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
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Abstract |
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Keywords: antigen presentation, T cells, tuberculosis
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Introduction |
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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-6CFP-10 complex is inferior to the individual components in triggering human T cells.
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Methods |
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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 ml1 and streptomycin 100 µg ml1 (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 ml1 ampicillin. The expression of ESAT-6 was induced in mid-log phase (corresponding to an optical density change at 600 nm of 0.60.7) 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 0.51.0 mg ml1. 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 BisTris 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 412% BisTris NuPAGE gel system) and electrospray mass spectrometry. A typical yield of purified ESAT-6 was 35 mg l1.
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 ml1 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 l1.
Generation of 1:1 complex of ESAT-6CFP-10.
The 1:1 ESAT-6CFP-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- 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 ml1 for ESAT-6 and CFP-10 individually and with 10 µg ml1 for the ESAT-6CFP-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 ml1 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 ml1 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-TNFallophycocyanin (catalog no. 554514, Beckman Coulter) and anti-IFN-PE (catalog no. IM 2717, BD Biosciences PharMingen) at 10 ng µl1 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- histograms.
The percentages of CD3+, CD4+ and CD8+ cells which are INF-+, TNF+ or IFN-
+/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-6CFP-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 mg1, 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 mg1, 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 412% acrylamide gradient, pre-cast NuPAGE® BisTris gels (Invitrogen) and gels were stained for 2030 min with Coomassie Brilliant Blue [2.5 g l1 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.
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Results |
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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- only (Fig. 1A). The signals detected are specific as shown by the isotype controls for IFN-
(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-
(12.0 ± 7.5%) or IFN-
only (5.0 ± 2.5%) (Table 1).
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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 ml1 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- only or both cytokines. When looking at CD4+ and CD8+ sub-populations, we could demonstrate a clear production of IFN-
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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Responses to ESAT-6 with at least 0.02% IFN--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-
-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-
-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- 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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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-6CFP-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-6CFP-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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Discussion |
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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- 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- were then determined using intracellular staining after at least another 4 h.
Using this assay we considered 0.02% IFN--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-
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-
-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-
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- 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 13
, 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-6CFP-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-6CFP-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-6CFP-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-6CFP-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-6CFP-10 in our studies is due to impaired processing is supported by our in vitro digestion experiments. Here ESAT-6CFP-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-6CFP-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).
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Acknowledgements |
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Abbreviations |
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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 |
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Notes |
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Received 12 January 2005, accepted 22 August 2005.
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References |
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