... Chang CC, Capra JD: Allelic polymorphism and transassociation of molecules encoded by the HLA-DQ subregion. Proc. Natl Acad Sci USA 82:1776-80, 1985.
Rapid Publications Transcomplementation of HLA Genes in IDDM HLA-DQ a- and (B-Chains Produce Hybrid Molecules in DR3/4 Heterozygotes BARBARA S. NEPOM, DAVID SCHWARZ, JERRY P. PALMER, AND GERALD T. NEPOM
SUMMARY The HLA association with insulin-dependent diabetes mellitus is highest among individuals heterozygous for DR3 and DR4. To investigate potential mechanisms to account for this association, we performed two-dimensional gel-electrophoretic analysis of HLA molecules from DR3/4 heterozygous patients. These studies demonstrated hybrid molecular dimers corresponding to products of HLA-DQ genes linked to DR3 and DR4, i.e., the DQw2 and DQw3 genes, respectively. Two types of OQ molecules were found: immunoprecipitation by DQw3-specific monoclonal antibody 17.15 identified a DQw3 p-chain associating with a DQw3 a-chain and a DQw3 p-chain associating with a DQw2 a-chain. The identity of a- and p-chains comprising these hybrid molecules was confirmed by HPLC peptide-map analysis. Several characteristic peptide peaks identified both DQw2 and DQw3 a-chains associated with DQw3 p-chains. The formation of such DQa(DQw2)DqP(DQw3) dimers potentially contributes a direct molecular mechanism for HLA-associated contributions to disease in DR3/DR4 heterozygotes. Diabetes 36:11417, 1987
T
he class II molecules of the human major histocompatibility region (HLA) are strongly associated with several autoimmune diseases (1). Several of these HLA-associated diseases show a dramatically increased relative risk for individuals possessing a particular heterozygous combination of class II antigens [e.g., DR3/4 in insulin-dependent diabetes mellitus (IDDM) (2) and Dw4/ Dw14 in seropositive juvenile rheumatoid arthritis (3)]. This
From Genetic Systems Corporation, Pacific Medical Center; Diabetes Research Center, Virginia Mason Research Center; and the Departments of Pediatrics, Medicine, and Pathology, University of Washington School of Medicine, Seattle, WA 98121. Address correspondence and reprint requests to Dr. Barbara Nepom, Genetic Systems, 3005 First Avenue, Seattle, WA 98121. Received for publication 15 September 1986 and accepted 29 September 1986.
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increased disease susceptibility may be related to an altered immune response resulting from the formation of hybrid class II molecules contributed from both haplotypes (3-6). We describe the identification of such hybrid molecules on cells from heterozygous DR3/4 IDDM patients, consisting of dimers containing products of genes from both DR3- and DR4associated haplotypes. Class II HLA molecules are dimers of a- and p-polypeptide chains encoded by separate genes and function as recognition signals for activation of immune responses. These HLA gene products include both DR and DQ molecules encoded in different but linked genes. DQ a- and p-molecules are polymorphic; i.e., the DQ a-chain linked to one DR gene, such as DR3, is different from the DQ a-chain linked to another, such as DR4, and the same is true for DQ (3chain. Thus, in a heterozygote, mixed ap-dimers potentially could form between DQ-chains from different haplotypes. Such transcomplementation of DQ a- and p-chains from opposite haplotypes would dramatically broaden the diversity of class II antigens available to participate in the immune response. The DR a-molecules are not polymorphic; i.e., DR a-molecules are invariant among haplotypes, and mixed DR ap-dimers would not result in novel HLA molecules. A DQ ap-dimer composed of transcomplementing chains would be unique to a heterozygous individual and not expressed by either parent. In the mouse, such transcomplementation has been demonstrated structurally, and epitopes newly formed on the resulting hybrid molecules allow for an altered functional immune response different from that of either parent (7,8). Recent structural studies suggest that similar transcomplementation can occur in humans, demonstrable by two-dimensional polyacrylamide gel electrophoresis (20PAGE) (9) and partial NH2-terminal protein sequencing (5). We report evidence for the existence of such hybrid DQmolecules among DR3/4 heterozygous IDDM patients, demonstrated by 2D-PAGE and confirmed by high-performance liquid chromatographic (HPLC) analysis. Furthermore, we examine HLA-identical siblings of IDDM patients to determine whether these hybrid molecules are unique to clinically affected individuals.
