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A. S. Mustafa, F. Oftung, H. A. Amoudy, N. M. Madi, A. T. Abal, F. Shaban, I. Rosen Krands, P. Andersen, Multiple Epitopes from the Mycobacterium tuberculosis ESAT-6 Antigen Are Recognized by Antigen-Specific Human T Cell Lines, Clinical Infectious Diseases, Volume 30, Issue Supplement_3, June 2000, Pages S201–S205, https://doi.org/10.1086/313862
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Abstract
A synthetic-peptide approach was used to map epitope regions of the Mycobacterium tuberculosis 6-kDa early secreted antigen target (ESAT-6) by testing human CD4+ T cell lines for secretion of IFN-γ in response to recombinant ESAT-6 (rESAT-6) and overlapping 20-mer peptides covering the antigen sequence. The results demonstrate that all of the ESAT-6 peptides screened were able to induce IFN-γ secretion from one or more of the T cell lines tested. Some of the individual T cell lines showed the capacity to respond to all peptides. Human leukocyte antigen (HLA-DR) typing of the donors showed that rESAT-6 was presented to T cells in association with multiple HLA-DR molecules. The results suggest that frequent recognition of the M. tuberculosis ESAT-6 antigen by T cells from patients with tuberculosis is due to the presence of multiple epitopes scattered throughout the ESAT-6 sequence.
Tuberculosis is ranked among the most important infectious diseases of worldwide prevalence. Since protection against tuberculosis is via cell-mediated immunity (CMI), a primary criterion for selecting candidate antigens for subunit vaccine design is their ability to induce protective T cell responses. Mycobacterium tuberculosis is rich in antigens that induce CMI, and the presence of such antigens in purified cell walls, in the cytosolic compartment, and culture filtrates (CFs) has been reported [1, 2]. However, recent studies suggest that antigens present in CFs are among the primary inducers of CMI that mediate protection against tuberculosis in mice and guinea pigs [3, 4].
The use of memory-immune mice has led to findings that CF-derived antigens with a molecular mass of 6–10 kDa, including the M. tuberculosis 6-kDa early secreted antigen target (ESAT-6), are strongly recognized by IFN-γ-secreting CD4+ T cells early during infection with M. tuberculosis [5]. By screening unselected peripheral blood mononuclear cells (PBMC) from tuberculosis patients for proliferative reactivity and IFN-γ secretion against recombinant ESAT-6 (rESAT-6), we demonstrated that this antigen is frequently recognized by human T cells after natural infection with M. tuberculosis [6].
In this work, we have mapped T cell epitopes of ESAT-6 by establishing and testing rESAT-6-reactive CD4+ T cell lines from tuberculosis patients for secretion of IFN-γ in response to 8 overlapping synthetic ESAT-6 peptides. The results showed that the M. tuberculosis ESAT-6 contains multiple T cell epitopes relevant to natural processing pathways. These epitopes are scattered throughout the entire sequence of ESAT-6.
Materials and Methods
Antigens and synthetic peptides. The short-term CF (ST-CF) antigen fraction was produced as described elsewhere [5]. In brief, M. tuberculosis were grown in modified Sauton's medium without Tween-80 on an orbital shaker for 7 days. The culture supernatants were sterile-filtered and were concentrated on an Amicon YM3 membrane (Amicon, Danvers, MA). rESAT-6 was expressed as a maltose-binding fusion protein in Escherichia coli, purified by preparative SDS-PAGE, and residual endotoxin was depleted by passage through a lipopolysaccharide-affinity column (Detoxi-Gel; Pierce, Oud-veijerland, the Netherlands) [7]. Recombinant maltose-binding protein (MBP; supplied by Dr. G. Singh through the World Health Organization Committee on Immunology of Leprosy) was produced in E. coli and used as control antigen. Eight overlapping 20-mer peptides (figure 1) covering the sequence of the ESAT-6 antigen were synthesized as described elsewhere [7].
Isolation of PBMC and genomic HLA-DR typing. Heparinized blood was collected from patients with culture-confirmed pulmonary tuberculosis at the Chest Diseases Hospital, Kuwait. PBMC were separated from the blood by density centrifugation on Lymphoprep gradients (Nycomed, Oslo) [8]. Genomic human leukocyte antigen (HLA-DR) typing of the donors was performed with use of DNA purified from PBMC and sequence-specific primers in PCR [9]. An HLA-DR “low-resolution” kit, containing the primers to type for DRB1, DRB3, DRB4, and DRB5 alleles, was purchased from Dynal AS (Oslo, Norway) and used according to the manufacturer's instructions.
