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The Journal of Immunology, 2006, 176: 1090-1097.
Copyright © 2006 by The American Association of Immunologists

Aspartic Acid Homozygosity at Codon 57 of HLA-DQ {beta} Is Associated with Susceptibility to Pulmonary Tuberculosis in Cambodia1

Julio C. Delgado*,{dagger}, Andres Baena2,*, Sok Thim§ and Anne E. Goldfeld3,*,{ddagger}

* CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA 02115; {dagger} Department of Pathology and {ddagger} Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and § The Cambodian Health Committee, Phnom Penh, Cambodia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
After infection with Mycobacterium tuberculosis, clinical disease usually remains latent, contained by the host immune response. Although polymorphisms of HLA loci have been hypothesized to play a major role in the breakdown of latency, a functional link has not been established. Molecular-based HLA-typing methods were used to test the association of sets of HLA alleles encoding an aspartic acid at codon 57 of the HLA-DQ {beta}-chain (HLA-DQ {beta}57-Asp) with susceptibility to tuberculosis in a cohort of 436 pulmonary tuberculosis patients and 107 healthy controls from Cambodia. HLA class II null cells were transduced with HLA-DQ {beta}57-Asp or HLA-DQ {beta}57-Ala and evaluated for their ability to bind peptides from two immunogenic M. tuberculosis specific proteins, ESAT-6 and CFP-10. In this study, we report a highly significant association between progressive pulmonary tuberculosis and homozygosity for HLA-DQ {beta}57-Asp alleles. The presence of HLA-DQ {beta}57-Asp resulted in a significantly reduced ability to bind a peptide from the central region of the ESAT-6 protein. Furthermore, when this peptide was presented by an HLA-DQ {beta}57-Asp allele, Ag-specific IFN-{gamma} production from CD4+ T cells from tuberculosis patients was significantly less than when this peptide was presented by an HLA-DQ-{beta} allele encoding an alanine at codon 57. Multiple genetic loci and ethnic-specific factors are likely involved in the human immune response to tuberculosis. The data presented here provide a functional explanation for a highly significant association between an HLA polymorphism and tuberculosis in a highly characterized group of patients with susceptibility to progressive tuberculosis infection in Cambodia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human beings have evolved on a background of infection and it has long been speculated that genetic variability among populations that confers resistance against infectious pathogens is favored by evolution (1). Numerous case-control studies have identified associations between susceptibility or resistance to human Mycobacterium tuberculosis infection and clinical disease with polymorphisms of candidate genes (2, 3, 4, 5, 6, 7). However, these polymorphisms are of unknown functional impact upon tuberculosis pathogenesis, and several of these candidate gene variations are ethnic-specific markers of other linked and unidentified genetic factors that may impact host immune responses to tuberculosis susceptibility or resistance in a given population (8).

The highly polymorphic HLA class II loci encode molecules responsible for Ag presentation to CD4+ T cells which are critical in containment of M. tuberculosis infection (9, 10, 11, 12). Several alleles of the HLA class II DR2 serotype (13, 14, 15, 16), and the HLA-DQB1*0503 allele (17) are associated with susceptibility to progression to clinical tuberculosis after infection in diverse populations. However, despite these multiple association studies, a functional link between HLA associations, Ag-driven T cell responses, and tuberculosis disease has not been demonstrated.

Crystal structures of HLA class II molecules have shown that peptides bind to a groove in the HLA class II molecule, and that Ag-binding specificity is determined by pockets formed by polymorphic side chains (18, 19). HLA-DQ molecules, encoded by polymorphic HLA-DQ {alpha} and {beta}-chain genes, bind peptides with certain amino acids that are anchored at specific positions within peptide-binding pockets termed P1, P4, and P9 in the HLA-DQ molecular groove (20, 21). P9-binding specificity for example, is critically dependent upon the specific amino acid at codon 57 of the HLA-DQ {beta}-chain (HLA-DQ {beta}57), because this position influences both the charge of pocket 9 and peptide-binding spatial constraints created by the specific amino acid side chains at this codon. Specifically, the P9 pocket has different peptide-binding specificities dependent upon whether there is an aspartic acid (HLA-DQ {beta}57-Asp) or a nonaspartic acid at this position (22, 23) (Fig. 1).



