Genetic control of murine IgD structural heterogeneity

Transcription

1 Occidental College From the SelectedWorks of Roberta Pollock 1980 Genetic control of murine IgD structural heterogeneity Roberta R Pollock, Occidental College Martin E Dorf Matthew F Mescher Available at:

2 Proc. Natl. Acad. Sd. USA Vol. 77, No. 7, pp , July 1980 Immunology Genetic control of murine IgD structural heterogeneity (surface immunoglobulin/igd allotypes/genetic mapping) ROBERTA R. POLLOCK, MARTIN E. DORF, AND MATTHEW F. MESCHER Department of Pathology, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts Communicated by Baruj Benacerraf, April 14,1980 ABSTRACT Murine B lymphocytes express two native forms of surface IgD: IgD1 consists of two a heavy chains and two light chains; IgDu consists of one heavy chain and one light chain. The relative amounts of IgD1 and IgDf present on spleen cells were found to vary significantly among different strains of mice. Genetic evidence demonstrated that the IgD /IgD11 ratio is linked to the Igh-5 allotype. (The a heavy chain is the roduct of the Igh-5 locus.) Mice bearing the Igh.5e allotype h~ave low ratio, and mice bearing the IghaS or Igb-Sb allotype have a high ratio. The Igh-5 locus and the gene controlling the IgDj/Igu ratio appear to map to the region between the Igh-6 and Ig-V loci. The antigen receptors of murine B lymphocytes are surface immunoglobulin molecules of predominantly two classes, or isotypes, IgM and IgD (reviewed in ref. 1). Recent studies have shown that surface IgD possesses a structural heterogeneity not seen for IgM (2, 3). IgM is found as a four-chain structure consisting of two Ai heavy chains and two light chains (4). In contrast, two structures of surface IgD occur on the B cell surface, IgDj and IgD1j (2, 3). IgD, is also a four-chain structure containing two a heavy chains and two light chains, but IgDjj is a two-chain structure composed of one a heavy chain and one light chain. The existence of two forms of IgD cannot be explained by proteolytic degradation or disulfide bond rearrangement (2, 3). Although all strains of mice studied have both IgDi and IgDn, the relative amounts of the two structures vary among strains. This strain variation may explain why some workers have seen two forms of IgD (5-7) and others have detected only one form (1). Furthermore, because IgD11 yields a relatively broad band upon electrophoresis, it may be difficult to detect in strains with a low amount of IgDn. Four-chain and two-chain IgD structures are also seen in human tonsil cells (8). The experiments described in this paper examined the relationship of the Igh-5 allotype to the variation in the relative amounts of IgD1 and IgDj1 seen in different strains. The data demonstrate that mice of the Igh-5e allotype have an IgDi/ IgD1j ratio that distinguishes them from mice of the Igh-5a or Igh-5b allotype. On the basis of IgDI/IgD11 ratios and analysis of IgD allotypic markers, two strains have been identified in which crossing over appears to have occurred within the Ig heavy chain constant region (Igh-C) gene cluster. Both recombinational events occurred between the Igh-5 locus, which encodes the a heavy chain, and the other Igh-C genes. MATERIALS AND METHODS Mice. B.C-8, C.B-20, CWB/20, and C.AL-20 mice were from the colony maintained at Harvard Medical School. All other mice were obtained from The Jackson Laboratory (Bar The 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 solely to indicate this fact. Harbor, ME), Charles River (Wilmington, MA), or Cumberland Farms (Clinton, TN). Antisera and Immunoabsorbents. F(ab')2 fragments of polyspecific rabbit anti-mouse Ig were coupled to Sepharose 4B (Pharmacia) as described (9), and will be referred to as RAMG-Sepharose. Alloantiserum specific for the IgDa allotype, defining IgD specificity 1, was a gift from E. R. Unanue and C. Sidman and is described elsewhere (9, 10). The hybrid cell line, which secretes antibody that recognizes IgDa molecules (11) defining specificity 4, was maintained in vitro and culture supernatants were used without purification. Ascites fluid containing the H6/31 HL monoclonal antibody which reacts with IgDb and IgDe molecules (12), defining IgD specificity 3, was purchased from Accurate Chemical and Scientific (Hicksville, NY). Purified monoclonal antibody from the hybrid cell line was obtained from Becton Dickinson; this antibody recognizes only IgDb molecules and determines IgD specificity 2 (11). Staphlococcus aureus protein A coupled to Sepharose 4B, referred to as protein A-Sepharose (Pharmacia), was used to precipitate immune complexes formed by the alloantiserum or the monoclonal antibody. Immune complexes formed by the H6/31 antibody were precipitated with protein A-Sepharose that had been preincubated with rabbit anti-mouse IgM antiserum (Litton Bionetics, Kensington, MD). Immune complexes containing the antibody were precipitated with protein A-Sepharose that had been preincubated with rabbit anti-mouse IgG1 (Litton Bionetics). Iodination of Spleen Cells and Isolation of Surface Ig. Spleen cells were purified and iodinated with 125I by using lactoperoxidase as described (3, 9). Iodinated, washed cells were lysed in 0.5% Nonidet P-40 in phosphate-buffered saline at ph 7.2, and total surface Ig was precipitated with RAMG-Sepharose as described (9). IgD was precipitated with antisera and protein A-Sepharose as described above. The Sepharose beads were washed with 0.5% Nonidet P-40 in phosphate-buffered saline and the bound Ig was eluted with NaDodSO4 at 100'C as described (9). NaDodSO4/Polyacrylamide Gel Electrophoresis. Electrophoresis was done on 3-13% acrylamide gradient gels as described previously (3, 9). Gels were stained and dried, and autoradiographs prepared as described (9). Bands were quantitated by cutting out the entire labeled band and assaying it for radioactivity; adjacent areas above and below the band were cut out and assayed to determine background. RESULTS Strain Variation of the IgD1/IgDu Ratio. The percentage of surface Ig found as IgD, 65-80% of the total surface Ig, did not vary significantly among the strains of mice examined. Electrophoresis of surface Ig under nonreducing conditions Abbreviations: RAMG-Sepharose, Sepharose 4B carrying F(ab')2 fragments of polyspecific rabbit anti-mouse Ig coupled to it. 4256