Antigen-induced proliferation and IFN-γ secretion from PBMC. Antigen-induced proliferation and IFN-γ secretion from PBMC were assayed according to procedures described elsewhere [10–12]. In brief, PBMC (2 × 105 cells) suspended in 50 µL of complete tissue-culture medium (RPMI 1640 + 10% human AB serum + penicillin [100 U/mL] + streptomycin [100 µg/mL] + gentamycin [40 µg/mL] + fungizone [2.5 µg/mL]) were seeded into the wells of 96-well tissue culture plates (Nunc, Roskilde, Denmark). Antigens in 50 µL of complete medium were added at optimal concentrations (5 µg/mL) to the wells in duplicates or triplicates [6, 7]. The final volume of the culture wells was adjusted to 200 µL.
The plates were incubated at 37°C in an atmosphere of 5% CO2 and 95% air. On day 6, 100 µL of supernatant from each well was aspirated and used for IFN-γ assay, as described below for T cell lines. The cultures were then pulsed with 1 µci of 3H-thymidine (Amersham Life Science, Amersham, UK) and were harvested on filter mats with a Skatron harvester (Skatron Instruments AS, Oslo, Norway), and the radioactivity incorporated was measured by liquid scintillation counting [13].
The radioactivity incorporated was determined as counts per minute (cpm). Average cpm values were calculated for the cultures stimulated with each antigen. The results were calculated as stimulation indices (SIs; [cpm in antigen-stimulated cultures]/[cpm in cultures lacking antigen]). An SI > 5 was considered to be a positive proliferative response [6].
Antigen-specific T cell lines. Antigen-specific T cell lines were established by stimulating 2 × 105 PBMC/well with rESAT-6 (5 µg/mL) in the wells of 96-well plates and were incubated at 37°C in an atmosphere of 5% CO2 and 95% air. After 6 days of incubation, human recombinant IL-2 (Amersham Life Sciences) was added to a final concentration of 100 U/mL twice a week, and the growing T cell lines were propagated in 24-well Costar plates (Costar, Cambridge, MA) [14, 15].
Antigen- and peptide-induced secretion of IFN-γ from T cell lines. The T cell lines (5 × 104 cells/well) were tested for antigen- and peptide (5 µg/mL)-induced IFN-γ secretion in the presence of irradiated autologous PBMC as antigen-presenting cells (1 × 105 cells/well) in 96-well plates. The plates were incubated at 37°C in an atmosphere of 5% CO2 and 95% air. On day 3, the culture supernatants (100 µL) from each well were collected, and the amount of IFN-γ released was quantitated with use of PREDICTA immunoassay kits (Genzyme, Cambridge, MA), according to the manufacturer's instructions. Secretion of IFN-γ was considered to be a positive response to the antigens and peptides tested when the concentration of ΔIFN-γ (IFN-γ in antigen- or peptide-stimulated cultures minus IFN-γ in control cultures without antigen or peptide) exceeded 500 pg/mL [6].
Results and Discussion
Our previous studies have shown that rESAT-6 is among the major antigens of M. tuberculosis recognized by PBMC- and CF-induced T cell lines from patients with tuberculosis in proliferation and IFN-γ assays [6]. The primary aim of this study was to map the T cell epitopes of ESAT-6 relevant to naturally processing pathways by first establishing ESAT-6-reactive T cell lines and then testing them with synthetic peptides covering the sequence of ESAT-6.
To identify donors for establishing ESAT-6-reactive T cell lines, PBMC from 48 tuberculosis patients were tested for antigen-induced proliferation in response to the ST-CF antigen fraction. Thirty patients responded to ST-CF antigens in the proliferation assays (mean, 20,900 cpm), and 29 of these patients also secreted IFN-γ (mean, 9700 pg/mL) in response to this antigen fraction (table 1). When tested for rESAT-6 reactivity, 23 (77%) and 27 (90%) of the 30 donors responded to rESAT-6 in proliferation assays (mean, 6600 cpm) and IFN-γ assays (mean, 4600 pg/mL), respectively (table 1).
Since rESAT-6 was expressed as a fusion protein with MBP, in order to determine the contribution of MBP in rESAT-6 responses we also tested PBMC from the same donors against MBP as a control antigen. The results showed that PBMC from only 5 (17%) and 3 (10%) of the 30 donors tested in proliferation assays (mean, 900 cpm) and IFN-γ assays (mean, 130 pg/mL), respectively, responded to MBP (table 1). These observations confirmed that MBP as a fusion protein partner did not significantly contribute to the positive responses obtained with rESAT-6.
