- Email: [email protected]
HLA-DRB1 and -DQB1 alleles in mestizo patients with Vogt-Koyanagi-Harada’s disease in Southern California
HLA-DRB1 and -DQB1 Alleles in Mestizo Patients With Vogt-Koyanagi-Harada’s Disease in Southern California Ralph D. Levinson, Robert F. See, Raja Rajalingam, Elaine F. Reed, Min S. Park, Narsing A. Rao, and Gary N. Holland ABSTRACT: We evaluated human leukocyte antigen (HLA)-DRB1 and -DQB1 alleles as genetic markers for Vogt-Koyanagi-Harada (VKH) disease in Mestizo patients in Southern California. Mestizo individuals with VKH disease (n ⫽ 29) at two institutions were evaluated. Typing of HLA-DRB1 and DQB1 genes was performed using DNA-based techniques. Gene frequencies were compared to Mestizo individuals living in Southern California. All patients had HLA-DRB1*01, DRB1*04, DQB1*03 or DQB1*05, or a combination of these genes. The gene frequency of combined HLA-DR4 alleles was increased when compared to controls. The frequencies of HLA-DRB1*0404 and DRB1*0407 were increased compared to controls, but were not significant after BonferABBREVIATIONS HLA human leukocyte antigen OR odds ratio
INTRODUCTION Vogt-Koyanagi-Harada’s (VKH) disease, a bilateral granulomatous panuveitis that also may affect the skin, meninges, and auditory system [1], is known to be strongly associated with the human leukocyte antigen
From the Ocular Inflammatory Disease Center, Jules Stein Eye Institute and Department of Ophthalmology (R.D.L., G.N.H.), Los Angeles; the UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine (R.R., E.F.R., M.S.P.), David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles; and the University of Southern California Keck School of Medicine, Los Angeles, CA, USA (R.F.S., N.A.R.). Address reprint requests to: Dr. Ralph D. Levinson, Jules Stein Eye Institute, 100 Stein Plaza, UCLA, Los Angeles, CA 90095-7002; Tel: (310) 794-7770; Fax: (310) 794-7906; E-mail: [email protected]. This research was supported in part by The MacDonald Family Foundation, Los Angeles, CA (R.D.L.); Research to Prevent Blindness, Inc, New York, NY (G.N.H.); and the David May II Endowed Professorship (G.N.H.). Dr. Holland is recipient of a Research to Prevent Blindness Physician-Scientist Award. Received April 1, 2004; revised July 8, 2004; accepted July 9, 2004. Human Immunology 65, 1477–1482 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
roni correction. Three patients had the HLA-DRB1*0410 allele; this allele was not found in controls. All HLADRB1*01 positive patients had the DRB1*0102 subtype. No HLA-DQB1 allele was significantly increased compared to controls. This study is the first to identify a possible association between HLA-DRB1*0404 and VKH disease, as well as to find DRB1*0102 and DRB1*0410 in Mestizo patients. Human Immunology 65, 1477–1482 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: HLA; immunogenetics; uveitis; VogtKoyanagi-Harada-disease; mestizo
UCLA VKH
University of California at Los Angeles Vogt-Koyangi-Harada
(HLA)-DR4 phenotype in Japanese patients [2]. A study looking at HLA associations with VKH disease in mestizo individuals in Southern California found either HLA-DR1 or HLA-DR4 in 84% of patients by means of serologic techniques [3]. Indeed, HLA-DR1 had a higher relative risk than HLA-DR4 (4.11 vs 1.96, respectively) in this population [3]. In contrast, although both HLADR1 and -DR4 serotypes were also found in mestizo patients with VKH disease in a study from Mexico City, only HLA-DR4 was significantly increased compared with controls [4]. Using DNA-based techniques to look at HLA alleles rather than HLA serotypes in the same population, HLA-DRB1*0101 was associated with disease and HLA-DRB1*01 or HLA-DRB1*04 subtypes were found in 89% [5]. Furthermore, while HLADRB1*0405 and HLA-DRB1*0410 alleles predominated in Japanese [6] and Korean [7] patients with VKH disease, no specific HLA-DRB1*04 alleles were associ0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.07.236
1478
ated with VKH disease among mestizo patients in the Mexico City study [5]. The differences between studies imply that the strength of the HLA association may differ between populations and may in part depend on whether serologic or DNA-based techniques are used. In the current study, we have expanded on the previous report of HLA types in mestizo patients with VKH disease in Southern California using DNA-based techniques to evaluate the HLA alleles that confer risk in this patient population. PATIENTS AND METHODS We evaluated 29 patients with VKH disease who volunteered for the study and who had not previously had HLA typing. Subjects were recruited from two institutions: the Jules Stein Eye Institute, David Geffen School of Medicine at UCLA (Los Angeles, CA) and the Doheny Eye Institute, University of Southern California (USC) Keck School of Medicine (Los Angeles, CA). This study was approved by the UCLA and USC institutional review boards and maintained strict adherence to the Declaration of Helsinki for research involving human subjects. Written informed consent was obtained from all participants prior to study enrollment. Medical records of patients participating in the study were reviewed to confirm the diagnosis of VKH disease, based on standardized criteria [1]. Patients were considered to be mestizo if they were self-identified as Hispanic and had at least one parent from Mexico or Central America. While self-identification is not an ideal method for classifying individuals on the basis of race [8], it has been used previously in studies of HLA gene frequencies, including the National Marrow Donor Program [9]. This approach allowed us to establish a range of HLA alleles associated with VKH disease in non-Asian population, and enabled us to compare our results with other populations of mestizo patients with VKH disease [4, 5]. All HLA typing was performed at the UCLA Immunogenetics Center. DNA was isolated from peripheral blood leukocytes using the Qiagen extraction method (Qiagen Inc., Valencia, CA, USA) and typed for HLA class II gene polymorphisms by a polymerase chain reaction (PCR) technique followed by hybridization with sequence-specific oligonucleotide probes, as published elsewhere [10]. The HLA-DRB1*01, HLA-DRB1*04, and HLA-DQB1 subtypes were further characterized at the allele level using sequence-specific primers (One Lambda, Canoga Park, CA, USA). Because it has been demonstrated that VKH disease is associated with HLADR1 and HLA-DR4 in previous studies [3–5], highresolution typing was limited to HLA-DRB1*01 and HLA-DRB1*04 alleles. Gene frequencies for HLA-DR and HLA-DQ alleles
