Proc. Nati. Acad. Sci. USA Vol. 84, pp. 6850-6853, October 1987 T-cell-receptor ,8- and I-An-chain of normal SWR mice are linked with the development of lupus nephritis in NZB x SWR crosses (T-celi-receptor genes/I-A genes/) SIBNATH GHATAK*, KRISHNA SAINIS*, FRANCES L. OWENt, AND SYAMAL K. DATTA** The Department of Medicine, *Tupper Research Institute and tDepartment of Pathology, Tufts University School of Medicine and New England Medical Center, Boston, MA 02111 Communicated by Michael Sela, June 4, 1987 (receivedfor review April 16, 1987)

ABSTRACT The incidence of nephritis in autoimmune to a variety offoreign and autoantigens has been shown to be New Zealand Black (NZB) mice is low, but when they are controlled by MHC-linked and immunoglobulin allotype- crossed with normal SWR mice, almost 100% of the female F1 linked (Ir) genes (6-8). Immune respon- hybrids (SNF1) develop lethal glomerulonephritis. To define siveness linked to TcR genes has not yet been studied. the contribution of the normal SWR strain to the development However, the TcRI3 chain of the normal SWR strain can be ofnephritis, we analyzed the association ofthe I-Afi-chain distinguished from other inbred mouse strains due to genetic of Ia-encoding region, the T-cell-receptor (3 (TcRfi)-chain polymorphisms and allotypic differences (9-11). Our studies gene, and immunoglobulin heavy-chain allotype (IgH) with the show that genes linked to TcR(3-chain and I-Af3-chain genes development oflupus nephritis in 165 NZB x SWR crosses. We of the normal SWR mice interact with corresponding genes found that genes linked to the TcR and Ir gene loci of the inherited from the autoimmune NZB strain to produce severe normal SWR mice interacted with NZB-derived genes, leading nephritis in the NZB x SWR crosses. to the development of accelerated and severe nephritis in the NZB x SWR crosses. MATERIALS AND METHODS In the New Zealand Black (NZB) mice, autoimmune hemo- NZB x SWR Crosses and Their Immunopathology. Here we lytic anemia occurs in high frequency, whereas the incidence used frozen samples of sera and tissues that were obtained in ofglomerulonephritis is only about 1% at 1 year of age (1, 2). a previous study (2) from NZB, SWR, and NZB x SWR However, when the autoimmune NZB mice are crossed with crosses. Reciprocal crosses were made to produce the F1 and the normal SWR strain, almost 100% ofthe female F1 hybrids backcross progeny mice, because the maternal direction of (SNF1) die from accelerated glomerulonephritis by 1 year of the cross did not affect the results (2). The (NZB x SWR)F1 age and the incidence oflupus nephritis is also increased and and (SWR x NZB)F1 mice are called SNF1, F1 x NZB are accelerated in the SNF1 male mice (1, 2). The results suggest backcrosses to NZB, and (NZB x SWR)F2 or (SWR x that the normal SWR parent makes a genetic contribution to NZB)F2 are called F2 mice. These animals were thoroughly the development of nephritis in the SNF1 mice. The SNF1 studied for the expression of immunologic abnormalities, progeny produces a select population of nephritogenic anti- production, and development of lupus glomer- DNA that are qualitatively different from the ulonephritis (2). Glomerulonephritis was documented by anti-DNA produced by their NZB parents (3-5). tests for proteinuria and uremia and by immunofluorescence The pathogenic autoantibodies, which are deposited initially and histopathology of kidney sections as described (2). All in the renal lesions of nephritic SNF1 mice, are of IgG class specimens were coded before examination and the severity of with highly cationic charge (4, 5); they also possess unique nephritis was graded as described (2). antigenic specificity patterns and share a distinct cross- Since NZB mice themselves