To define the impact of major histocompatibility complex (MHC)-encoded glycoproteins on the selection of the T-cell receptor repertoire, we have determined the frequency with which T-cell receptor variable region (V. and Vat) genes are expressed in T cells from MHC disparate mice. Approximately 500 T-cell hybridomas were generated from each of three strains of MIIC congenic mice [B1O (H-2b), (H-2b), three B10.BR (H-2k), and five B10.Q (H-2q)] were separately cultured for 2 days in the presence of Con A (3 jug/ml) and separately fused to the thymoma BW5147 by standard methods (19). All fusions were plated at a low density such that >90% of the hybridomas were clonal. Hybridomas expressing >2 Va, or >2 V,3 genes were assumed to be nonclonal and were not considered into the data base. These fusions generated 555, 583, and 491 hybrids from B10, B1O.BR, and B1O.Q Con A blasts, respectively. Cells (107) from each hybrid were grown, washed in balanced salt solution, and frozen for RNA extraction at a later time.RNA Extraction and Hybridization Analyses. RNA was isolated by a slight modification of the protocol described by Cheley and Anderson (20); instead of passage through a 22-g needle, lysates were sonicated for 10 sec with a fine probe to shear genomic DNA. Then, 3 x 105 cell equivalents of each RNA was applied to a nitrocellulose filter (12 x 8 cm) using a %-well blotting manifold and a 12-channel pipetter. Twenty replicate nitrocellulose filters were prepared from each set of 94 hybridoma RNAs. BW5147 RNA was applied to each filter and served as a positive control for constant region (Cal) Statistical Analysis of the Data. The entire distribution of V gene expression in the three panels of T-cell hybridomas was subjected toX2 analysis (36) and the Vgenes (V1a3, V,35, V11I, and V,312) making a major contribution to the total x2 value were identified. The data for the Va3, V1,5, Vp1,1 genes were Abbreviations: MHC, major histocompatibility complex; TCR, Tcell receptor; V, variable; D, diversity; J, joining; C, constant. tTo whom reprint requests should be sent at * address. 9184The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We have studied the genetic diversity of the TCR repertoire to the murine alloantigen I-Abm12 by generating a panel of 178 C57BL/10-derived I-Abm12-reactive T cell hybridomas. The expression of V alpha and V beta gene families was examined in this panel and the frequency of expression of V beta, but not ofV alpha, gene families differed significantly from that observed in a companion panel of random C57BL/10-derived hybridomas. The V beta 5 gene family was expressed significantly less frequently while the V beta 14, V beta 15, and V beta 16 genes were expressed significantly more frequently in the panel of I-Abm12-reactive than in the panel of random hybridomas. The junctional regions (VJ alpha and VDJ beta) of TCR V alpha and V beta genes from selected I-Abm12-specific hybridomas were amplified using the polymerase chain reaction, and directly sequenced. Surprisingly, no conserved J alpha, D beta, J beta, or N region-encoded sequences among these selected I-Abm12-reactive TCRs were identified. Thus, the T cell response to an I-A alloantigen that differs by only three amino acid residues from the I-A molecule of the responding strain is genetically complex but nonrandom. We have estimated that the repertoire to this alloantigen is comprised of at least 37 different TCRs.
Proteinuria, an indicator of renal disease, was evaluated monthly by using tetrabromphenol paper, and was graded 0-3+. Trace proteinuria is <30 mg/dl, 1+ detects 30 mg/dl, 2+ detects 100 mg/dl, and 3+ detects >500 mg/dl. Severe proteinuria was defined as >2+, and animals with this level of proteinuria at 11 months of age or earlier were classified as having severe renal disease (2, 8). These mice usually died from renal failure within 4-8 weeks after development of severe proteinuria. Animals with negative or trace proteinuria at 12 months of age were classified as having no evidence of renal disease.Southern Analysis of Genomic DNA. Liver and kidney DNA from each animal was isolated as described (9). DNA (10 pkg) was digested with restriction enzymes at a concentration of 2 units per ,ug of DNA for 1 hr at 370C. This procedure was repeated once. Various enzymes were utilized depending on the probe used: Pvu II for I-Aa, BamHI for C3, Pvu II for TU66, and Bgl II for both TU169 and the a-globin pseudogene 4 (a-q4). Digested DNAs were subjected to electrophoresis through agarose gels (10). A 0.7% gel was used for MHC, TU66, TU169, and a-+4, and the gels were run for 20 hr. To separate the fragments hybridizing to the C3 probe, a 1.5% gel was run for 48 hr. The DNA was transferred to nitrocellulose filters, baked, and hybridized with a 32p labeled probe as described (10), except that the depurination step was omitted and the gel was irradiated for 7 min with shortwave UV light before denaturation to facilitate transfer ofDNA to nitrocellulose (11). Hybridized filters were washed in 2x SSC/1% SDS at room temperature followed by 0.1X SSC/1% SDS at 550C for 45 min (lx SSC = 0.15 M Abbreviation: MHC, major histocompatibility complex.fTo whom reprint requests should be addressed. 7552The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Unlike parental New Zealand Black (NZB) or New Zealand White (NZW) mice, (NZB × NZW)F1 mice exhibit a lupus-like disease characterized by IgG autoantibody production and severe immune complex-mediated nephritis. In studies of the genetic susceptibility to disease in this F1 model, the NZW MHC (H2z) has been strongly linked with the development of disease, and it was hypothesized that class II MHC genes, particularly Ez genes, may underlie this genetic contribution. In the present study, we bred transgenic B6 mice expressing I-Ez or congenic B6 mice carrying H2z with NZB mice and used a backcross analysis to test the hypothesis that Eaz and/or Ebz genes account for the effect of H2z on disease. The genetic analysis of different backcross combinations showed that unlike mice carrying H2z, mice inheriting Ez transgenes do not demonstrate increased IgG autoantibody production or increased incidence of nephritis. Surprisingly, in the same transgenic backcross mice, inheritance of the endogenous H2b from the B6 strain was strongly linked with the production of IgG autoantibodies, but not with disease. Additional experiments suggested that the level of IgG3 autoantibody production, which is controlled by H2, may be important in the pathogenesis of renal disease. Contributions to autoantibody production were also detected from an NZB locus on distal chromosome 1 (previously named Nba2). Together, these studies provide new insight into the role of MHC in lupus-like autoimmunity.
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