3 IgM * IgD1 IgDi1 q0~ A B C D _m - Po _M f FIG. 1. Strain variation in the relative amounts of IgD1 and IgDu. Spleen cells were iodinated with 12'I and surface Ig was isolated on RAMG-Sepharose. Samples were electrophoresed on an NaDodSO4 gradient gel under nonreducing conditions. Surface Ig was from: A, AKR mice; B, C.AL-20 mice; C, A mice; and D, BALB/c mice. showed that, although all strains had both IgD1 and IgD11, the relative amounts of the two forms varied with strain (Fig. 1). The relative amounts of IgD, and IgDn are expressed as the IgD1/IgDug ratio, determined by dividing the net cpm in the IgD1 band by the net cpm in the IgDH band. Strain A mice had a low ratio, 0.4, which was significantly different from the IgD1/IgDuj ratios of all the other strains tested (Table 1). The ratios of the other strains did not differ significantly from one another. Immunology: Pollock et al. Linkage of the IgDI/IgDuj Ratio to the Lgh.Aiotype. The results shown in Table 1 suggested that a low IgDI/IgDrh ratio may be linked to the Igh-5e allotype. To test this possibility, surface Ig was isolated from several Igh congeneic lines and their IgDi/IgDu ratios were determined (Table 2). Congeneic mice having the Igh-5a (BALB/c) or Igh-5b (C.B-20) allotype had high IgDI/IgDil ratios. The C.AL-20 line, which is also an Igh congeneic strain, had the Igh-5e allotype (see below and Proc. Natl. Acad. Sci. USA 77 (1980) 4257 Table 1. Strain variation of IgDvIgDnj ratio Experiments, IgD IgDl/IgDui Strain no. allotype (mean 4 SEM) AKR 6 a BALB/c 14 a 1.4 d 0.1 CBA 3 a C3H 5 a 1.6b0.3 DBA/2 2 a NZB 5 a C57BL/6 11 b SJL 3 b A 16 e 0.4±0.03 Table 4) and the low IgD1/IgD11 ratio characteristic of the A strain (Fig. 1). The Igh genes of the C.AL-20 line came from the AL/N mouse, which derived most of its Igh-C genes from the AKR strain and its heavy chain variable region (Igh-V) genes from the A strain (13). The a chain gene of AL/N mice was derived from the A strain, as will be discussed below. The results obtained from G.AL-20 mice indicate that the low IgDh/IgD1l ratio of A mice is closely linked to the Igh-5 locus and the Igh-V genes of A mice. IgD1/IgDu Ratios of F1 Hybrid Mice. Surface IgD exhibits the phenomenon of allelic exclusion. A given B cell of an F1 hybrid can express only one of its two a heavy chain genes (1). F1 hybrid mice, therefore, are mosaics for Ig expression; half of their IgD-bearing B cells will express the a chain gene derived from one parent and the remaining cells will express the gene obtained from the other parent. If F1 hybrids have two populations of IgD molecules, each with its own IgD1/IgD11 ratio, the expected ratio of the surface Ig from an F1 hybrid would be the average of the ratios of the two parental strains. The observed ratios agreed well with the predicted ratios (Table 3). Further evidence supporting the conclusion that the IgD of F1 hybrids exists as two distinct populations was obtained by precipitating the surface IgD of a (BALB/c X A)F1 hybrid with the antibody. This antibody is specific for the IgD molecules of the a allotype expressed by BALB/c mice. The IgD molecules precipitated by this antibody had the same high IgD1/IgD1l ratio as did the BALB/c parent (not shown), in contrast to the intermediate ratio obtained from RAMG- Sepharose was used to precipitate surface Ig from the same labeled F1 cells. Two Recombinants in the Igb-C Region. The C.AL-20 line has the IgD1/IgDr1 ratio of the A strain, rather than that of the AKR strain. Because the other Igh-C genes of the C.AL-20 mouse are similar to those of the AKR strain (14), this finding suggested that a recombination event occurred between the 6 chain gene and the other Igh-C genes. The C.AL-20 line, and presumably the AL/N strain, derived their S chain genes and their Igh-V genes from the A strain and their remaining Igh-C genes from the AKR strain (13, 14). The involvement of the,u chain gene in the presumed recombination event that gave rise to the C.AL-20 Igh complex cannot be determined because the Igh-6 allotypes of AL/N and AKR mice are not known. The high IgDI/IgDin ratio of the NZB mouse suggested that it does not possess the Igh-5e allotype, although its other Igh-C genes are serologically identical to those of the A strain (14, 15). The NZB mouse has been reported to be of the Igh-5a allotype (15X, although no data were presented, suggesting that a recombination event occurred between the Igh-5 locus and the remaining Igh-C genes.