develop a low incidence of reactive (CRI) called IdLNF1 (idiotype-lupus nephritis later in life (2), only those NZB x SWR crosses that nephritis-SNF,). The IgH allotypes of the normal SWR and developed accelerated and severe (2+ to 4+) glomerulone- the autoimmune NZB parents are equally represented among phritis were considered to have SNF1-like disease in this the SNF1-derived nephritogenic autoantibodies that share the study. SNF1 females develop severe nephritis between 4 and IdLNF, marker; however, the IdLNF, marker is not expressed 7 months and SNF1 males develop nephritis between 8 and 13 in the serum immunoglobulins of the parents themselves months ofage, whereas only afew NZB female and rare NZB (3-5). Thus, pathogenic anti-DNA antibodies with distinct male mice develop nephritis between 13 and 21 months and CRI markers that are "dormant" in the normal SWR and the 18 and 24 months of age, respectively (2). The SWR mice do autoimmune NZB parents become expressed and expanded not produce any autoantibodies nor do they develop nephritis in the SNF1 progeny due to an interaction of genes inherited (1, 2). from both parents. Determination of Aflotypes. heavy-chain allo- To define these genetic interactions, we investigated here types of the NZB x SWR crosses were determined here by the influence ofgenes linked to the I-A subregion ofthe major testing their sera with two monoclonal antibodies (mAb) histocompatibility complex (MHC), the immunoglobulin specific for mouse IgM allotypes using an enzyme-linked heavy-chain allotype, and the T-cell-receptor 13 (TcRf3)-chain immunosorbent assay (ELISA) as described (3-5). The mAb gene loci of the NZB and SWR strains on the rapid devel- opment of nephritis in the NZB x SWR crosses. We decided Abbreviations: NZB, New Zealand Black; SNF1, (NZB x SWR)FI to investigate the role of these genes because responsiveness or (SWR x NZB)F1; B/W, (NZB x NZW)FI; TcR, T-cell receptor; mAb, (ies); RFLP, restriction fragment length polymorphism; MHC, major histocompatibility complex. 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Bet 1 (12) was specific for SWR, and the other mAb, N N AF6-78.25 (13), was specific for the NZB allotype (3-5). Sera CAz c,, Z c,, co z kb from age-matched NZB, SWR, and SNF1 mice, the k.b MOPC104E myeloma IgM, and a NZB-derived anti-DNA 10.0- : mAb of IgM class (3) were used as control standards in all 7.2- i, < assays. 117- Southern Blot Hybridization Analysis. To detect genomic _0 ven..4~.X 6.9- qub_ DNA polymorphism of TcR3-chain genes, the 86T5 cDNA 6.3n- _ _- probe (11) was used. The 86T5 cDNA probe defines an EcoRI restriction site polymorphism in the intron between the Jf31 and CP1 coding regions ofTcR (11). This restriction site allele is linked to the TcRf3-chain allotypic marker defined by the 2.4- _ KJ16-133 antibody and is associated with genetic polymor- phisms in the TcR,8 variable region gene pool (9-11). To FIG. 1. (Left) Southern blot analysis of genomic DNA showing study polymorphisms of genes encoding Ia , another EcoRI restriction site polymorphisms of I-A3-chain gene. (Right) probe called pla.3-1 (14) was used. This cDNA clone is Southern analysis of genomic DNA showing EcoRI restriction site encoded by a gene in the I-A subregion and extensive polymorphisms of TcR3-chain gene. restriction fragment length polymorphism (RFLP) is present around this genetic region (14). Analysis of DNA from the have the NZB-type I-AP or TcRJ RFLP pattern and the other frozen tissues of NZB x SWR crosses was done as described half are expected to have the F1 type, whereas the F2 crosses with slight modifications (11, 14). DNA was extracted from are expected to contain 50%o of the progeny with the F1 type, liver nuclei by the phenol/chloroform method; this was 