R. Levinson et al.
found in patients with VKH disease in the current study were compared with mestizo individuals living in Southern California. Seventy-three mestizo individuals in Southern California were originally typed at the UCLA Immunogenetics Center as standard controls for DNA typing methods for HLA polymorphisms. Comparisons were made using a two-sided Fisher’s exact test. Because multiple comparisons were made in the evaluation of individual HLA-DR and HLA-DQ alleles, a simple Bonferroni’s correction was performed, and a p value ⬍ 0.005 was chosen as the threshold for statistical significance. Because previous studies had indicated a high prevalence of HLADR1 and HLA-DR4 genes in mestizo patients with VKH disease [3–5], combined data for HLA-DR1 and HLADR4 alleles were evaluated as a global measure for comparisons; as such, no Bonferroni’s correction was necessary for this evaluation, and a p value ⬍ 0.05 was chosen as the threshold for statistical significance. RESULTS All 29 patients had either HLA-DRB1*01, HLADRB1*04, HLA-DQB1*03, or HLA-DQB1*05 genes, or a combination of these genes (Table 1). Table 2 shows gene frequencies for study subjects and for the Southern California mestizo control population. There were no significant differences in HLA gene frequencies between the mestizo control group from Southern California and a previously published Mexican mestizo population [11] (data not shown). Either HLA-DRB1*01, HLADRB1*04 alleles, or both were present in 27 patients (93.1%); the gene frequency for combined HLA-DR1 and HLA-DR4 alleles was greater than that for controls (p ⫽ 0.0006, odds ratio [OR] ⫽ 3.17). All patients who were HLA-DR1–positive had the HLA-DRB1*0102 subtype (p ⫽ 0.01, OR ⫽ 4.60; not significant after Bonferroni’s correction). HLA-DRB1*04 alleles were found in 22 patients (75.9%); the gene frequency of combined HLA-DR4 alleles was increased when compared with controls (p ⫽ 0.005, OR ⫽ 2.60). Three patients were homozygous for HLA-DRB1*04 alleles. Only one HLA-DRB1*04 allele was found for three patients; it is not known if they were homozygous for that allele; for all evaluations it was considered one allele. Gene frequencies for HLA-DRB1*0404 (p ⫽ 0.035, OR 3.23), HLA-DRB1*0407 (p ⫽ 0.028, OR ⫽ 3.23) and HLA-DRB1*0410 (p ⫽ 0.022) were increased compared with controls, but these were not significant after Bonferroni’s correction. Because HLA-DRB1*0410 was not found in mestizo controls from either Southern California or a previously published control population [11], an OR could not be calculated. Patients in the current study had a higher frequency of HLA-DRB1*0102, HLADRB1*0404, and HLA-DRB1*0410 than previously re-
HLA in VKH Disease in Mestizos in California
1479
TABLE 1 HLA subtypes in mestizo patients with VKH disease in Southern California Patient
HLA-DRB1
HLA-DQB1
1 2 3 4 5 6 7 8 9* 10† 11 12 13 14 15* 16* 17† 18 19 20 21 22 23 24 25 26† 27 28 29
0102,07 0102,08 0102,08 0102,11 0102,13 0102, 0404 0102, 0407 0102, 0407 04031, 0407 0404‡ 0404‡ 0404, 0701 0404, 08 0404, 13 0404, 0406 0404, 0407 0405, 1501 04051, 15021 0407‡ 0407, 08 0407, 0802 0407, 1104 0407, 14 0407, 1406 0410, 03 0410, 14 0410, 1402 0301, 10 08021, 1402
0303, 0501 0301/13, 0501 0402, 0501 0301/13, 0501 0303, 0501 0302, 0501 0302, 0501 0302, 0501 0302, 0202 — 0302, 0202 0302, 0202 0302, 0402 0402, 0603 0302, 0402 0302‡ — 0302, 0601 0302, 0402 0302, 0402 0302, 0402 0302, 0301 0302, 0503 0302, 0301 0302, 0201 — 0302, 0301 0202, 0501 0301/13, 0402
High-resolution typing performed only for HLA-DRB1*01, HLA-DRB1*04, and HLA-DQB1 alleles. Abbreviation: VKH ⫽ Vogt-Koyanagi-Havada. * Homozygous for HLA-DRB1*04 alleles. † HLA-DQB1 typing was not obtained for these subjects. ‡ Only one allele was found; it cannot be determined if the subject was homozygous or heterozygous for the HLA subtype.
ported Mexican mestizo patients with VKH disease [5], but the differences were not statistically significant (data not shown). Although 25 (96.2%) of 26 patients had either HLADQB1*03 or HLA-DQB1*05 genes, no HLA-DQB1 gene was found to be significantly more frequent in study subjects than in controls (Table 2). DISCUSSION We found HLA-DRB1*04 to be associated with VKH disease in mestizo patients living in Southern California. There was also an increased frequency of HLA-DRB1*01 and HLA-DRB1*04 alleles combined, but this was primarily from the increase in HLA-DRB1*04. This is consistent with previous studies that reported either HLA-DR1 or HLA-DR4 to be associated with VKH disease in mestizo patients [3–5].