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  		FIGURE 1. A model of the peptide-binding groove of an HLA-DQ molecule showing pocket P9 formed by the side chains of amino acids encoded by the HLA-DQ {alpha} and {beta} chains. Peptide-binding specificity of the P9 pocket depends on the interaction between the invariant amino acid arginine at position 76 of the HLA DQ {alpha}-chain and whether there is an aspartic acid (HLA-DQ {beta}57-Asp) or a nonaspartic acid (HLA-DQ {beta}57-non-Asp) at position 57 of the HLA DQ {beta}-chain. The figure illustrates that HLA-DQ {beta} alleles encoding an aspartic acid at position 57 (including HLA-DQB1*0503, shown in red), result in a P9 pocket that has a neutral charge because the negatively charged aspartic acid forms a salt bridge with the positively charged arginine at position 76 (shown in yellow). The HLA-DQ {beta} alleles encoding HLA-DQ {beta}57-non-Asp are also noted.

 
The presence of an alanine at this position (HLA-DQ {beta}57-Ala), which is encoded by a variety of HLA-DQ alleles, favors the binding of large branched amino acids (24), and is associated with susceptibility to autoimmune type 1 diabetes in humans (25, 26). By contrast, HLA-DQ {beta}57-Asp, which is also encoded by a number of HLA-DQ alleles, favors the binding of small hydrophobic peptides (24). Notably, the HLA-DQB1*0503 allele we previously demonstrated to be associated with tuberculosis susceptibility in Cambodia (17) encodes an aspartic acid at HLA-DQ {beta}57. This led us to hypothesize that the presence of an aspartic acid at codon 57 of HLA-DQ may influence P9-binding specificity, and thus impact the functional binding of HLA-DQ and subsequent host immune responses to immunogenic M. tuberculosis-derived peptides.

Thus, we performed HLA association studies investigating whether sets of alleles that form a similar P9 peptide-binding pocket, secondary to shared binding specificities resulting from the presence of an aspartic acid or a nonaspartic acid at codon 57, were associated with susceptibility to tuberculosis. In this study, we show a highly significant association between homozygosity for alleles encoding HLA-DQ {beta}57-Asp and active tuberculosis disease. Furthermore, as compared with alleles which encode an alanine at this position, HLA-DQ {beta}57-Asp binds a peptide from the central region of the M. tuberculosis immunogenic protein ESAT-6 with 5-fold less affinity and is significantly inferior to HLA-DQ {beta}57-Ala in stimulating effector T cell responses when presenting this same peptide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Study site and subjects

The study patients were Cambodian individuals over 14 years old who were randomly recruited between 1995 and 2002 from the Cambodian Health Committee tuberculosis treatment program in southeastern rural Cambodia (27). The diagnosis of clinical pulmonary tuberculosis was made as described previously (8). A total of 436 HIV-1-negative pulmonary tuberculosis patients were recruited in this study. Their mean age was 42.2 ± 14.1 years, and 62.7% were female.

Control subjects were recruited in 1995 from tuberculin-positive patients visiting the same hospitals for minor complaints. Based on detailed clinical history, control subjects did not have a history of tuberculosis or current signs or symptoms consistent with tuberculosis. A total of 117 tuberculin-positive control subjects with no history of tuberculosis were followed up for at least 7 years to verify their control status. Ten control subjects who developed clinical tuberculosis disease over the 7-year follow-up period were excluded. The mean age of the remaining 107 control individuals studied was 37.5 ± 12.9 years, and 56.1% were female. All individuals gave appropriate written consent and the study was approved by the Institutional Review Boards at the CBR Institute for Biomedical Research and in Cambodia.

All patients and control subjects were screened with tuberculin. Briefly, 5 tuberculin units of Tubersol (Aventis-Pasteur) was injected intradermally in the forearm of patients and controls and was evaluated for induration 48 h later by a trained tuberculosis heath care worker (S.T.) under the supervision of a physician with subspecialty training in infectious disease (A.E.G.). Patients were classified as tuberculin-positive if they had an induration of >10 mm as evaluated by the ballpoint method (28). Although bacillus Calmette-Guérin (BCG)4 vaccination was not routinely performed in Cambodia before 1990 because of the social breakdown of the country, and thus, is not a usual cause of false-positive tuberculin results in adults, we did however screen all our control subjects and patients for the presence of a BCG scar to rule out previous BCG vaccination. We note that we have previously demonstrated that BCG vaccination or infection by other environmental mycobacterial species are not common causes of false-positive tuberculin results in this population by correlating Ag-specific IFN-{gamma} assays to M. tuberculosis Ags with tuberculin positivity (29).