DIABETES, VOL. 36, JANUARY 1987
B. S. NEPOM AND ASSOCIATES
MATERIALS AND METHODS
Cells and Antibodies. B-lymphoblastoid cell lines were prepared by Epstein-Barr virus transformation of peripheral blood leukocytes from families of two HLA-DR3/4, DQw2/ w3 diabetic patients. Immunoprecipitations were performed with monoclonal antibody 17.15, a DQw3-specific antibody (10), and P17.1, and DQw2-reactive monoclonal antibody. Immunoprecipitations and 2D-PAGE. Cells were cultured and labeled with [3H]leucine as previously described (11). Membranes were extracted with NP-40 and then immunoprecipitated with Affigel-coupled antibodies (Bio-Rad, Richmond, CA) after preclearing with affigel-coupled bovine serum albumin. Sialic acid residues were removed by neuraminidase treatment (0.75 U/25x10 6 cell equivalents). 20PAGE was modified from the procedure of Shackelford et al. (12), separating the samples by molecular weight via sodium dodecyl sulfate-PAGE in the first dimension and by pi via isoelectric focusing in the second dimension, with a pH gradient formed by 1:1:1 proportions of 3.5-10, 4 - 6 , and 6-8 ampholytes. Samples for Fig. 1 were loaded at the anode and run for 10,000 volt-hours (V-h); samples for Fig. 3 were loaded at the cathode and run for 2200 V-h. Gels for autoradiography were dried, fluorographed, and exposed to Kodak XAR film for 3-7 days.
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FIG. 1. Electrophoretic analysis of HLA-DQ a- and p-chains. Radiolabeled HLA class II molecules were immunoprecipitated with DQreactive monoclonal antibodies and analyzed by 2-dimensional polyacrylamide electrophoresis. Acidic end of gel is on left, and basic end is on right, with higher molecular wieght at fop of each autoradiograph. A: parental DQw3-associated a- {large arrowhead) and p{small arrowhead) chains immunoprecipitated from cell line HA with monoclonal antibody 17.15. B: heterozygous diabetic cell line F1D (DR3/4, DQw2/w3) was immunoprecipitated with monoclonal antibody 17.15; only the DQw3 p-chain is seen, although both DQw2 and DQw3 a-chains coprecipitate. C: parental DQw2-associated a- (large arrow) and p- (small arrow) chains from cell F1F precipitated with monoclonal antibody P17.1. Antibody 17.15 does not react with DQw2 parental cell F1F(D).
DIABETES, VOL. 36, JANUARY 1987
1
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FIG. 2. Peptide map analysis of DQ a-chains purified by 2-dimensional gel electrophoresis. DQ a-chain spots were purified by 2-dimensional gel electrophoresis. DQ a-chain spots were recovered by elution from polyacrylamide gels (Fig. 1) and analyzed by reverse-phase HPLC. Tryptic peptides from purified a-chains were eluted from octadecyl silica column with ascending acetonitrile gradient (dashed line in A). Peptide maps from parental DQw3 and DQw2 a-chains are shown in A and C, respectively. Peptide maps from DQw3 and DQw2 a-chains precipitated from heterozygous diabetic cell line F1D with monoclonal antibody 17.15 are shown in B and D. Open arrow, peak at fraction 266 characteristic of DQw3 a-chains; closed arrow, peak at fraction 215 unique to DQw2 a-chains.
Tryptic peptide mapping. HPLC analysis was performed on gel-purified a-chains recovered from nonfluorographed gels. a-Chain spots to be analyzed were excised from the dried gel, rehydrated,'and eluted. Samples were then reduced with dithiothreitol, alkylated with 50 mM iodoacetamide, and digested with TPCK-trypsin (11). Precipitated peptides were analyzed by reverse-phase HPLC as previously described (11) on an IBM 9533 ternary gradient liquid chromatograph equipped with a 5-|xm, 4.5 x 250-mm octadecyl silica column. Samples were separated by a gradient elution of 2 70% CH3CN containing 0.08% trifluoracetic acid. Fractions were collected at 20-s intervals and the cycles per minute determined. RESULTS AND DISCUSSION
To investigate the question of transcomplementation resulting in hybrid molecule formation among heterozygous IDDM patients, we immunoprecipitated DQw3+ class II polypeptides and investigated the presence of DQw3/DQw2 hybrid dimers. DR and DQ genes are closely linked, such that
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TRANSCOMPLEMENTATION OF HLA GENES IN IDDM