Although the frequency of HLA-DRB1*0102 allele was increased compared with controls, it did not remain significant after Bonferroni’s correction for multiple comparisons. HLA-DRB1*0102 is of interest because it is strongly associated with tubulointerstitial nephritis and uveitis syndrome, another disease often complicated by chronic and recurrent anterior uveitis [12]. Although no specific HLA-DRB1*04 subtype was increased when compared with controls after Bonferroni’s correction for multiple comparisons, there was a suggestion that HLA-DRB1*0404, HLA-DRB1*0407, and HLA-DRB1*0410 may be associated with disease in our patients. HLA-DRB1*0410 has been associated with VKH disease in Asian patients, but not previously in mestizo patients [5–7]. Although the association with HLA-DRB1*0410 lost statistical significance after Bonferroni’s correction, no individual in two mestizo control populations had that allele, suggesting that it may indeed be associated with VKH disease in our patients. Mestizo populations have genes derived from Caucasian as well as Native American and black ancestors. In light of the clinical and pathologic similarities between sympathetic ophthalmia and VKH disease [13], it is interesting to compare the specific HLA-DR subtypes associated with these diseases in different populations. Similar to the findings in our mestizo patients with VKH disease, HLADRB1*0404 was associated with sympathetic ophthalmia in Caucasians, whereas in Japanese patients HLADRB1*0405 has been associated with both VKH disease and sympathetic ophthalmia [6, 7, 14]. Because no HLA-DRB1*01 or HLA-DRB1*04 allele predominated with a particularly large OR in either our study or a previous study of mestizo patients with VKH disease from Mexico City [5], it is likely that a range of HLA-DR1 and HLA-DR4 alleles may act as markers for disease risk. It has been suggested that HLADRB1*0405 has specific peptide-binding characteristics for residues 4 and 9 that may confer particular risk for the development of VKH disease [15]. In fact, the HLA associations with VKH disease in mestizo individuals are similar to those described in patients with rheumatoid arthritis, which is also associated with both HLA-DR1 and HLA-DR4. Interestingly, as appears to be the case for VKH disease and sympathetic ophthalmia, HLADRB1*0404 was associated with rheumatoid arthritis in Caucasians, whereas HLA-DRB1*0405 was found to be associated with rheumatoid arthritis in Asians [16]. It is possible that different HLA types could play a role in disease pathogenesis by presenting similar immunogenic peptides to T cells. The HLA-DR1 and HLADR4 molecules that are associated with VKH disease in mestizo patients and in patients with rheumatoid arthritis share the amino acid sequence glutamine, arginine, arginine, alanine in positions 70 to 73 of the HLA-DR
1480
R. Levinson et al.
TABLE 2 Gene frequencies of HLA-DRB1 and HLA-DQB1 alleles in mestizo patients with VKH disease versus controls Study subjects (n ⫽ 29)
Allele
Number of patients
Number of genes*
HLA DR DR1 DRB1*0101 DRB1*0102 DR4 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0406 DRB1*0407 DRB1*0410 DRB1*0411 DR1 or DR4 HLA DQ DQ5 DQB1*0501 DQB1*0502 DQB1*0503 DQ3 DQB1*0301 DQB1*0302 DQB1*0303
29 8 0 8 22 0 1 8 2 1 10 3 0 27 26 10 9 0 1 23 6 18 2
55 8 0 8 25 0 1 8 2 1 10 3 0 33 51 10 9 0 1 26 6 18 2
Controls (n ⫽ 73)
GF (%)
Number of individuals tested
Number of genes†
GF (%)
p value‡
OR
— 14.5 — 14.5 45.5 — 1.8 14.5 3.6 1.8 18.2 5.5 — 60.0 — 19.6 17.6 — 2.0 51.0 11.8 35.3 3.9
73 11 4 5 30 1 7 7 4 0 9 0 6 37 73 13 11 2 1 58 33 29 2
140 11 4 5 34 1 7 7 4 0 9 0 6 45 133 14 11 2 1 64 33 29 2
— 7.9 2.9 3.6 24.3 0.7 5.0 5.0 2.9 — 6.4 — 4.3 32.1 — 10.5 8.3 1.5 0.8 48.1 24.8 21.8 1.5
— 0.182 0.579 0.010 0.005 1.000 0.445 0.035 0.675 0.282 0.028 0.022 0.187 0.0006 — 0.140 0.109 1.000 0.479 0.745 0.069 0.088 0.308
— 2.00 — 4.60 2.60 — 0.35 3.23 1.28 — 3.23 — — 3.17 — 2.07 2.38 — 2.64 1.12 0.40 1.96 2.67
Abbreviations: GF ⫽ gene frequency (the proportion of the given gene in the population studied, given as a percentage); HLA ⫽ human leukocyte antigen; OR ⫽ odds ratio; VKH ⫽ Vogt-Koyahagi-Harada. * Number of alleles with the HLA type in study subjects. † Number of alleles with the HLA type in individuals from the control population. In some cases, there are more genes than subjects for a given HLA type because some individuals were homozygous. ‡ Comparing GF in patients with a control group consisting of mestizo individuals living in Southern California using two-sided Fisher’s exact tests.
molecule; HLA-DRB1*0407 has a glutamate in position 74, whereas the other HLA-DR subtypes in our patients have an alanine in position 74 [17], implying that this difference in amino acids in position 74 is not critical. These and related HLA amino acid sequences have been termed “shared epitopes” and are thought to confer risk for the development of rheumatoid arthritis, as reviewed elsewhere [18]. Furthermore, peptide-binding repertoires are shared by HLA-DR1 and HLA-DR4, again calling into question whether the unique binding characteristics of HLA-DRB1*0405 are critical for the development of VKH disease [19]. Binding of an immunodominant peptide of type II collagen [20] and an epitope of the influenza hemagglutinin peptide [21] to both HLA-DR1 and HLA-DR4 molecules has been examined by x-ray crystallography. The same anchor residues were found to be involved in the presentation of antigens by both HLA-DR1 and HLA-DR4 molecules, with only a slight conformational difference noted when the same peptide was bound to either HLA-DR1 or