25% with the NZB type, and 25% with the SWR type ofRFLP followed by ethanol precipitation. DNA was next dissolved patterns. in water and 20 Aug was digested with 400 units ofEcoRI (New DNA from 90 F1 x NZB backcross mice were analyzed for England Biolabs) for 5 hr at 37TC. Agarose gel (0.9o, I-Ap and TcR,3 genotypes and the results were correlated Seakem, FMC Bioproducts, Rockland, ME) electrophoresis with the development of accelerated and severe glomerulo- of digested DNA was carried out in Tris/acetate/EDTA nephritis in these mice (Table 1). In the backcross progeny buffer at 70 mA for 16 hr. X DNA digested with HindIl (New with the heterozygous F1 type of either I-Afl or TcRP England Biolabs) was used as a molecular weight marker. genotypes, a highly significant association with the develop- Electrophoresed DNA was then blotted onto nitrocellulose ment of accelerated lupus nephritis was observed, in marked paper (BA85, 0.45 um, Schleicher & Schuell) according to contrast to the backcross mice with the homozygous NZB Southern (15). The paper was baked for 2 hr at 80°C and then type of I-AP or TcR/3 genotypes (Table 1). Moreover, those prehybridized for 2 hr at 42°C in 50%o formamide/10%o backcrosses that simultaneously inherited the I-Aj3 and TcR,3 dextran sulfate/0.2% NaDodSO4/0.6 M NaCVI60 mM sodium genotypes of the F1 type developed a markedly higher citrate/0.01 M Tris, pH 7.6/1% Denhardt's solution (0.02% incidence of lupus nephritis as compared to the rest of the bovine serum albumin/0.02% polyvinylpyrrolidone) contain- backcross mice with other combinations of I-Af and TcRP ing herring sperm DNA at 100 ,ug/ml. Hybridization was (Table 1). carried out for 17 hr at 42°C in the same solution with 0.2 ,g genotypes (-10 x 106 cpm) of each nick-translated probe (radionucle- Analysis of 75 F2 progeny mice supported the findings in ides from New England Nuclear; nick-translation kit from the F1 x NZB backcrosses (Table 2). The F2 mice with either Bethesda Research Laboratories). Following hybridization, I-AP or TcR8 genotype of the heterozygous F1 type devel- the paper was washed twice at room temperature with 0.3 M oped a significantly higher incidence of accelerated lupus NaCVI30 mM sodium citrate/1% NaDodSO4, each wash for nephritis than those inheriting the NZB type of I-Af3 or TcRf3 30 min. Then it was washed twice again for 30 min each, at genotype. Moreover, like the F1 x NZB backcrosses (Table 55°C with 30 mM NaCV/3 mM sodium citrate/1% NaDodSO4. 1), the F2 progeny that simultaneously possessed I-A/ and After drying, the blot was exposed with Kodak XAR-5 film TcR/3 genes of the F1 type developed a markedly higher with intensifying screens at -70°C for 3-5 days. incidence of nephritis than the rest of the F2 mice with other combinations of I-AP3 and TcR/ genotypes (Table 2). How- RESULTS ever, the incidence ofnephritis in the F2 mice with I-Af genes of the SWR type was not significantly different from those Inheritance of I-A13- and TcR3-Chain Genes and Develop- with the I-AP genes of the F1 type (X2 = 0.05, P > 0.8). On ment of Accelerated Nephritis in NZB X SWR Crosses. the other hand, the incidence of nephritis was considerably Southern blotting analysis of liver DNA with the pIAf3-1 less in the F2 mice with TcRP genes ofthe SWR type vs. those cDNA probe for I-A,8 gene revealed that NZB-derived DNA with the F1 type of TcRP genes (X2 = 3.31, 0.1 > P > 0.05). was like other H-2d strains (14) showing two EcoRI fragments Moreover, none of the F2 mice that possessed the combined [11.7 and 6.3 kilobases (kb)], and SWR-derived DNA was like I-AP/TcRP genotype of the SWR/SWR type developed other H-2q strains (14) yielding two EcoRI fragments (7.9 and accelerated nephritis. 