HLA and gene polymorphism typing

HLA alleles were identified in PCR-amplified products of exons 2 and 3 (HLA-A, -B, and -Cw) and exon 2 (HLA-DRB1 and -DQB1) by sequence-specific oligonucleotide probe hybridization as previously described (17).

Cell lines

The EBV-transformed human B cell line TEM expressing HLA-DQA1*0104 and HLA-DQB1*0503 described in the 10th International Histocompatibility Workshop (November 18–21, 1987, New York, NY) was obtained from the ASHI Cell Repository (Minneapolis, MN). BLS-1 cells, an HLA class II-null EBV-transformed B cell line generated from the cells of a patient with bare lymphocyte syndrome (30) was a gift from J. Blum (Indiana University School of Medicine, Indianapolis, IN). Human embryonic kidney 293 T cells were a gift from R. Van Etten (Tufts-New England Medical Center, Boston, MA). Murine fibroblast NIH-3T3 cells were purchased from the American Type Culture Collection.

Generation of BLS-1 cell lines expressing HLA-DQ {beta}57-Asp and HLA-DQ {beta}57-Ala

BLS-1 cell lines expressing HLA-DQA1*0104 and HLA-DQ {beta}57-Asp or {beta}57-Ala, were generated by retroviral-mediated gene transfer. Briefly, the HLA-DQA1*0104 and HLA-DQB1*0503 cDNAs were isolated from TEM cells. In addition, a mutagenic primer was used to change the codon (GAC) specifying aspartic acid at position {beta}57 to create an isogenic molecule containing the codon specifying alanine (GCC) at this position. The wild-type (HLA-DQ {beta}57-Asp) and mutated (HLA-DQ {beta}57-Ala) cDNAs were then cloned into the retroviral vector pLPCX and the HLA-DQA1*0104 cDNA was cloned into the pLXSN vector (BD Clontech). 293 T cells (4 x 106) were plated in 60-mm plates and the next day, 10 µg of the retroviral vector DNA containing the cDNA of interest was cotransfected by calcium phosphate precipitation with 5 µg of MCV-amphopack, a gift from R. Van Etten. Titers of viral supernatants obtained from the transfected 293 T cells were determined by transduction of NIH-3T3 cells, and colonies resistant to G-418 sulfate (pLXSN) or puromycin (pLPCX) were generated. All viruses had titers of 2.5–5.0 x 106 G-418 sulfate or puromycin-resistant PFU/ml. Viral supernatants were then used to infect BLS-1 cells and 500 µg/ml G-418 sulfate (pLXSN vector) or 200 ng/ml puromycin (pLPCX vector) was used for selection. BLS-1 cell lines stably expressing HLA-DQ {beta}57-Asp or {beta}57-Ala were verified by FACS with the anti-HLA-DQ SPVL3 mAb (BD Pharmingen).

Whole-cell peptide-binding assays

ESAT-6, a secreted protein specific to the M. tuberculosis complex (31, 32), is recognized by a large proportion of tuberculosis patients and tuberculin-positive controls from a variety of populations, including Cambodia (29), Ethiopia (33), Kuwait (34), and the United States and Germany (35). CFP-10, another M. tuberculosis-specific secreted protein, has also been shown to elicit an immune response in tuberculosis patients (36, 37). Thus, peptides from these two proteins allowed us to compare the binding affinity of HLA-DQ {beta}57-Asp and HLA-DQ {beta}57-Ala alleles to peptides from immunogenic M. tuberculosis proteins and their ability to elicit T cell activation.

Peptides spanning the length of the ESAT-6 and CFP-10 proteins were synthesized by F-moc chemistry at Mixture Sciences. Each peptide was 15 aa in length and overlapped the adjacent peptide by 10 residues. Purity (>95%) and identity of each peptide were characterized using mass spectrometry. A total of 2 x 105 BLS-1 cells expressing HLA-DQA1*0104 and HLA-DQ {beta}57-Asp or {beta}57-Ala were incubated in 96-well plates with 100 µM of the biotinylated peptide (VSKMRMATPLLMQAL) derived from the CLIP peptide sequence and with increasing concentrations (0.1–100 µM) of nonbiotinylated specific competitor ESAT-6 or CFP-10 peptides at 37°C for 4 h. At the end of the incubation, the cells were washed then incubated for 30 min at 4°C with 10 µg/ml FITC-avidin (Vector Laboratories), washed again, followed by a 30-min incubation at 4°C with 10 µg/ml biotinylated anti-avidin (Vector Laboratories), washed again and finally followed by a 30-min incubation at 4°C with 10 µg/ml FITC-avidin. Ultimately, the cells were resuspended in FACS buffer and analyzed by FACS. All binding experiments using different concentrations of competitor peptide were performed at least six times. The average concentration at which 50% inhibition of the mean fluorescent intensity was obtained with the biotinylated CLIP peptide alone was determined for each nonbiotinylated competitor peptide by plotting a curve for each peptide examined and extrapolating from the curve the concentration of peptide at which 50% inhibition of CLIP binding occurs.