HLA-DR4. Although slight conformational changes could alter immunogenicity [22], thereby resulting in different pathogenic potential for peptides bound by different HLA subtypes, the observation that HLA-DR1 and HLA-DR4 molecules could present the same immunogenic peptides is consistent with both HLA-DR1 and HLA-DR4 subtypes having a role in the immunopathogenesis of VKH disease. There may also be a role for HLA-DQ genes in the pathogenesis of VKH disease. We found either HLADQB1*03 or HLA-DQB1*05 alleles in all but one patient tested for HLA-DQ alleles. The one patient without these HLA-DQ alleles had the HLA-DQB1*04 allele, which has been associated with VKH disease in Asian VKH patients [6, 7]. In addition, two patients in the current study did not have either the HLA-DR1 or the HLA-DR4 genotype; one had an HLA-DQB1*03 allele and the other an HLADQB1*05 allele. The lack of a statistically significant association between VKH disease and any specific DQB1 gene in our patients may be a result of the range of
HLA in VKH Disease in Mestizos in California
HLA-DQ3 and HLA-DQ5 alleles that are able to play a role in disease pathogenesis. It cannot be determined from the current data whether the HLA-DQB1 genes may make a unique contribution to disease risk or, rather, are in linkage disequilibrium with critical HLA-DR genes or other genes on chromosome 6 that may play a role in the immune response. In tubulointerstitial nephritis and uveitis syndrome, different HLA-DR associations found could be explained by linkage disequilibrium to HLA-DQ genes [12]. Interactions between HLA-DR and HLA-DQ molecules may also be important; for example, there is evidence from in vitro studies that peptides derived from the HLA-DR glycoprotein itself can be presented by HLA-DQ molecules [23]. In conclusion, we found that HLA-DR4 confers modest risk for VKH disease in mestizo individuals without a strong association with any specific subtype. This provides further evidence that multiple HLA-DR alleles confer risk for VKH disease. The observation that HLA-DRB1*0404 is associated with sympathetic ophthalmia and rheumatoid arthritis in Caucasians, and HLA-DRB1*0405 confers risk for these diseases in Asians, is further evidence for the possibility that these different HLA subtypes may play a similar role in the pathogenesis of VKH disease in different populations. A better understanding of the similarities, as well as the differences, in the immunogenetics and pathogenesis of these diseases has important implications for further research and the design of novel therapies. Examining HLA associations with disease in diverse populations is important for evaluating the range of HLA subtypes that may contribute to disease risk and provides additional insights into possible disease mechanisms. REFERENCES 1. Read RW, Holland GN, Rao NA, Tabbara KF, Ohno S, Arellanes-Garcia L, Pivetti-Pezzi P, Tessler HH, Usui M. Revised diagnostic criteria for Vogt-Koyanagi-Harada disease: report of an international committee on nomenclature. Am J Ophthalmol 131:647, 2001. 2. Ohno S. Immunological aspects of Behcet’s and VogtKoyanagi-Harada’s diseases. Trans Ophthalmol Soc U K 101:335, 1981. 3. Weisz JM, Holland GN, Roer LN, Park MS, Yuge AJ, Moorthy RS, Forster DJ, Rao NA, Terasaki PI. Association between Vogt-Koyanagi-Harada syndrome and HLA-DR1 and -DR4 in Hispanic patients living in southern California. Ophthalmology 102:1012, 1995. 4. Arellanes-Garcia L, Bautista N, Mora P, Ortega-Larrocea G, Burguet A, Gorodezky C. HLA-DR is strongly associated with Vogt-Koyanagi-Harada disease in Mexican Mestizo patients. Ocul Immunol Inflamm 6:93, 1998.
1481
5. Alaez C, del Pilar MM, Arellanes L, Cano S, Perez-Luque E, Vazquez MN, Olivo A, Burguete A, Hernandez A, Pedroza M, Gorodezky C. Strong association of HLA class II sequences in Mexicans with Vogt-Koyanagi-Harada’s disease. Hum Immunol 60:875,1999 6. Shindo Y, Ohno S, Yamamoto T, Nakamura S, Inoko H. Complete association of the HLA-DRB1*04 and -DQB1*04 alleles with Vogt-Koyanagi-Harada’s disease. Hum Immunol 39:169, 1994. 7. Kim MH, Seong MC, Kwak NH, Yoo JS, Huh W, Kim TG, Han H. Association of HLA with Vogt-KoyanagiHarada syndrome in Koreans. Am J Ophthalmol 129:173, 2000. 8. Kaplan JB, Bennett T. Use of race and ethnicity in biomedical publication. JAMA 289:2709, 2003. 9. Cao K, Hollenbach J, Shi X, Shi W, Chopek M, FernandezVina MA. Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations. Hum Immunol 62:1009, 2001. 10. Erlich H, Bugawan T, Begovich AB, Scharf S, Griffith R, Saiki R, Higuchi R, Walsh PS. HLA-DR, DQ and DP typing using PCR amplification and immobilized probes. Eur J Immunogenet 18:33,1991 11. Olivo A, Alaez C, Debaz H, Vazquez M, de la Rosa G, Perez Luque E, Hernandez A, Gorodezky C. Mexican mestizo normal. In Terasaki PI, Gjertson DW (Eds): HLA 1997. Los Angeles, CA: UCLA Tissue Typing Laboratory, 1997:295. 12. Levinson RD, Park MS, Rikkers SM, Reed EF, Smith JR, Martin TM, Rosenbaum JT, Foster CS, Sherman MD, Holland GN. Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome. Invest Ophthalmol Vis Sci 44:653, 2003. 13. Rao NA. Mechanisms of inflammatory response in sympathetic ophthalmia and VKH syndrome. Eye 11:213, 1997. 14. Kilmartin DJ, Wilson D, Liversidge J, Dick AD, Bruce J, Acheson RW, Urbaniak SJ, Forrester JV. Immunogenetics and clinical phenotype of sympathetic ophthalmia in British and Irish patients. Br J Ophthalmol 85:281, 2001. 15. Goldberg AC, Yamamoto JH, Chiarella JM, Marin ML, Sibinelli M, Neufeld R, Hirata CE, Olivalves E, Kalil J. HLA-DRB1*0405 is the predominant allele in Brazilian patients with Vogt-Koyanagi-Harada disease. Hum Immunol 59:183, 1998. 16. Ollier W, Thomson W. Population genetics of rheumatoid arthritis. Rheum Dis Clin North Am 18:741, 1992. 17. Marsh SGE, Parham P, Barber LD. The HLA Factsbook. San Diego, CA, Academic Press, 2000:332.
1482
18. Buckner JH, Nepom GT. Genetics of rheumatoid arthritis. is there a scientific explanation for the human leukocyte antigen association? Curr Opin Rheumatol 14:254, 2002. 19. Southwood S, Sidney J, Kondo A, del Guercio MF, Appella E, Hoffman S, Kubo RT, Chesnut RW, Grey HM, Sette A. Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol 160:3363, 1998. 20. Rosloniec EF, Whittington KB, Zaller DM, Kang AH. HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen. J Immunol 168:253, 2002.
