in upper, middle and lower right-hand panels).



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Fig. 5. Digestion of ESAT-6 and CFP-10 and ESAT-6–CFP-10 by lysosomal enzymes. Purified proteins were subjected to digestion at dilutions 1:200, 1:2000 and 1:20 000 relative to the Mycobacterium tuberculosis proteins and samples were separated by gel electrophoresis after 0, 15 and 30 min. Lane 1 gives molecular weight markers with 17.0, 14.2, 6.5 and 3.5 kDa from top to bottom. One of three similar experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the present report we have analyzed T cell responses to RD1 proteins ESAT-6 and CFP-10 individually and as a 1:1 complex. For this we have used the CFC assay that was shown to have some advantages such as being very rapid, requiring no processing of cells before stimulation and giving higher numbers of responding T cells than ELISPOT (8). Also, CFC has the advantage of allowing the determination of the phenotype of responding cells.

Initially, we have looked at polyclonal stimulation with TPA/ionomycin and we could show that among T cells there were cells that produce either TNF or IFN-{gamma} and there are T cells producing both cytokines.

We then studied responses to RD1-encoded proteins ESAT-6 and CFP-10. We have used incubation of whole blood with recombinant protein and added a protein export blocker only after 2 h in order to allow for transport of the MHC peptide complexes to the cell surface. T cells and their expression of TNF and/or IFN-{gamma} were then determined using intracellular staining after at least another 4 h.

Using this assay we considered 0.02% IFN-{gamma}-positive cells to indicate a positive response. This cut-off point was used since it is based on at least 20 positive events when analyzing 100 000 T cells in the flow cytometer. It may well be that the other patients do have specific T cells with numbers being below the sensitivity of our assay. When using such an assay for the purpose of diagnosis, it may be necessary to acquire more events by FACS. On the other hand, the ESAT-6 ELISPOT, which easily scores the response in >1 000 000 cells, could be considered for diagnostics.

The response rate in our control group was higher than expected for a European population but we have selected donors based on likely exposure to M. tuberculosis, be they medical personnel or colleagues coming from countries with high prevalence of M. tuberculosis. The response rate in our patient cohort was in the range of what has been reported previously. Ravn et al. (9) have shown that ~70% of patients with minimal disease responded to ESAT-6 with IFN-{gamma} release in a 5-day culture assay and the response rate decreased to ~25% in those with extensive disease. Also, Pathan et al. (10) noted that patients with active pulmonary tuberculosis had clearly lower levels of IFN-{gamma}-producing T cells in response to ESAT-6 in the ELISPOT assay. This may indicate that low numbers of responder T cells may favor disease or, more likely, that these cells were sequestered at the sites of active disease. Also, it is possible that infection with M. tuberculosis induces anergy of T cells leading to failure of IFN-{gamma} production (11).

The CFC assay has been used previously to analyze the pattern of cytokines expressed by individual T cells. Waldrop et al. (12) could show that the response to cytomegalovirus antigen was dominated by CD4 T cells, which produced both IFN-{gamma} and TNF. In our studies we also noted some T cells co-expressing the two cytokines, but for most T cells, expression of these two cytokines was mutually exclusive (Figs 1–3GoGo, Table 1). The reasons for this different response pattern between the studies of Waldrop et al. and the present report are unclear but it is likely that the nature of the antigen and the APCs involved determine which type of T cell expresses which type of cytokine. On the other hand, infection with M. tuberculosis may affect the cytokine pattern expressed by leukocytes (13).

The nature of the peptides recognized by the ESAT-6 responsive T cells has been demonstrated to be dominated by the N-terminal sequences (14) but obviously the process of antigen digestion in the lysosome and selection by the MHC class II alleles will determine what is presented to CD4 T cells when a complete protein or a protein complex is employed. Also, the types of peptides entered into the MHC class I pathway during cross-presentation to CD8 cells will be determined by the proteasome and the transporter of antigenic peptides within the APCs.

Both ESAT-6 and CFP-10 have been used in in vitro tests for diagnosis of infection with M. tuberculosis and combining the data from assays with peptides from the two antigens was shown to increase diagnostic sensitivity (15). We have combined the antigens as a complex and added them together to the assay. We have reported earlier that ESAT-6 and CFP-10 form a stable 1:1 complex when combined in solution or co-expressed in E. coli (7, 16) and it can be expected that the same will happen when the two are expressed from the RD1 of the M. tuberculosis genome (6, 17). Being two different proteins with unique protein sequences and no matching nonapeptides between them, there will be different T cell clones responding to the peptides derived from the two antigens and the T cell responses can be expected to be additive. We noted, however, for both patients and controls, that stimulation with the ESAT-6–CFP-10 complex did not lead to increased numbers of responding T cells (Fig. 4A). Specifically, in the standard 6-h assay, the response to the ESAT-6–CFP-10 complex was as low as the response to CFP-10 alone, and in the overnight assay, that allows for a longer period of time for antigen processing, the response to the complex still did not exceed the response to CFP-10 alone. Our finding that the response to the ESAT-6–CFP-10 complex is lower or similar to the response to ESAT-6 alone indicates that there must be a mechanism which prevents a maximum activation of the T cell response to the two components.

The results of combined stimulation of blood mononuclear cells for 5 days with ESAT-6 and CFP-10 by van Pinxteren et al. (18) did show an additive effect of the two antigens. This discrepancy to our findings could be due to the two proteins not being complexed in those studies. Alternatively, the longer period of culture may allow for complete processing and presentation of the complexed proteins. It will be important to analyze proliferative responses to our ESAT-6–CFP-10 complex in order to demonstrate that the relative inefficiency in T cell stimulation of the complex as compared with the individual proteins is also relevant at this level.

The hypothesis that the impaired T cell response to ESAT-6–CFP-10 in our studies is due to impaired processing is supported by our in vitro digestion experiments. Here ESAT-6–CFP-10 resisted digestion with cathepsin L and S under conditions where the individual proteins were readily digested.

These studies on digestion with lysosomal enzymes are relevant to presentation to CD4 cells via the MHC class II pathway. We assume that similar rules may apply to cross-presentation to CD8 via the MHC class I pathway. Here, processing by the proteasome may be impaired for the ESAT-6–CFP-10 complex as compared with the individual components because of the higher stability of the complex.

Whatever the explanation, these findings show that the combination of ESAT-6 and CFP-10 provides an inferior stimulus for T cells. This may have to be taken into consideration when co-expressing the two for generation of a superior vaccine (6).


    Acknowledgements
 
This work was supported by a grant from MediSearch, Leicester (to L.Z.-H., UK) and the Wellcome Trust (066047 to M.C.). We thank B. W. Kiernan for excellent assistance with the preparation of the manuscript.


    Abbreviations
 
APC   antigen-presenting cell
BCG   Bacille-Calmette-Guerin
CFC   cytokine flow cytometry
CFP-10   culture filtrate protein-10
DTT   dithiothreitol
ELISPOT   enzyme-linked immunospot
ESAT-6   early secretory antigenic target-6
PMSF   phenylmethylsulfonylfluoride
PPD   purified protein derivative
RD1   region of difference 1
TNF   tumor necrosis factor
TPA   12-O-tetradecanoylphorbol 13-acetate

    Notes
 
Transmitting editor: S. Kaufmann

Received 12 January 2005, accepted 22 August 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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