Determination of HLA-DQ {beta}57-Asp and {beta}57-Ala T cell-restricted response to M. tuberculosis peptides

After appropriate consent was obtained, PBMC were isolated from Cambodian individuals positive for HLA-DQB1*0503 with prior history of tuberculosis disease but without current clinical or laboratory evidence of active disease. CD4+ T cell fractions were separated using paramagnetic methods (Miltenyi Biotec). The purity of each CD4+ T cell separation was analyzed by FACS and was consistently >95%. The HLA-DQ {beta}57-Asp and {beta}57-Ala T cell-restricted response to M. tuberculosis peptides were then studied by IFN-{gamma} ELISPOT assays. Briefly, 5 x 104 APC (BLS-1 cells expressing HLA-DQ {beta}57-Asp or {beta}57-Ala) were plated in Immunobilon-P membrane plates (Millipore) previously coated with IFN-{gamma} capture Ab (BD Pharmingen). Where indicated, transduced BLS-1 cells were pulsed with 20 µg/ml individual peptides for which we previously established an optimal saturating concentration to minimize variations in loading for T cell stimulation in ELISPOT assays (data not shown). Furthermore, lower concentrations of peptides (5–15 µg/ml) did not produce more significant differences in the production of IFN-{gamma} by T cells in response to peptide-loaded HLA-DQ {beta}57-Asp- or {beta}57-Ala-transduced BLS-1 cells.

The cells were cultured at 37°C for 2 h and, subsequently, 2.5 x 105 CD4+ T cells were added to the transduced Ag-presenting BLS-1 cells and incubated at 37°C for 16 h. After addition of IFN-{gamma} detection Ab (BD Pharmingen), followed by the streptavidin-HRP conjugate (DakoCytomation) HRP-substrate 3-amino-9-ethylcarbazole (BD Pharmingen), IFN-{gamma} spots were scored using a Series 1 ImmunoSpot Analyzer (Cellular Technology). All conditions were performed in triplicate. To rule out T cell stimulation from self- or EBV-derived peptides from the EBV-transformed BLS-1 transfectants or the participation of HLA class I-restricted peptide presentation as the stimulatory sources of IFN-{gamma}, we also cocultured CD4+ T cells with HLA-DQ {beta}57-Asp- or HLA-DQ {beta}57-Ala-transduced cells without peptide or with CD4+ T cells matched for HLA class I alleles expressed on BLS-1 cells and found no detectable IFN-{gamma} spots (data not shown).

Measurement of IFN-{gamma} levels in culture supernatants by ELISA gave the same pattern of results as did IFN-{gamma} ELISPOT assays in our standardization experiments of the T cell stimulation assays (data not shown). However, because IFN-{gamma} ELISPOT assays are a more sensitive measure of T cell stimulation as compared with ELISA measurement of IFN-{gamma} levels in culture supernatants (38, 39), we used the ELISPOT method to determine the HLA-DQ {beta}57-Asp and {beta}57-Ala T cell-restricted response to each one of the peptides.