R. Levinson et al.
21. Hennecke J, Wiley DC. Structure of a complex of the human alpha/beta T cell receptor (TCR) HA1.7, influenza hemagglutinin peptide, and major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRB1*0401): insight into TCR cross-restriction and alloreactivity. J Exp Med 195:571, 2002. 22. Neveu R, Auriault C, Angyalosi G, Georges B. Evidences of conformational changes in class II major histocompatibility complex molecules that affect the immunogenicity. Mol Immunol 38:661, 2002. 23. Snijders A, Elferink DG, Geluk A, Der Zanden AL, Vos K, Schreuder GM, Breedveld FC, de Vries RR, Zanelli EH. An HLA-DRB1-derived peptide associated with protection against rheumatoid arthritis is naturally processed by human APCs. J Immunol 166:4987, 2002.
INTRODUCTION Vogt-Koyanagi-Harada’s (VKH) disease, a bilateral granulomatous panuveitis that also may affect the skin, meninges, and auditory system [1], is known to be strongly associated with the human leukocyte antigen
From the Ocular Inflammatory Disease Center, Jules Stein Eye Institute and Department of Ophthalmology (R.D.L., G.N.H.), Los Angeles; the UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine (R.R., E.F.R., M.S.P.), David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles; and the University of Southern California Keck School of Medicine, Los Angeles, CA, USA (R.F.S., N.A.R.). Address reprint requests to: Dr. Ralph D. Levinson, Jules Stein Eye Institute, 100 Stein Plaza, UCLA, Los Angeles, CA 90095-7002; Tel: (310) 794-7770; Fax: (310) 794-7906; E-mail: [email protected]. This research was supported in part by The MacDonald Family Foundation, Los Angeles, CA (R.D.L.); Research to Prevent Blindness, Inc, New York, NY (G.N.H.); and the David May II Endowed Professorship (G.N.H.). Dr. Holland is recipient of a Research to Prevent Blindness Physician-Scientist Award. Received April 1, 2004; revised July 8, 2004; accepted July 9, 2004. Human Immunology 65, 1477–1482 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
roni correction. Three patients had the HLA-DRB1*0410 allele; this allele was not found in controls. All HLADRB1*01 positive patients had the DRB1*0102 subtype. No HLA-DQB1 allele was significantly increased compared to controls. This study is the first to identify a possible association between HLA-DRB1*0404 and VKH disease, as well as to find DRB1*0102 and DRB1*0410 in Mestizo patients. Human Immunology 65, 1477–1482 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: HLA; immunogenetics; uveitis; VogtKoyanagi-Harada-disease; mestizo
UCLA VKH
University of California at Los Angeles Vogt-Koyangi-Harada
(HLA)-DR4 phenotype in Japanese patients [2]. A study looking at HLA associations with VKH disease in mestizo individuals in Southern California found either HLA-DR1 or HLA-DR4 in 84% of patients by means of serologic techniques [3]. Indeed, HLA-DR1 had a higher relative risk than HLA-DR4 (4.11 vs 1.96, respectively) in this population [3]. In contrast, although both HLADR1 and -DR4 serotypes were also found in mestizo patients with VKH disease in a study from Mexico City, only HLA-DR4 was significantly increased compared with controls [4]. Using DNA-based techniques to look at HLA alleles rather than HLA serotypes in the same population, HLA-DRB1*0101 was associated with disease and HLA-DRB1*01 or HLA-DRB1*04 subtypes were found in 89% [5]. Furthermore, while HLADRB1*0405 and HLA-DRB1*0410 alleles predominated in Japanese [6] and Korean [7] patients with VKH disease, no specific HLA-DRB1*04 alleles were associ0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.07.236
1478
ated with VKH disease among mestizo patients in the Mexico City study [5]. The differences between studies imply that the strength of the HLA association may differ between populations and may in part depend on whether serologic or DNA-based techniques are used. In the current study, we have expanded on the previous report of HLA types in mestizo patients with VKH disease in Southern California using DNA-based techniques to evaluate the HLA alleles that confer risk in this patient population. PATIENTS AND METHODS We evaluated 29 patients with VKH disease who volunteered for the study and who had not previously had HLA typing. Subjects were recruited from two institutions: the Jules Stein Eye Institute, David Geffen School of Medicine at UCLA (Los Angeles, CA) and the Doheny Eye Institute, University of Southern California (USC) Keck School of Medicine (Los Angeles, CA). This study was approved by the UCLA and USC institutional review boards and maintained strict adherence to the Declaration of Helsinki for research involving human subjects. Written informed consent was obtained from all participants prior to study enrollment. Medical records of patients participating in the study were reviewed to confirm the diagnosis of VKH disease, based on standardized criteria [1]. Patients were considered to be mestizo if they were self-identified as Hispanic and had at least one parent from Mexico or Central America. While self-identification is not an ideal method for classifying individuals on the basis of race [8], it has been used previously in studies of HLA gene frequencies, including the National Marrow Donor Program [9]. This approach allowed us to establish a range of HLA alleles associated with VKH disease in non-Asian population, and enabled us to compare our results with other populations of mestizo patients with VKH disease [4, 5]. All HLA typing was performed at the UCLA Immunogenetics Center. DNA was isolated from peripheral blood leukocytes using the Qiagen extraction method (Qiagen Inc., Valencia, CA, USA) and typed for HLA class II gene polymorphisms by a polymerase chain reaction (PCR) technique followed by hybridization with sequence-specific oligonucleotide probes, as published elsewhere [10]. The HLA-DRB1*01, HLA-DRB1*04, and HLA-DQB1 subtypes were further characterized at the allele level using sequence-specific primers (One Lambda, Canoga Park, CA, USA). Because it has been demonstrated that VKH disease is associated with HLADR1 and HLA-DR4 in previous studies [3–5], highresolution typing was limited to HLA-DRB1*01 and HLA-DRB1*04 alleles. Gene frequencies for HLA-DR and HLA-DQ alleles
