The Journal of Immunology Redundancy in Antigen-Presenting Function of the HLA-DR and -DQ Molecules in the Multiple Sclerosis-Associated HLA-DR2 Haplotype1 Mireia Sospedra,2* Paolo A. Muraro,* Irena Stefanova´,† Yingdong Zhao,‡ Katherine Chung,* Yili Li,§ Marc Giulianotti,¶ Richard Simon,‡ Roy Mariuzza,§ Clemencia Pinilla,¶ʈ and Roland Martin3* The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequi- librium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4؉ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction. The Journal of Immunology, 2006, 176: 1951–1961. ultiple sclerosis (MS)4 is considered a T cell-mediated fect, inbreeding, and admixture, as well as gene-specific factors autoimmune disease of the CNS, and both genetic and such as rates of mutation and recombination, and also natural se- M environmental factors contribute to disease expres- lection (2–4). The last one is particularly pertinent to explain al- sion. The HLA-DR2 haplotype, which is dominant in Caucasian lelic associations over large genetic distances and brings the pos- MS populations, confers most of the genetic risk for MS (1). Three sibility that coexpression of HLA class II loci in the DR2 HLA class II molecules are expressed in this haplotype in strong haplotype can provide a survival advantage via efficient immune linkage disequilibrium: DRB1*1501 (DR2b), DRB5*0101 responses to common infections; but at the same time, it might (DR2a), and DQA1*0102/DQB1*0602 (DQw6). Levels and pat- increase the risk for MS. terns of linkage disequilibrium depend on several demographic Our current understanding as to how the presence of certain factors including population size, population structure, founder ef- HLA class II molecules confers susceptibility to autoimmune dis- eases is limited, and several mechanisms have been considered. Disease-associated HLA-DR and -DQ molecules could possess binding grooves that lead to preferential presentation of specific *Cellular Immunology Section, Neuroimmunology Branch, National Institute of Neu- rological Disorders and Stroke, and †Lymphocyte Biology Section, Laboratory of sets of self peptides (e.g., myelin peptides in MS) (5), but com- Immunology, National Institute of Allergy and Infectious Diseases, National Insti- parisons of polymorphic residues in the HLA-DR and DQ binding ‡ tutes of Health, Bethesda, MD 20892; Computational and System Biology Group, pockets of MS-associated DR molecules have been inconclusive Biometric Research Branch, National Cancer Institute, National Institutes of Health, Rockville, MD 20852; §Center for Advanced Research in Biotechnology, University (6, 7). As a variation of the above, it was speculated that disease- of Maryland, Rockville, MD 20850; and ¶Mixture Sciences and ʈTorrey Pines Insti- associated HLA class II molecules could have binding character- tute for Molecular Studies, San Diego, CA 92121 istics that allow only a limited number of peptides to bind, leading Received for publication June 9, 2005. Accepted for publication November 2, 2005. to incomplete thymic negative selection (8). Diabetes-prone NOD The costs of publication of this article were defrayed in part by the payment of page mice are considered an example for this situation (9). As another charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. possibility, polymorphic residues of the TCR-exposed surfaces of ␣ ␣ ␤ 1 This work was supported in part by the Multiple Sclerosis National Research In- the -helical regions of DR/DQ- - and - -chains such as the stitute (to C.P.). M.S. was a recipient of a National Institute of Neurological Disorders shared motif in rheumatoid arthritis-associated class II molecules and Stroke Fellowship. could select autoreactive T cells (10). 2 Current address: Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Unitat de Neu- The ability of some TCRs to use different restriction elements, roimmunologia Clinica, Hospital Universitari Vall d’Hebron, Pg Vall d’Hebron 119- 129, 08035 Barcelona, Spain. promiscuous restriction, has been demonstrated previously (11– 3 Address correspondence and reprint requests to Dr. Roland Martin at the current 16) and likely plays a role in improving responses to infectious address: Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Unitat de Neuroimmu- agents. During an infection, T cell recognition of the same viral pep- nologia Clinica, Hospital Universitari Vall d’Hebron, Pg Vall d’Hebron 119-129, tide by one TCR in the