Aspartic Acid Homozygosity at Codon 57 of HLA-DQ  Is Associated with Susceptibility to Pulmonary Tuberculosis in Cambodia 1 Julio C. Delgado,* † Andres Baena, 2 * Sok Thim, § and Anne E. Goldfeld 3 * ‡ 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 -chain (HLA-DQ 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 57-Asp or HLA-DQ 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 57-Asp alleles. The presence of HLA-DQ 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 57-Asp allele, Ag-specific IFN- production from CD4  T cells from tuberculosis patients was significantly less than when this peptide was presented by an HLA-DQ- 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. The Journal of Immunology, 2006, 176: 1090 –1097. H uman beings have evolved on a background of infection and it has long been speculated that genetic variability among populations that confers resistance against infec- tious pathogens is favored by evolution (1). Numerous case-con- trol studies have identified associations between susceptibility or resistance to human Mycobacterium tuberculosis infection and clinical disease with polymorphisms of candidate genes (2–7). 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 re- sponses 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 –12). Several alleles of the HLA class II DR2 serotype (13–16), and the HLA- DQB1*0503 allele (17) are associated with susceptibility to pro- gression to clinical tuberculosis after infection in diverse popula- tions. However, despite these multiple association studies, a functional link between HLA associations, Ag-driven T cell re- sponses, 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 poly- morphic side chains (18, 19). HLA-DQ molecules, encoded by polymorphic HLA-DQ  and -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 -chain (HLA-DQ 57), because this position in- fluences 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 57-Asp) or a nonaspartic acid at this position (22, 23) (Fig. 1). The presence of an alanine at this position (HLA-DQ 57-Ala), which is encoded by a variety of HLA-DQ alleles, favors the bind- ing of large branched amino acids (24), and is associated with susceptibility to autoimmune type 1 diabetes in humans (25, 26). By contrast, HLA-DQ 57-Asp, which is also encoded by a num- ber 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 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 *CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA 02115; † Department of Pathology and ‡ Department of Medicine, Brigham and Wom- en’s Hospital, Harvard Medical School, Boston, MA 02115; and § The Cambodian Health Committee, Phnom Penh, Cambodia Received for publication August 8, 2005. Accepted for publication October 31, 2005. 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.). 2 Current address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461. 3 Address correspondence and reprint requests to Dr. Anne E. Goldfeld, CBR Institute for Biomedical Research, 800 Huntington Avenue, Boston, MA 02115. E-mail ad- dress: goldfeld@cbrinstitute.org. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00