R. Levinson et al.
found in patients with VKH disease in the current study were compared with mestizo individuals living in Southern California. Seventy-three mestizo individuals in Southern California were originally typed at the UCLA Immunogenetics Center as standard controls for DNA typing methods for HLA polymorphisms. Comparisons were made using a two-sided Fisher’s exact test. Because multiple comparisons were made in the evaluation of individual HLA-DR and HLA-DQ alleles, a simple Bonferroni’s correction was performed, and a p value ⬍ 0.005 was chosen as the threshold for statistical significance. Because previous studies had indicated a high prevalence of HLADR1 and HLA-DR4 genes in mestizo patients with VKH disease [3–5], combined data for HLA-DR1 and HLADR4 alleles were evaluated as a global measure for comparisons; as such, no Bonferroni’s correction was necessary for this evaluation, and a p value ⬍ 0.05 was chosen as the threshold for statistical significance. RESULTS All 29 patients had either HLA-DRB1*01, HLADRB1*04, HLA-DQB1*03, or HLA-DQB1*05 genes, or a combination of these genes (Table 1). Table 2 shows gene frequencies for study subjects and for the Southern California mestizo control population. There were no significant differences in HLA gene frequencies between the mestizo control group from Southern California and a previously published Mexican mestizo population [11] (data not shown). Either HLA-DRB1*01, HLADRB1*04 alleles, or both were present in 27 patients (93.1%); the gene frequency for combined HLA-DR1 and HLA-DR4 alleles was greater than that for controls (p ⫽ 0.0006, odds ratio [OR] ⫽ 3.17). All patients who were HLA-DR1–positive had the HLA-DRB1*0102 subtype (p ⫽ 0.01, OR ⫽ 4.60; not significant after Bonferroni’s correction). HLA-DRB1*04 alleles were found in 22 patients (75.9%); the gene frequency of combined HLA-DR4 alleles was increased when compared with controls (p ⫽ 0.005, OR ⫽ 2.60). Three patients were homozygous for HLA-DRB1*04 alleles. Only one HLA-DRB1*04 allele was found for three patients; it is not known if they were homozygous for that allele; for all evaluations it was considered one allele. Gene frequencies for HLA-DRB1*0404 (p ⫽ 0.035, OR 3.23), HLA-DRB1*0407 (p ⫽ 0.028, OR ⫽ 3.23) and HLA-DRB1*0410 (p ⫽ 0.022) were increased compared with controls, but these were not significant after Bonferroni’s correction. Because HLA-DRB1*0410 was not found in mestizo controls from either Southern California or a previously published control population [11], an OR could not be calculated. Patients in the current study had a higher frequency of HLA-DRB1*0102, HLADRB1*0404, and HLA-DRB1*0410 than previously re-
HLA in VKH Disease in Mestizos in California
1479
TABLE 1 HLA subtypes in mestizo patients with VKH disease in Southern California Patient
HLA-DRB1
HLA-DQB1
1 2 3 4 5 6 7 8 9* 10† 11 12 13 14 15* 16* 17† 18 19 20 21 22 23 24 25 26† 27 28 29
0102,07 0102,08 0102,08 0102,11 0102,13 0102, 0404 0102, 0407 0102, 0407 04031, 0407 0404‡ 0404‡ 0404, 0701 0404, 08 0404, 13 0404, 0406 0404, 0407 0405, 1501 04051, 15021 0407‡ 0407, 08 0407, 0802 0407, 1104 0407, 14 0407, 1406 0410, 03 0410, 14 0410, 1402 0301, 10 08021, 1402
0303, 0501 0301/13, 0501 0402, 0501 0301/13, 0501 0303, 0501 0302, 0501 0302, 0501 0302, 0501 0302, 0202 — 0302, 0202 0302, 0202 0302, 0402 0402, 0603 0302, 0402 0302‡ — 0302, 0601 0302, 0402 0302, 0402 0302, 0402 0302, 0301 0302, 0503 0302, 0301 0302, 0201 — 0302, 0301 0202, 0501 0301/13, 0402
High-resolution typing performed only for HLA-DRB1*01, HLA-DRB1*04, and HLA-DQB1 alleles. Abbreviation: VKH ⫽ Vogt-Koyanagi-Havada. * Homozygous for HLA-DRB1*04 alleles. † HLA-DQB1 typing was not obtained for these subjects. ‡ Only one allele was found; it cannot be determined if the subject was homozygous or heterozygous for the HLA subtype.
ported Mexican mestizo patients with VKH disease [5], but the differences were not statistically significant (data not shown). Although 25 (96.2%) of 26 patients had either HLADQB1*03 or HLA-DQB1*05 genes, no HLA-DQB1 gene was found to be significantly more frequent in study subjects than in controls (Table 2). DISCUSSION We found HLA-DRB1*04 to be associated with VKH disease in mestizo patients living in Southern California. There was also an increased frequency of HLA-DRB1*01 and HLA-DRB1*04 alleles combined, but this was primarily from the increase in HLA-DRB1*04. This is consistent with previous studies that reported either HLA-DR1 or HLA-DR4 to be associated with VKH disease in mestizo patients [3–5].
Although the frequency of HLA-DRB1*0102 allele was increased compared with controls, it did not remain significant after Bonferroni’s correction for multiple comparisons. HLA-DRB1*0102 is of interest because it is strongly associated with tubulointerstitial nephritis and uveitis syndrome, another disease often complicated by chronic and recurrent anterior uveitis [12]. Although no specific HLA-DRB1*04 subtype was increased when compared with controls after Bonferroni’s correction for multiple comparisons, there was a suggestion that HLA-DRB1*0404, HLA-DRB1*0407, and HLA-DRB1*0410 may be associated with disease in our patients. HLA-DRB1*0410 has been associated with VKH disease in Asian patients, but not previously in mestizo patients [5–7]. Although the association with HLA-DRB1*0410 lost statistical significance after Bonferroni’s correction, no individual in two mestizo control populations had that allele, suggesting that it may indeed be associated with VKH disease in our patients. Mestizo populations have genes derived from Caucasian as well as Native American and black ancestors. In light of the clinical and pathologic similarities between sympathetic ophthalmia and VKH disease [13], it is interesting to compare the specific HLA-DR subtypes associated with these diseases in different populations. Similar to the findings in our mestizo patients with VKH disease, HLADRB1*0404 was associated with sympathetic ophthalmia in Caucasians, whereas in Japanese patients HLADRB1*0405 has been associated with both VKH disease and sympathetic ophthalmia [6, 7, 14]. Because no HLA-DRB1*01 or HLA-DRB1*04 allele predominated with a particularly large OR in either our study or a previous study of mestizo patients with VKH disease from Mexico City [5], it is likely that a range of HLA-DR1 and HLA-DR4 alleles may act as markers for disease risk. It has been suggested that HLADRB1*0405 has specific peptide-binding characteristics for residues 4 and 9 that may confer particular risk for the development of VKH disease [15]. In fact, the HLA associations with VKH disease in mestizo individuals are similar to those described in patients with rheumatoid arthritis, which is also associated with both HLA-DR1 and HLA-DR4. Interestingly, as appears to be the case for VKH disease and sympathetic ophthalmia, HLADRB1*0404 was associated with rheumatoid arthritis in Caucasians, whereas HLA-DRB1*0405 was found to be associated with rheumatoid arthritis in Asians [16]. It is possible that different HLA types could play a role in disease pathogenesis by presenting similar immunogenic peptides to T cells. The HLA-DR1 and HLADR4 molecules that are associated with VKH disease in mestizo patients and in patients with rheumatoid arthritis share the amino acid sequence glutamine, arginine, arginine, alanine in positions 70 to 73 of the HLA-DR