context of two or more HLA molecules could 08035 Barcelona, Spain. E-mail address: [email protected] assure more efficient activation if both or all HLA molecules are ex- 4 Abbreviations used in this paper: MS, multiple sclerosis; DR2b, DRB1*1501; DR2a, DRB5*0101; DQw6, DQA1*0102/DQB1*0602; PS-SCL, positional scanning pressed. Under conditions in which expression of one restriction el- synthetic combinatorial peptide library; TCC, T cell clone; CSF, cerebrospinal fluid; ement is limited (e.g., the brain), promiscuous restriction could pro- RR-MS, relapsing remitting MS; SI PS-SCL, stimulation index to the PS-SCL; SI, stimulation index to peptides; BLS, bare lymphocyte syndrome; hrIL-2, human re- vide a safeguard and would still allow T cell activation. Finally, combinant IL-2; AUC, area under the curve; MBP, myelin basic protein. recognition of different peptides on more than one restriction element Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 1952 REDUNDANT ANTIGEN PRESENTATION IN MULTIPLE SCLEROSIS may increase the possibilities for cross-reactivity. HLA class II mol- acterize the TCC by flow cytometry. T cell cultures were considered for ϩ ecules with structures that allow their recognition by a single TCR and further analysis if Ͼ97% of the CD4 T cells stained with a single TCRBV are able to present identical peptides to single T cells would be mAb. In the case in which none of the 22 TCRBV Abs stained the cells, probably consistent with the presence of a monoclonal or oligoclonal pop- optimal in such a scenario. Coexpression of these molecules could ulation not covered by the Ab panel, TCRBV expression was assessed by guarantee more efficient immune responses against infectious or- PCR (see below). ganisms, but it may, at the same time, increase the risk for auto- RT-PCR and sequencing of TCR rearrangements immunity. The dose effect demonstrated in genetic studies in which it was shown that homozygosity for the DR2 haplotype TCC TCRV gene usage was analyzed by PCR using 21 TCRAV and 23 increases MS risk (17) and the evidence that the DQw6 molecule, TCRBV family-specific oligonucleotide primers (24). Nucleotide sequenc- ing of PCR products was performed as described previously (23). V␤ usage together with DR2a and DR2b or independently, contributes to MS designations are in accord with Arden’s nomenclature (25), and TCR gene susceptibility in some MS populations (18, 19) support this hy- is in accord with IMGT (ImMunoGeneTics database) (26). pothesis. In this study, we examined whether autoreactive T cells CDR3 spectratyping can recognize peptides with several or all MS-associated HLA- DR/DQ molecules and particularly whether such promiscuous re- To assure clonality and assess in vivo clonal expansion, high-resolution striction occurs with identical peptides. TCR ␤-chain CDR3 spectratyping was performed. PCR product (2.5 ␮l) from each TCRBV was used as a template in a 12.5-␮l primer-extension An unbiased strategy to study promiscuous restriction of T cell (runoff) reaction containing 1.25 ␮lof5ЈFAM-labeled BV primer, 0.25 ␮l recognition is testing positional scanning synthetic combinatorial of 10 mM dNTP, 0.06 ␮l/PFU DNA polymerase, 1.25 ␮l/PFU reaction ␮ peptide libraries (PS-SCLs) with B cell lines expressing only a buffer, and 7.2 lofH2O. After thermal cycling (95°C for 2 min, followed single class II restriction element as an APC. Decapeptide PS- by 10 cycles of 94°C for 20 s, 55°C for 45 s, and 72°C for 45 s, and a final ␮ SCLs, containing 6 trillion (1910) peptides, represent all possible extension of 72°C for 10 min), 2 l of runoff product were mixed with loading buffer containing four Cy-5-labeled DNA size markers, heat- peptides of a given length in systematically arranged mixtures treated at 80°C for 2 min and run on a 6% polyacrylamide gel on an (20). The recognition of these mixtures by T cell clones (TCCs) OpenGene (Visible Genetics) sequencer. Electropherograms were analyzed suggests that many peptides can contribute to the T cell response, for peak size (bp), peak height, and area under the curve (AUC). The because single-peptide species are present at only extremely low percentage represented by each CDR3 peak in a BV spectrum (correspond- Ϫ10 ␮ 8 ing to the representation of clonal populations with a given CDR3 length) concentrations, 10 M, that is 10 -fold lower than typically ϭ ⌺ ϩ was calculated according to the formula %AUC BVn (AUC BVn/ AUC required for CD4 TCC activation.