6.3 kb) that hybridized with the probe (Fig. 1 Left). DNA Accelerated Nephritis from SNF1 mice exhibited the combined RFLP pattern ofthe Antibody Heavy-Chain Allotypes and two parental strains. Southern blotting analysis of liver DNA Development in NZB x SWR Crosses. Among the 90 F1 X with the 86T5 probe for the TcRf3 gene showed that the NZB NZB backcrosses tested, 17/50 (34%) mice with the hetero- strain was like BALB/c and differed from SWR (which was zygous F1-like allotype developed nephritis as compared to like SJL mice, ref. 11) by the loss of one EcoRI site and the 18/40 (45%) mice with NZB allotype; the difference in gain of another (Fig. 1 Right). NZB liver DNA yielded two incidence was not significant (P > 0.2). Among the 75 F2 EcoRI fragments of 10 kb and 2.4 kb that hybridized with the progeny 19/41 (46%) mice with F1-like allotype, 8/19 (42%) 86T5 probe, whereas SWR-derived DNA showed a RFLP with SWR allotype, and 7/15 (47%) mice with NZB allotype pattern ofthree fragments, 10, 7.2, and 3 kb. Liver DNA from developed accelerated and severe nephritis. There were no the SNF1 progeny had the combined RFLP pattern of these significant differences in susceptibility to glomerulonephritis two parental strains (Fig. 1 Right). Thus, among the F1 x among the F2 progeny with the different IgH allotypes (P > NZB backcross progeny, half of the animals are expected to 0.8). Downloaded by guest on September 25, 2021 6852 Immunology: Ghatak et al. Proc. Natl. Acad. Sci. USA 84 (1987) Table 1. Development of accelerated glomerulonephritis in SNF1 x NZB backcross progeny in relation to I-AP and TcR,3 genotypes Total number Mice with nephritis* Mice tested Number % x2 (P value) All F1 x NZB 90 (489, 426) 35 (169, 196) 38.8 I-A.3 genotype F1 type 48 (229, 266) 32 (159, 176) 66.7| 33.40 (<0.001) NZB type 42 (269, 166) 3 (19, 26) 7.1 TCR,8 genotype F, type 48 (279, 216) 26 (139, 136) 54.21 10.10 <0.005 NZB type 42 (219, 216) 9 (39, 66) 21.4 101 (<.0) I-Ap/TcR, genotype F1/F, type 28 (159, 136) 23 (129, 116) 82.11 32 00 <0 Other typest 62 (339, 296) 12 (49, 86) 19.4 32. (<.001) *Rapidly developing glomerulonephritis, 2+ to 4+ in severity. tAIl other combinations (types) of I-AB/TcRB genotypes that were found among the F1 x NZB backcross progeny: NZB/NZB, F1/NZB, and NZB/Fl.

DISCUSSION NZW strain could have contributed to the autoimmune manifestations in the B/W hybrids (20). Our studies show The rapid development of severe lupus nephritis in NZB x that the TcR(-chain gene of the normal SWR strain can also SWR crosses is a multistep process resulting from the contribute to severe lupus nephritis development in the NZB interaction of multiple genes inherited from both parental x SWR crosses. In the nonautoimmune SWR strain only strains (1, 2). Our present study shows that genes linked to certain Vp subfamilies of TcR genes have been deleted (10), the TcRf3- and I-A(3-chain genes of the normal SWR mice which is different from the extensive deletion of TcR genes interact with NZB-derived genes to contribute to the devel- found in the autoimmune NZW strain (19, 20) and yet both opment of severe nephritis in the NZB x SWR crosses. types of TcR gene deletions can be associated with autoim- Unlike the NZB x SWR cross, most other crosses ofNZB mune disease. One possibility suggested by these results is with other nonautoimmune strains do not develop lupus that an abnormality in the TcR gene product itself may not be nephritis (16). The other well-studied NZB cross that does contributing to the development ofnephritis but that a certain a incidence of severe lupus nephritis is the (NZB develop high gene or genes linked to the TcRf-chain genes of the SWR or x NZW)F1 or B/W hybrid. The NZW parents of this cross, the NZW strain may be playing a role. These TcR-linked unlike