1480
R. Levinson et al.
TABLE 2 Gene frequencies of HLA-DRB1 and HLA-DQB1 alleles in mestizo patients with VKH disease versus controls Study subjects (n ⫽ 29)
Allele
Number of patients
Number of genes*
HLA DR DR1 DRB1*0101 DRB1*0102 DR4 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0406 DRB1*0407 DRB1*0410 DRB1*0411 DR1 or DR4 HLA DQ DQ5 DQB1*0501 DQB1*0502 DQB1*0503 DQ3 DQB1*0301 DQB1*0302 DQB1*0303
29 8 0 8 22 0 1 8 2 1 10 3 0 27 26 10 9 0 1 23 6 18 2
55 8 0 8 25 0 1 8 2 1 10 3 0 33 51 10 9 0 1 26 6 18 2
Controls (n ⫽ 73)
GF (%)
Number of individuals tested
Number of genes†
GF (%)
p value‡
OR
— 14.5 — 14.5 45.5 — 1.8 14.5 3.6 1.8 18.2 5.5 — 60.0 — 19.6 17.6 — 2.0 51.0 11.8 35.3 3.9
73 11 4 5 30 1 7 7 4 0 9 0 6 37 73 13 11 2 1 58 33 29 2
140 11 4 5 34 1 7 7 4 0 9 0 6 45 133 14 11 2 1 64 33 29 2
— 7.9 2.9 3.6 24.3 0.7 5.0 5.0 2.9 — 6.4 — 4.3 32.1 — 10.5 8.3 1.5 0.8 48.1 24.8 21.8 1.5
— 0.182 0.579 0.010 0.005 1.000 0.445 0.035 0.675 0.282 0.028 0.022 0.187 0.0006 — 0.140 0.109 1.000 0.479 0.745 0.069 0.088 0.308
— 2.00 — 4.60 2.60 — 0.35 3.23 1.28 — 3.23 — — 3.17 — 2.07 2.38 — 2.64 1.12 0.40 1.96 2.67
Abbreviations: GF ⫽ gene frequency (the proportion of the given gene in the population studied, given as a percentage); HLA ⫽ human leukocyte antigen; OR ⫽ odds ratio; VKH ⫽ Vogt-Koyahagi-Harada. * Number of alleles with the HLA type in study subjects. † Number of alleles with the HLA type in individuals from the control population. In some cases, there are more genes than subjects for a given HLA type because some individuals were homozygous. ‡ Comparing GF in patients with a control group consisting of mestizo individuals living in Southern California using two-sided Fisher’s exact tests.
molecule; HLA-DRB1*0407 has a glutamate in position 74, whereas the other HLA-DR subtypes in our patients have an alanine in position 74 [17], implying that this difference in amino acids in position 74 is not critical. These and related HLA amino acid sequences have been termed “shared epitopes” and are thought to confer risk for the development of rheumatoid arthritis, as reviewed elsewhere [18]. Furthermore, peptide-binding repertoires are shared by HLA-DR1 and HLA-DR4, again calling into question whether the unique binding characteristics of HLA-DRB1*0405 are critical for the development of VKH disease [19]. Binding of an immunodominant peptide of type II collagen [20] and an epitope of the influenza hemagglutinin peptide [21] to both HLA-DR1 and HLA-DR4 molecules has been examined by x-ray crystallography. The same anchor residues were found to be involved in the presentation of antigens by both HLA-DR1 and HLA-DR4 molecules, with only a slight conformational difference noted when the same peptide was bound to either HLA-DR1 or
HLA-DR4. Although slight conformational changes could alter immunogenicity [22], thereby resulting in different pathogenic potential for peptides bound by different HLA subtypes, the observation that HLA-DR1 and HLA-DR4 molecules could present the same immunogenic peptides is consistent with both HLA-DR1 and HLA-DR4 subtypes having a role in the immunopathogenesis of VKH disease. There may also be a role for HLA-DQ genes in the pathogenesis of VKH disease. We found either HLADQB1*03 or HLA-DQB1*05 alleles in all but one patient tested for HLA-DQ alleles. The one patient without these HLA-DQ alleles had the HLA-DQB1*04 allele, which has been associated with VKH disease in Asian VKH patients [6, 7]. In addition, two patients in the current study did not have either the HLA-DR1 or the HLA-DR4 genotype; one had an HLA-DQB1*03 allele and the other an HLADQB1*05 allele. The lack of a statistically significant association between VKH disease and any specific DQB1 gene in our patients may be a result of the range of
HLA in VKH Disease in Mestizos in California
HLA-DQ3 and HLA-DQ5 alleles that are able to play a role in disease pathogenesis. It cannot be determined from the current data whether the HLA-DQB1 genes may make a unique contribution to disease risk or, rather, are in linkage disequilibrium with critical HLA-DR genes or other genes on chromosome 6 that may play a role in the immune response. In tubulointerstitial nephritis and uveitis syndrome, different HLA-DR associations found could be explained by linkage disequilibrium to HLA-DQ genes [12]. Interactions between HLA-DR and HLA-DQ molecules may also be important; for example, there is evidence from in vitro studies that peptides derived from the HLA-DR glycoprotein itself can be presented by HLA-DQ molecules [23]. In conclusion, we found that HLA-DR4 confers modest risk for VKH disease in mestizo individuals without a strong association with any specific subtype. This provides further evidence that multiple HLA-DR alleles confer risk for VKH disease. The observation that HLA-DRB1*0404 is associated with sympathetic ophthalmia and rheumatoid arthritis in Caucasians, and HLA-DRB1*0405 confers risk for these diseases in Asians, is further evidence for the possibility that these different HLA subtypes may play a similar role in the pathogenesis of VKH disease in different populations. A better understanding of the similarities, as well as the differences, in the immunogenetics and pathogenesis of these diseases has important implications for further research and the design of novel therapies. Examining HLA associations with disease in diverse populations is important for evaluating the range of HLA subtypes that may contribute to disease risk and provides additional insights into possible disease mechanisms. REFERENCES 1. Read RW, Holland GN, Rao NA, Tabbara KF, Ohno S, Arellanes-Garcia L, Pivetti-Pezzi P, Tessler HH, Usui M. Revised diagnostic criteria for Vogt-Koyanagi-Harada disease: report of an international committee on nomenclature. Am J Ophthalmol 131:647, 2001. 2. Ohno S. Immunological aspects of Behcet’s and VogtKoyanagi-Harada’s diseases. Trans Ophthalmol Soc U K 101:335, 1981. 3. Weisz JM, Holland GN, Roer LN, Park MS, Yuge AJ, Moorthy RS, Forster DJ, Rao NA, Terasaki PI. Association between Vogt-Koyanagi-Harada syndrome and HLA-DR1 and -DR4 in Hispanic patients living in southern California. Ophthalmology 102:1012, 1995. 4. Arellanes-Garcia L, Bautista N, Mora P, Ortega-Larrocea G, Burguet A, Gorodezky C. HLA-DR is strongly associated with Vogt-Koyanagi-Harada disease in Mexican Mestizo patients. Ocul Immunol Inflamm 6:93, 1998.