DQw2 is associated with DR3, and DQw3 is associated with DR4. As previously reported with genomic restriction fragment analysis, specific DQ genetic polymorphisms correlate with a higher HLA-associated disease risk in IDDM than the DR markers themselves (13,14). Figure 1 shows 2D-PAGE analysis of HLA-DQ polypeptides; panel B shows a cell line from a DR3/4, DQw2/w3 diabetic patient, and panels A and C illustrate the electrophoretic patterns obtained with the parental specificities DQw3 and DQw2, respectively. The parental DQw2 a-chain in panel C is much more basic than the DQw3 a-chain in panel A. Similarly, the DQw2 (3chain spot is more basic than the DQw3 p-chain. We then investigated which polypeptide chains were immunoprecipitated by 17.15 in the DQw2/w3 heterozygous cell line F1D. If transcomplementation occurs, monoclonal antibody 17.15, although reactive only with DQw3, would immunoprecipitate polypeptides from both DQw2 and DQw3 in the heterozygote. In fact, this was demonstrated: panel B shows a 17.15 immunoprecipitate from F1D and reveals both the DQw2 and DQw3 a-chains. At the same time, only the DQw3 (3-chain is seen, confirming that the appeara i ~:e of two different DQ a-chains does not merely represent • oprecipitated class II antigens. This indicates that two types of molecules are formed—the DQw3 (3-chain associating with the DQw3 achain and a new hybird molecule formed by the DQw3 pchain associating with the DQw2 a-chain. Panel D confirms the specificity of 17.15 for DQw3, because no DQ a- or (3chains were precipitated with this monoclonal antibody from the DQw2 parental cell. To confirm the identity of the a-chain spots seen in Fig. 1, we performed HPLC analysis of [3H]leucine- and [35S]methionine-labeled a-chains from each cell line. Immunoprecipitates were run on 2D-PAGE as in Fig. 1; single polypeptide "spots" were eluted from the gel, digested with trypsin, and analyzed by reverse-phase HPLC. Representative chromatographs are shown in Fig. 2. The DQw3 a-chain peptide chromatograph from cell line HA is shown in panel A. Several 3 H-labeled peptides are visualized, and a peak at fraction 266 is unique to the DQw3 a-chain pattern. Panel C shows the pattern of the DQw2 a-chain after precipitation with antibody P17.1 from cell line F1F. In this case, a characteristic peak is seen at fraction 215, and the peak at 266 is absent.
Other peaks are similar between the two a-chains. To confirm that the a-chain spots from F1D immunoprecipitated with monoclonal antibody 17.15 in fact do represent the same achains as seen on the two parental haplotypes, we next performed HPLC on both a-chains from this heterozygous cell line. Panel B displays a chromatograph of the DQw3 aspots from F1D, showing the DQw3-associated peak at 266 and with a pattern similar to that in panel A. Similarly, panel D shows a chromatograph of the DQw2 spots from F1D, which shows an identical pattern to that of parental cell F1F. Peaks at approximately fractions 121 and 128 appear to be consistent markers for DQ a-chains from both haplotypes and distinguish these molecules from DR a-chains (data not shown). Such HPLC analyses confirm our interpretation of the 2D-PAGE data that DQ a-and p-dimers from different haplotypes produce hybrid molecules in DQw2/w3 heterozygous diabetic patients. Finally, we investigated whether this transcomplementation phenomenon is unique to diabetic patients or whether it occurs in genotypically identical nondiabetic siblings. Figure 3 shows autoradiographs from 2D-PAGE analysis of cell lines from HLA-identical siblings of two DQw2/w3 diabetic patients. In both families, immunoprecipitation with antibody 17.15 shows that both DQw2 and DQw3 a-chains are precipitated in all cases, indicating that transcomplementation occurs in these heterozygotes, regardless of clinical status. We therefore conclude that these hybrid molecules occur in nondiseased siblings as well, indicating that this may contribute to disease expression but that additional factors must also be involved. The existence of such DQ hybrid molecules in heterozygous diabetic patients supports the hypothesis that novel epitopes are formed that may control an immune response critical to disease expression, either by altering a normal immune response to an environmental agent or by allowing an aberrant immune response to a "self" antigen. Such novel determinants are potentially created by the association of polymorphic DQ a- and p-gene products from different haplotypes, present in heterozygous individuals as demonstrated here. The resulting class II dimer [DQ a (DQw2) and DQ p (DQw3)] forms an expressed HLA molecule not found on either parental haplotype.
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FIG. 3. Electrophoretic analysis of HLA-DQ molecules from siblings in 2 families of diabetic patients. Radiolabeled DQ a- and p-chains were immunoprecipitated with monoclonal antibody 17.15 (anti-DQw3) and analyzed by 2-dimensional gel electrophoresis as described for Fig. 1. A shows 3 siblings from 1 family, all are HLA-DR3/ 4, DQw2/w3. B shows another family of 3 genotypically identical siblings, all are HLA-DR3/4, DQw2/w3. Asterisks, siblings with IDDM. Large arrowhead, DQw3 a-chains; small arrowheads, DQw3 p-chains; arrows, DQw2 a-chains.