Statistical analysis

The statistical significance of the difference in the frequency of individual HLA loci between the two groups was calculated by the 2 x 2 Fisher’s exact test. The frequencies of homozygous HLA-DQ {beta}57 genotypes were each compared with the more common heterozygous reference genotype using two separate 2 x 2 {chi}2 tests. Normally distributed variables were analyzed by the paired t test. Levels of significance are reported as two-tailed p values. A p value <0.05 was considered significant. These data analyses were performed with the aid of INSTAT software (GraphPad). Testing for the Hardy-Weinberg equilibrium was performed by use of SAS/Genetics Software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Homozygosity for aspartic acid at HLA-DQ {beta}57 is associated with susceptibility to pulmonary tuberculosis

Among genotypes derived from 436 tuberculosis patients in this study, we found that individuals homozygous for HLA-DQ {beta}57-Asp alleles were at a significantly higher risk for developing pulmonary tuberculosis as compared with 107 tuberculin-positive control individuals with no history or symptoms consistent with tuberculosis (p = 0.001; odds ratio, 3.05; 95% confidence interval, 1.53–6.07) (Table I). Consistent with our previous study (17), we detected 41 HLA-DQB1*0503 alleles among tuberculosis patients and detected none in the 107 tuberculin-positive control individuals (p = 0.0002). Furthermore, we found no association between alleles of class II HLA-DR or class I HLA-A, -B, or -Cw loci with susceptibility or resistance to pulmonary tuberculosis (data not shown).


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  		Table I. Comparison of the genotype frequency of the presence or absence of aspartic acid at position 57 of HLA-DQ {beta} chaina

 
Because HLA-DQB1*0503 occurs with higher frequency in Asians in comparison with Caucasian and African-derived populations (40), we also performed the analysis excluding HLA-DQ {beta}57-Asp homozygous genotypes carrying an HLA-DQB1*0503 allele to rule out that a Cambodian-specific HLA population marker was influencing our finding of HLA-DQ {beta}57-Asp homozygosity and susceptibility to pulmonary tuberculosis. When 26 HLA-DQ {beta}57-Asp homozygous genotypes carrying an HLA-DQB1*0503 allele were excluded from the analysis, the frequency of HLA-DQ {beta}57-Asp homozygosity remained significantly increased in tuberculosis patients (p = 0.02; odds ratio, 2.35; 95% confidence interval, 1.17–4.71), indicating the robustness of the association of HLA-DQ {beta}57-Asp homozygosity and tuberculosis in this population.

HLA-DQ {beta}57-Asp impairs binding of tuberculosis-derived peptides relative to HLA-DQ {beta}57-Ala

To determine the functional impact of this genetic association upon binding of M. tuberculosis peptides and subsequent T cell stimulation, we constructed retroviral vectors expressing either HLA-DQ {beta}57-Asp or HLA-DQ {beta}57-Ala using retroviral-mediated gene transfer into the HLA class II-null BLS-1 cell line. We isolated an HLA-DQ-{beta} gene encoding aspartic acid at codon 57 and, using site-directed mutagenesis, changed the aspartic acid to an alanine. Thus, these two cell lines expressed an isogenic HLA class II DQ molecule differing by only a single amino acid at position 57 of the HLA-DQ {beta}-chain. We confirmed that HLA-DQ {beta}57-Asp and HLA-DQ {beta}57-Ala were successfully transduced and equivalently expressed into the BLS-1 cells by flow cytometric analysis (Fig. 2A) and RT-PCR analysis of the DQ {beta}-chain (Fig. 2B) with subsequent sequencing of the PCR products (Fig. 2C).



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  		FIGURE 2. Expression of HLA-DQ molecules in BLS-1 cells. A, FACS analysis of HLA-DQ allelic proteins expressed on BLS-1 cells (HLA class II-null) before and after retroviral-mediated gene transfer as indicated in the figure. Cells were stained with anti-HLA-DQ SPVL3 mAb demonstrating that the BLS wild-type cells did not express HLA-DQ whereas the HLA-DQ {beta}57-Asp and HLA-DQ {beta}57-Ala did express surface HLA-DQ. B, RT-PCR analysis of DQ {beta} transcription. First-strand cDNA was synthesized from total RNA and the HLA-DQ {beta}-chain was amplified using PCR demonstrating that HLA-DQ {beta}57-Asp and HLA-DQ {beta}57-Ala transcripts were being synthesized in both transduced BLS-1 cells. {beta}-actin was amplified as an internal control. MW, DNA molecular weight marker. C, Sequence chromatogram results of the HLA-DQ {beta}-chain transcription products shown in B from the transduced BLS-1 cells demonstrating that the appropriate allele contains either an alanine or an aspartic acid at codon 57.