1481
5. Alaez C, del Pilar MM, Arellanes L, Cano S, Perez-Luque E, Vazquez MN, Olivo A, Burguete A, Hernandez A, Pedroza M, Gorodezky C. Strong association of HLA class II sequences in Mexicans with Vogt-Koyanagi-Harada’s disease. Hum Immunol 60:875,1999 6. Shindo Y, Ohno S, Yamamoto T, Nakamura S, Inoko H. Complete association of the HLA-DRB1*04 and -DQB1*04 alleles with Vogt-Koyanagi-Harada’s disease. Hum Immunol 39:169, 1994. 7. Kim MH, Seong MC, Kwak NH, Yoo JS, Huh W, Kim TG, Han H. Association of HLA with Vogt-KoyanagiHarada syndrome in Koreans. Am J Ophthalmol 129:173, 2000. 8. Kaplan JB, Bennett T. Use of race and ethnicity in biomedical publication. JAMA 289:2709, 2003. 9. Cao K, Hollenbach J, Shi X, Shi W, Chopek M, FernandezVina MA. Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations. Hum Immunol 62:1009, 2001. 10. Erlich H, Bugawan T, Begovich AB, Scharf S, Griffith R, Saiki R, Higuchi R, Walsh PS. HLA-DR, DQ and DP typing using PCR amplification and immobilized probes. Eur J Immunogenet 18:33,1991 11. Olivo A, Alaez C, Debaz H, Vazquez M, de la Rosa G, Perez Luque E, Hernandez A, Gorodezky C. Mexican mestizo normal. In Terasaki PI, Gjertson DW (Eds): HLA 1997. Los Angeles, CA: UCLA Tissue Typing Laboratory, 1997:295. 12. Levinson RD, Park MS, Rikkers SM, Reed EF, Smith JR, Martin TM, Rosenbaum JT, Foster CS, Sherman MD, Holland GN. Strong associations between specific HLA-DQ and HLA-DR alleles and the tubulointerstitial nephritis and uveitis syndrome. Invest Ophthalmol Vis Sci 44:653, 2003. 13. Rao NA. Mechanisms of inflammatory response in sympathetic ophthalmia and VKH syndrome. Eye 11:213, 1997. 14. Kilmartin DJ, Wilson D, Liversidge J, Dick AD, Bruce J, Acheson RW, Urbaniak SJ, Forrester JV. Immunogenetics and clinical phenotype of sympathetic ophthalmia in British and Irish patients. Br J Ophthalmol 85:281, 2001. 15. Goldberg AC, Yamamoto JH, Chiarella JM, Marin ML, Sibinelli M, Neufeld R, Hirata CE, Olivalves E, Kalil J. HLA-DRB1*0405 is the predominant allele in Brazilian patients with Vogt-Koyanagi-Harada disease. Hum Immunol 59:183, 1998. 16. Ollier W, Thomson W. Population genetics of rheumatoid arthritis. Rheum Dis Clin North Am 18:741, 1992. 17. Marsh SGE, Parham P, Barber LD. The HLA Factsbook. San Diego, CA, Academic Press, 2000:332.
1482
18. Buckner JH, Nepom GT. Genetics of rheumatoid arthritis. is there a scientific explanation for the human leukocyte antigen association? Curr Opin Rheumatol 14:254, 2002. 19. Southwood S, Sidney J, Kondo A, del Guercio MF, Appella E, Hoffman S, Kubo RT, Chesnut RW, Grey HM, Sette A. Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol 160:3363, 1998. 20. Rosloniec EF, Whittington KB, Zaller DM, Kang AH. HLA-DR1 (DRB1*0101) and DR4 (DRB1*0401) use the same anchor residues for binding an immunodominant peptide derived from human type II collagen. J Immunol 168:253, 2002.
R. Levinson et al.
21. Hennecke J, Wiley DC. Structure of a complex of the human alpha/beta T cell receptor (TCR) HA1.7, influenza hemagglutinin peptide, and major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRB1*0401): insight into TCR cross-restriction and alloreactivity. J Exp Med 195:571, 2002. 22. Neveu R, Auriault C, Angyalosi G, Georges B. Evidences of conformational changes in class II major histocompatibility complex molecules that affect the immunogenicity. Mol Immunol 38:661, 2002. 23. Snijders A, Elferink DG, Geluk A, Der Zanden AL, Vos K, Schreuder GM, Breedveld FC, de Vries RR, Zanelli EH. An HLA-DRB1-derived peptide associated with protection against rheumatoid arthritis is naturally processed by human APCs. J Immunol 166:4987, 2002.