DIABETES, VOL. 36, JANUARY 1987
B. S. NEPOM AND ASSOCIATES
The evidence for transcomplementation in IDDM has been indirect. In nondisease states, evidence for transcomplementation comes from structural (5,9) and functional (15,16) studies, the latter demonstrating the restriction of T-lymphocyte clones specific for PPD (purified protein derivative) in the context of heterozygous stimulator cells, possibly with hybrid class II antigens. In disease states, evidence comes from the fact that the relative risk for IDDM is 5.8 for DR3/3 and 4.1 for DR4/4 but rises to 20.2 for the heterozygous combination DR3/4 (2). We demonstrate hybrid HLA molecules among diabetic patients whose disease is strongly associated with a heterozygous state and provide direct evidence for a mechanism potentially responsible for this dramatically increased susceptibility.
4. 5.
6. 7. 8. 9. 10. 11.
ACKNOWLEDGMENTS
We thank Holly Chase for manuscript preparation. This work was supported by Genetic Systems Corporation and Grants AM-37296, AM-17047, and AM-30780 from the National Institutes of Health.
12. 13. 14.
REFERENCES 1. Tiwari JL, Terasaki PI (eds): HLA and Disease Associations. New York, Springer-Verlag, 1985 2. Bertrams J, Baur MP: Insulin-dependent diabetes mellitus. In Histocompatibility Testing 1984. Albert ED, Baur MP, Mayr WR, Eds. Berlin, Springer-Verlag, 1984, 348-58 3. Nepom BS, Nepom GT, Mickelson E, Schaller JG, Antonelli P, Hansen JA: Specific HLA-DR4-associated histocompatibility molecules charac-
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terize patients with seropositive juvenile rheumatoid arthritis. J Clin Invest 74:287-91, 1984 Bodmer WF: The HLA system. In Histocompatibility Testing 1984. Albert ED, Baur MP, Mayr WR, Eds. Berlin, Springer-Verlag, 1984, p. 11-22 Giles RC, DeMars R, Chang CC, Capra JD: Allelic polymorphism and transassociation of molecules encoded by the HLA-DQ subregion. Proc Natl Acad Sci USA 82:1776-80, 1985 Svejgaard A, Ryder LP: HLA genotype distribution and genetic models of insulin-dependent diabetes mellitus. Ann Hum Genet 45:293-98, 1981 Fathman CG, Kimoto M, Melvold R, David C: Reconstitution of Ir genes, la antigens, and mixed lymphocyte reaction determinants by gene complementation. Proc Natl Acad Sci USA 78:1853-57, 1981 Lafuse W, McCormick J, Corser P, David C: Gene complementations to generate la antigens (la.23) on hybrid molecules. Transplantation 30:341-46, 1980 Charron DJ, Lotteau V, Turmel P: Hybrid HLA-DC antigens provide molecular evidence for gene trans-complementation. Nature (Lond) 132:157-59, 1984 Hansen JA, Martin P, Kamoun M, Nisperos B, Thomas ED: A supertypic HLA-DR specificity (DR4 + 5) defined by a murine monoclonal antibody. Hum Immunol 2:103-11, 1981 Seyfried CE, Gregerson P, Nepom BS, Nepom GT: Limited peptide diversity accounts for functional polymorphisms among HLA-DR4-positive DRp chains. Mol Immunol. In press Shackelford DA, Lampson LA, Strominger JL: Analysis of HLA-DR antigens by using monoclonal antibodies: recognition of conformational differences in biosynthetic intermediates. J Immunol 127:1403-10, 1981 Nepom BS, Palmer J, Kim SJ, Hansen JA, Holbeck SL, Nepom GT: Specific genomic markers for the HLA-DQ subregion discriminate between DR4-positive IDDM and DR4-positive JRA. J Exp Med 164:345-50, 1986 Kim SJ, Holbeck SL, Nisperos B, Hansen JA, Maeda H, Nepom GT: Identification of a polymorphic variant associated with HLA-DQw3 and characterized by specific restriction sites within the DQ p-chain gene. Proc Natl Acad Sci USA 82:8139-43, 1985 Gomard E, Henin Y, Sterkers G, Masset M, Fauchet R, Levy JP: An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes. J Immunol 136:3961-67, 1986 Hansen GS, Svejgaard A, Claesson MH: T cell clones restricted to "hybrid" HLA-D antigens? J Immunol 128:2497-99, 1982
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