 
To test the binding affinity of ESAT-6 and CFP-10 peptides, we first established that HLA-DQ {beta}57-Ala- and HLA-DQ {beta}57-Asp-transduced cells equivalently bound a peptide derived from the CLIP protein (Fig. 3A). The CLIP peptide binds newly synthesized HLA class II molecules and directs them to acidified intracellular vesicles where peptide loading occurs (reviewed in Ref.41). Thus, our demonstration that the CLIP peptide binds equivalently to HLA-DQ {beta}57-Ala- and HLA-DQ {beta}57-Asp-expressing cells, shown in Fig. 3A, allowed us to use this peptide to perform competition assays with M. tuberculosis-derived peptides from ESAT-6 and CFP-10. Using increasing concentrations of these peptides, we were therefore able to calculate their binding affinity based upon their ability to inhibit binding of prebound CLIP to HLA-DQ {beta}57-Ala- and HLA-DQ {beta}57-Asp-transduced cells.



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  		FIGURE 3. Relatively reduced binding of HLA-DQ {beta}57-Asp to the ESAT46–60 peptide. A, Binding of biotinylated CLIP peptide at various concentrations to HLA-DQ {beta}57-Ala- or {beta}57-Asp-transduced cells. The cells were incubated with increasing concentrations of CLIP peptide and binding was determined by FACS using fluorescent avidin. The dotted line shows the concentration of CLIP peptide (100 µM) used for the peptide-binding competition experiments. MFI, mean fluorescent intensity. B, The binding affinities of ESAT-6 and CFP-10 peptides to HLA-DQ {beta}57-Asp- and HLA-DQ {beta}57-Ala-transduced BLS-1 cells. IC50 represents the concentration of unlabeled competitor M. tuberculosis (ESAT-6 or CFP-10) peptide (0.1–100 µM) required to inhibit 50% of the binding of indicator CLIP peptide. Synthetic peptides ESAT6–20, ESAT71–85, CFP1–15, and CFP31–45 were not tested due to poor quality synthesis. C–I, Binding of ESAT-6 and CFP-10 peptides to HLA-DQ {beta}57-Ala- or {beta}57-Asp-transduced cells at different concentrations expressed as percent binding of the indicator CLIP peptide. The average concentration of each peptide at which 50% inhibition of CLIP binding occurs at 100 µM is indicated in the figure.

 
Increasing concentrations of 31 competitor peptides spanning ESAT-6 and CFP-10 were thus incubated with HLA-DQ {beta}57-Ala- or HLA-DQ {beta}57-Asp-transduced cells and their ability to compete with CLIP for binding to the two cell lines was measured (Fig. 3B). Strikingly, when the ESAT46–60 peptide was presented by HLA-DQ {beta}57-Ala-transduced BLS-1 cells, 50% inhibition of CLIP binding was achieved at an ~5-fold lower concentration of ESAT46–60 than when the peptide was presented by HLA-DQ {beta}57-Asp-transduced cells (Fig. 3, B and C). We note that six other peptides (ESAT31–45, ESAT36–50, ESAT41–55, ESAT51–65, CFP16–30, and CFP41–55) minimally competed with CLIP for binding to one or the other of the HLA-DQ molecules (Fig. 3, B and D–I).

Presentation of ESAT46–60 by HLA-DQ {beta}57-Asp vs HLA-DQ {beta}57-Ala elicits significantly decreased IFN-{gamma} from CD4+ T cells

To determine the functional impact of this differential binding of M. tuberculosis-derived peptides, we next isolated CD4+ T cells from six former pulmonary tuberculosis patients from Cambodia who carried an HLA-DQB1*0503 allele and cocultured their CD4+ T cells with HLA-DQ {beta}57-Asp- or HLA-DQ {beta}57-Ala-transduced cells that had been loaded with one of the seven peptides shown to have differential binding to HLA-DQ {beta}57-Ala or HLA-DQ {beta}57-Asp. As a measure of CD4+ T cell activation, we calculated IFN-{gamma} levels subsequent to peptide presentation by the HLA-DQ {beta}57-Asp- or HLA-DQ {beta}57-Ala-transduced cells.