the SWR strain, are not normal: they produce anti- DNA antibodies and develop nephritis late in life (17, 18). genes could function like other Ir genes that have been They also produce high levels of retroviruses and retroviral classically linked to the MHC and antibody heavy-chain gp7O antigens that participate in the immune-complex-medi- allotype loci (6-8). Indeed, in certain organ-specific autoim- ated nephritis of B/W mice, unlike the NZB x SWR crosses mune diseases an interactive effect of MHC and antibody (1). The TcRf3-chain genes ofNZW mice have a large deletion allotype genes has been observed (8). We did find here that of 8.8 kb spanning the Cf31, Df32, and J2 clusters, but the T genes linked to I-Af8-chain gene of the I region of normal cells of NZW mice are still capable of making a functional SWR mice also contributed to the nephritis development in TcR with the remaining TcR genes (19). While our studies NZB x SWR crosses. In a previous study with NZB x NZW were in progress, a report linking the TcRB gene deletion of crosses, the development of proteinuria was also linked with the NZW strain with IgG anti-DNA autoantibody production the H-2 complex genes of the autoimmune NZW parent (21). in B/W mice appeared, but development ofnephritis was not In contrast to certain organ-specific autoimmune diseases, studied in that investigation (20). Since the NZW parent itself we did not find a linkage ofthe antibody allotype genes ofthe is autoimmune (17, 18), an abnormal TcR gene product ofthe parental strains to the development of lupus nephritis. This Table 2. Development of accelerated glomerulonephritis in (NZB x SWR)F2 progeny in relation to I-AP and TcR,8 genotypes Total number Mice with nephritis* Mice tested Number % x2 (P value) All F2 75 (319, 446) 34 (189, 166) 45.3 I-AB genotype F1 type 39 (179, 226) 21 (109, 116) 53.8 } 5.57 (<0.025) NZB type 22 (89, 146) 5 (49, 16) 22.7 (< ) SWR type 14 (69, 86) 8 (49, 46) 57.1 } 4.39 (<0.05) TcRp genotype F1 type 37 (169, 216) 22 (109, 126) 59.5 } 4.51 (<0.05) NZB type 20 (79, 136) 6 (49, 26) 30.0 SWR type 18 (89, 106) 6 (49, 26) 33.3 1 0.05 (>0.8) I-A,B/TcR,B genotype Fl/Fl type 20 (89, 126) 15 (59, 106) 75.0 1 Other typest 55 (239, 326) 19 (139, 66) 34.5 J 9.69 (<0.005) *Rapidly developing glomerulonephritis, 2+ to 4+ in severity. tAll other combinations (types) of I-A3/TcRP genotypes that were found among the F2 mice: NZB/NZB, SWR/SWR, F1/NZB, NZB/F1, F1/SWR, SWR/F1, SWR/NZB, and NZB/SWR. Downloaded by guest on September 25, 2021 Immunology: Ghatak et al. Proc. Natl. Acad. Sci. USA 84 (1987) 6853 result is consistent with previous genetic studies (22) and also 1. Datta, S. K., Manny, N., Andrzejewski, C., Andre-Schwartz, with the observation that the select population of IdLNFl- J. & Schwartz, R. S. (1978) J. Exp. Med. 147, 854-871. positive nephritogenic autoantibodies produced by the SNF1 2. Eastcott, J. W., Schwartz, R. S. & Datta, S. K. (1983) J. hybrids could possess either parental allotype (3-5). Besides Immunol. 131, 2232-2239. 3. Gavalchin, J., Nicklas, J. A., Eastcott, J. W., Madaio, M. P., these genetic linkage studies in B/W mice and NZB x SWR Stollar, B. D., Schwartz, R. S. & Datta, S. K. (1985) J. crosses, the MRL-lpr lupus strain has also been analyzed for Immunol. 134, 885-894. TcRf-chain gene expression (23, 24). Those studies may 4. Gavalchin, J., Seder, R. A. & Datta, S. K. (1987) J. Immunol. reflect peculiarities of the abnormal T-cell populations that 138, 128-137. accumulate in the massively enlarged lymph nodes of the Ipr 5. Gavalchin, J. & Datta, S. K. (1987) J. Immunol. 