To identify T cell epitopes of the ESAT-6 antigen relevant to natural processing pathways, we established human CD4+ T cell lines from PBMCs of 20 antigen-responding patients by using rESAT-6 as the primary stimulating antigen in vitro. In addition, HLA-DR typing of these donors showed that they covered a large spectrum of HLA-DR molecules encoded by HLA-DRB1, -DRB3, -DRB4, and -DRB5 genes, including HLA-DR1, -DR2, -DR3, -DR4, -DR5, -DR6, -DR7, -DR8, DR9, -DR51, -DR52, and -DR53. When tested for antigen-induced IFN-γ secretion, all of the 20 T cell lines secreted IFN-γ in response to rESAT-6 (figure 2). Only 2 T cell lines responded to MBP (data not shown), confirming the PBMC results showing that the T cell responses to rESAT-6 were primarily due to ESAT-6 sequences. Since HLA-DR molecules are the main presenters of mycobacterial antigens to T cells [16–20], the recognition of ESAT-6 by T cell lines from HLA-DR-heterogeneous donors suggests that this antigen can be presented to T cells in association with several HLA-DR molecules.
To map the T cell epitopes of ESAT-6, the same rESAT-6-reactive T cell lines were subjected to cytokine analysis with respect to secretion of IFN-γ in response to the synthetic peptides of ESAT-6 (figure 1). Seventeen (85%) of the 20 rESAT-6 T cell lines responded to ≥1 of the ESAT-6 peptides. Three T cell lines secreted IFN-γ in response to all the ESAT-6 peptides tested. These responses were not due to nonspecific stimulation of the T cell lines, because none of these T cell lines responded to MBP in control experiments (data not shown).
The frequency of T cell lines responding (percentage of positive response) to individual peptides ranged between 30% and 65% (figure 2). However, the c-terminal peptide P8 (aa 72–95) induced the strongest IFN-γ responses (mean, 4100 pg/mL), and it was the most frequently presented and recognized peptide by antigen-specific T cells, with 65% of the donors responding to this peptide (figure 2). Again, all of the ESAT-6 peptides were recognized by T cell lines from a HLA-DR-heterogeneous group of individuals, a finding suggesting that each peptide was presented to T cells by multiple HLA-DR molecules.
Our findings from Kuwait are consistent with previous studies showing frequent recognition of ESAT-6 by PBMC of tuberculosis patients in 3 different geographic locations (i.e., Germany [21], Denmark, and Ethiopia [22]) and the presence of multiple T cell epitopes scattered throughout the protein sequence [22]. However, with respect to the predominant recognition of ESAT-6 peptides, our results, although similar to Danish findings of predominant recognition of the C-terminal peptide (aa 72–95) [22], are at variance with reports from Ethiopia [22] and Germany [21] of the predominant recognition of the central region (aa 42–75) and N-terminal (aa 1–30) peptides of ESAT-6, respectively.
The differences observed between different ethnic groups with respect to predominant recognition of ESAT-6 peptides suggest a genetic influence of peptide recognition in terms of major histocompatibility complex restriction. This suggestion is supported by the findings of Brandt et al. [7], whose studies in mice showed exclusive recognition of N-terminal (aa 1–20) and central region (aa 51–70) peptides of ESAT-6 by T cells in the context of H-2b,d and H-2a,k, respectively.
In conclusion, our results in humans show that both PBMC and T cell lines obtained from an HLA-DR-heterogeneous group of donors responded to rESAT-6 by secretion of IFN-γ, the cytokine important for protective immunity against mycobacterial infections. In addition, the results demonstrate that the M. tuberculosis ESAT-6 antigen contains multiple naturally derived T cell epitopes that are present throughout the sequence of the ESAT-6 protein and are recognized by IFN-γ-secreting T cell lines from donors expressing a spectrum of HLA-DR phenotypes.
Recent studies with the mouse model of tuberculosis have shown that immunization with ESAT-6 protects the animals against challenge with viable M. tuberculosis [23, 24]. ESAT-6 therefore may be considered for inclusion as a subunit vaccine, either by itself or in combination with other relevant antigens of M. tuberculosis inducing protective immunity. However, since not all subjects infected with M. tuberculosis respond to single antigens, including ESAT-6 (table 1 and [6, 21, 22]), a combination of several promising antigens may constitute a better candidate vaccine. This view is strengthened by a recent report showing the greater protective efficacy of DNA vaccines expressing multiple antigens (i.e. ESAT-6, MPT-64, and Ag-85B) in comparison with the protection afforded by immunization of mice with DNA vaccines expressing any single antigen [24].
References
Grant support: Kuwait University Research Administration (MI114) and the European Union (grants for sciences and technologies for developing countries; projects TS3* CT94-0313 and ERBICC18CT970254).
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