Presentation of ESAT31–45, ESAT36–50, ESAT41–55, ESAT51–65, CFP16–40, or CFP41–55 peptides by HLA-DQ {beta}57-Asp- or HLA-DQ {beta}57-Ala-transduced cells resulted in an erratic pattern of IFN-{gamma} production in each of the six patients (Fig. 4), consistent with the minor differences detected in the binding of these peptides by HLA-DQ {beta}57-Asp or HLA-DQ {beta}57-Ala molecules. Remarkably, however, in every case using CD4+ T cells from each of the six patients tested, we detected increased numbers of IFN-{gamma}-secreting cells when they were stimulated with HLA-DQ {beta}57-Ala-transduced cells loaded with ESAT46–60 as compared with stimulation with HLA-DQ {beta}57-Asp-transduced cells loaded with ESAT46–60 (Fig. 4). Furthermore, the difference in mean number of IFN-{gamma} spots per 500,000 CD4+ cells observed in CD4+ T cells stimulated with HLA-DQ {beta}57-Ala (106.5 ± 7.9) vs HLA-DQ {beta}57-Asp (101.8 ± 9.8) when the six patients were grouped (p = 0.049), reached the accepted critical value of statistical significance in biology (p = 0.049). Furthermore, the difference in the mean number of IFN-{gamma} spots per 500,000 CD4+ cells observed in the CD4+ T cells stimulated with HLA-DQ {beta}57-Ala (106.5 ± 7.9) vs HLA-DQ {beta}57-Asp (101.8 ± 9.8) in the six patients reached statistical significance (p = 0.049). We note that the presentation of the complete set of peptides spanning the length of the ESAT-6 or CFP-10 proteins by the HLA-DQ {beta}57-Ala or HLA-DQ {beta}57-Asp cells elicited a nonstatistically significant increase in the number of IFN-{gamma}-secreting T cells when stimulated with HLA-DQ {beta}57-Ala cells as compared with HLA-DQ {beta}57-Asp cells (p = 0.08). Peptides presented by HLA class II molecules are generated from exogenous native proteins and only a few epitopes of specific length and sequence are ultimately presented to the immune system (reviewed in Ref.41). Because each of the HLA DQ-transduced cells were loaded with overlapping peptides of ESAT-6, the observed effect of ESAT46–60 upon T cell stimulation would be expected to be diluted by other peptides.



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  		FIGURE 4. HLA-DQ {beta}57-Asp reduces host CD4+ T cell response to the ESAT46–60 peptide. The figure shows the number of ESAT-6 peptide-specific IFN-{gamma}-secreting T cells in samples from six HLA-DQB1*0503 individuals with a history of tuberculosis after coculture with HLA-DQ {beta}57-Ala (•) or HLA-DQ {beta}57-Asp- ({circ}) transduced BLS-1 cells loaded with 20 µg/ml peptide. Each individual is identified by a number (1–6) and the panel of peptides tested (ESAT31–45, ESAT36–50, ESAT41–55, ESAT46–60, ESAT51–65, CFP16–40, and CFP41–55) can thus be compared for each individual’s T cell response to Ag presentation by each transduced BLS-1 cell. Dotted and solid lines represent a decrease or increase, respectively, in the number of IFN-{gamma}-secreting T cells using HLA-DQ {beta}57-Asp-transduced cells compared with HLA-DQ {beta}57-Ala to present peptide. In each of the six patients, IFN-{gamma} production increased when ESAT46–60 was presented by HLA-DQ {beta}57-Ala.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that HLA alleles that encode an aspartic acid at codon 57 of the HLA-DQ {beta}-chain are significantly associated with susceptibility to tuberculosis. The presence of a single change, an aspartic acid vs an alanine at this position, results in a 5-fold difference in binding to a central portion of the immunogenic M. tuberculosis peptide, ESAT46–60, and to a significant difference in T cell effector response. These results thus provide a functional rationale for our genetic data showing that individuals homozygous for HLA-DQ {beta}57-Asp are more susceptible to pulmonary tuberculosis progression.

We note that HLA loci are in linkage disequilibrium with several neighboring genes. Among these, the TNF gene has been implicated in the host response to M. tuberculosis (42, 43). We previously ruled out an association between tuberculosis susceptibility and polymorphisms of the TNF gene, as well as other polymorphisms of candidate genes including NRAMP, IL-10, IL-1, and the vitamin D receptor (8). Linkage disequilibrium with other HLA loci is also unlikely in our study due to our finding of no significant association between tuberculosis susceptibility and the HLA-DRB1*1404 allele, an allele previously linked to HLA-DQB1*0503 in southeastern Asians (40).