138, 138-148. strain and may be relevant to its lymphoproliferative disor- 6. McDevitt, H. 0. & Sela, M. (1965) J. Exp. Med. 122, 517-531. der. However, the expansion of the abnormal T cells that is 7. Benncerraf, B. (1981) Science 212, 1229-1233. induced by the lpr gene plays a secondary accelerating role 8. Whittingham, S., Mathews, J. D., Schanfield, M. S., Tait, B. D. & Mackay, I. R. (1981) Clin. Exp. Immunol. 43, 80-86. in lupus; the underlying mechanism ofautoimmune disease in 9. Roehm, N., Carbone, A., Kushnir, E., Taylor, B., Riblet, R., the MRL model lies in the congenic MRL-+/+ background Marrack, P. & Kappler, J. (1985) J. Immunol. 135, 2176-2182. (25), which lacks the lymphoproliferation component. 10. Behlke, M., Chou, H., Huppi, K. & Loh, D. Y. (1986) Proc. The TcRf3-chain and the I-Af3-chain-linked genes of SWR Natl. Acad. Sci. USA 83, 767-771. mice could interact with NZB genes to influence the devel- 11. Epstein, R., Roehm, N., Marrack, P., Kappler, J., Davis, M., opment and function of T cells in the NZB x SWR crosses. Hedrick, S. & Cohn, M. (1985) J. Exp. Med. 161, 1219-1224. 12. Kung, J. T., Sharrow, S. O., Sieckmann, D. G., Lieberman, Select populations of activated T-helper cells with classical R. & Paul, W. E. (1981) J. Immunol. 127, 873-876. and novel induce the production of pathogenic 13. Stall, A. M. & Loken, M. R. (1984) J. Immunol. 132, 787-795. IgG anti-DNA antibodies with highly cationic charge in mice 14. Robinson, R. R., Germain, R. N., McKean, D. J., Mescher, prone to develop lupus nephritis (26). Similar T-helper cells M. & Seidman, J. G. (1983) J. Immunol. 131, 2025-2031. are also responsible for the augmented production of a 15. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. restricted population ofnephritogenic autoantibodies bearing 16. Howie, J. B. & Helyer, B. J. (1968) Adv. Immunol. 9, 215-268. 17. Kelly, V. E. & Winkelstein, A. (1980) Clin. Immunol. Immu- the IdLNF, marker in the NZB x SWR crosses (27). These nopathol. 16, 142-150. nephritogenic are dormant in the autoimmune NZB 18. Hahn, B. H. & Shulman, L. E. (1969) Arthritis Rheum. 12, and the normal SWR parents but are switched on due to 355-364. certain immunoregulatory defects in the NZB x SWR cross. 19. Kotzin, B. L., Barr, V. L. & Palmer, E. (1985) Science 229, Those immunoregulatory imbalances in the NZB x SWR 167-171. crosses could result from the of and 20. Yanagi, Y., Hirose, S., Nagasawa, R., Shirai, T., Mak, T. W. complementation TcRf3- & Tada, T. (1986) Eur. J. Immunol. 16, 1179-1182. I-A(-chain genes inherited from the SWR and the NZB 21. Knight, J. G. & Adams, D. D. (1978) J. Exp. Med. 147, parents. An expansion of autoreactive T-helper cells that 1653-1660. augment the production of pathogenic autoantibodies could 22. Datta, S. K., Owen, F. L., Womack, J. E. & Riblet, R. J. result from the combined action of hybrid Ia and TcR (1982) J. Immunol. 129, 1539-1544. molecules that might be expressed in the progeny ofthe NZB 23. Singer, P. A., McEvilly, R. J., Noonan, D. J., Dixon, F. J. & x SWR crosses that possess the F1 or heterozygous genotype Theofilopoulos, A. N. (1986) Proc. Natl. Acad. Sci. USA 83, 7018-7022. for I-AB and TcR,8 genes. 24. Hashimoto, Y., Maxam, A. M. & Green, M. I. (1986) Proc. Natl. Acad. Sci. USA 83, 7865-7869. We thank Dr. John G. Seidman (Harvard Medical School) for 25. Kelly, V. E. & Roths, J. B. (1985) Clin. Immunol. Immu- giving us the plasmids containing the 86T5 and pIA,8 probes and Ms. nopathol. 37, 220-229. Stefanie Woodward for word processing. This work was supported 26. Datta, S. K., Patel, H. & Berry, D. (1987) J. Exp. Med. 165, by National Institutes of Health Grant CA31789 (to S.K.D.) and 1252-1268. American Cancer Society Grant IM-394 (to F.L.O.). F.L.O. is the 27. Sainis, K. & Datta, S. K. (1987) Fed. Proc. Fed. Am. Soc. recipient of Research Career Development Award K04-00546. 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