Our data also demonstrate that the central region of ESAT-6 (spanning aa 46–60), but not the complete ESAT-6 protein, is the region of the molecule restricted by HLA-DQB1*0503. Data from Ethiopia demonstrated that the predominant immune recognition region of ESAT-6 is its central region, including aa 42–75 (33). By contrast, reports from Germany and the United States (35) have suggested that the N-terminal of ESAT-6 is the predominant peptide recognition region. Reports from Kuwait (34) and Denmark (33) indicated that the C-terminal is the major ESAT-6 immune recognition domain in association with HLA-DR. Thus, ethnic-specific genetic variations in HLA class II loci likely influence T cell epitope selection with respect to the predominant ESAT-6 immune recognition epitopes involved in maintenance of latent tuberculosis infection, which should be considered in peptide selection in vaccine design.

Greater than 80% of the estimated 1.7 million new cases of tuberculosis that occur annually are in Africa, Southeast Asia, and in Central and South America (44, 45). Natural selection for resistance to tuberculosis has been debated as an explanation for the current uneven epidemiological distribution of tuberculosis (46, 47), because during the last millennium, before adequate chemotherapy, tuberculosis claimed over a billion lives in European Caucasians (48). Thus, it is reasonable to speculate that alleles that provided some host advantage in containing tuberculosis disease would have been favored in individuals who did not die of progressive tuberculosis in highly exposed populations (47, 48). In this regard, it is interesting that susceptibility to human immune-mediated type-1 diabetes, which has the highest incidence in Caucasians from European descent (49) is correlated with two alleles encoding HLA-DQ-{beta}57-Ala (HLA-DQ2 and -DQ8) (25, 26), which we have shown to bind the ESAT46–60 with higher affinity. Strikingly, and consistent with this hypothesis, in mice, the presence of an aspartic acid at position 57 of H2 I-A{beta} (the homologue of HLA-DQ{beta} in humans), diminishes the spontaneous incidence of immune-mediated type-1 diabetes (50).

Although multiple pathogens and many other environmental factors are certainly responsible for selecting polymorphisms in the HLA system, it is interesting to speculate that the respective association of HLA-DQ {beta}57-Ala and HLA-DQ {beta}57-Asp with susceptibility to type-1 diabetes and tuberculosis is not random. We note that before the discovery of insulin in the beginning of the 20th century, autoimmune diabetes was universally fatal. Perhaps similar to the maintenance of sickle cell trait in populations of African ancestry, which confers resistance to falciparum malaria (51, 52), and in the homozygous state was fatal, the increased distribution of HLA-DQ {beta}57-Ala in Caucasians conferred an advantage over tuberculosis, which was gained at the expense of increased susceptibility to autoimmune type-1 diabetes. A recent report demonstrated an opposite association of TNF polymorphisms, which are highly linked to HLA genes (53, 54), between tuberculosis and three autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren’s syndrome (55). This finding supports the hypothesis that increased susceptibility to autoimmunity is a consequence of natural selection for enhanced host immunity to tuberculosis.

Multiple genetic loci are surely involved in the optimal immune response to any specific pathogen and similarly render individuals susceptible to autoimmune diseases. Thus, to obtain any functional data to support the role of a specific genetic difference such as that presented here represents a significant advance, because it is undoubtedly the sum of a number of differences in distinct genetic loci which ultimately determines the host immune response, and thus susceptibility or resistance to a pathogen. Finally, the correlation between genetic association, functional HLA binding, and T cell activation presented here also suggests strategies to identify individuals at particular risk of progressive tuberculosis and candidate peptides for tuberculosis vaccines.


   Acknowledgments
 
We thank Rick Van-Etten for providing the CMV-amphopack vector and advice on retroviral gene delivery methods and Janice Blum for providing the BLS-1 cell line. We thank Olga Clavijo for expert technical assistance, Edmond Yunis for helpful discussions, and Chester Alper, Anne Cooke, Paulo Vieira, and Anne O’Garra for critical comments on the manuscript. We thank the Cambodian Health Committee staff for assistance in Cambodia and Jean-Marc Reynes and Jean-Louis Sarthou of the Institut Pasteur du Cambodge for laboratory support in Cambodia.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants HL67471 (to J.C.D.) and HL59838 (to A.E.G.). Back

2 Current address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461. Back

3 Address correspondence and reprint requests to Dr. Anne E. Goldfeld, CBR Institute for Biomedical Research, 800 Huntington Avenue, Boston, MA 02115. E-mail address: goldfeld{at}cbrinstitute.org Back

4 Abbreviation used in this paper: BCG, bacillus Calmette-Guérin. Back

Received for publication August 8, 2005. Accepted for publication October 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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