4 4258 Immufnology: Pollock et al. Proc. Nati. Acad. Sci. USA 77 (1980) Table 2. IgDI/IgDu ratios of Igh congeneic strains Parental strain Donor strain IgD Experiments, IgDvflgDn Strain (IgDvflgDu) (IgDdIgDII) allotype no. (mean s SEM) B.C-8 C57BL (1.4) BALB/c (1.4) a C.B-20 BALB/c (1.4) C57BL (1.4) b CWB/20 C3H.SW (1.6) C57BL (1.4) b C.AL-20 BALB/c (1.4) AL/N * e * Not tested. The IgD allotypes of C.AL-20 and NZB mice were examined to determine if these proposed recombinations could be confirmed by serology. The three Igh-5 alleles, a, b, and e, can be defined on the basis of four distinct antigenic specificities (11). Each specificity is determined by an alloantiserum or a monoclonal antibody. The Igh-5a gene product is defined by two unique specificities. Specificity 1 is defined by the C57BL anti-cba alloantiserum (10) and specificity 4, by the monoclonal antibody (11). The Igh-5b gene product is also defined by two specificities. Specificity 2 is defined by the monoclonal'antibody (11) and specificity 3, by the H6/31 monoclonal antibody (12). The Igh-5e gene product has no unique specificities but shares specificity 3 with the Igh-5b gene product. Surface IgD from each strain was precipitated with these antibodies and then electrophoresed under reducing and nonreducing conditions. The anti-igd allotype reagents specifically precipitated both native heavy chains, 61 and 62 (9), which are detected by electrophoresis under reducing conditions, and both IgD structures, IgD1 and IgD][, which are detected by electrophoresis under nonreducing conditions. The C.AL-20 line had the Igh-5e allotype and the NZB strain had the Igh-5a allele (Table 4). C.AL-20 and NZB mice therefore appear to represent recombinants between the Igh-5 gene and the other Igh-C genes (Table 5). The above results suggest that at least in the NZB strain, Igh-5 maps to the left of Igh-6 (the gene coding for IgM). The inbred Igh congeneic line BAB/14 was also examined. BAB/14 is a spontaneous recombinant which has the Igh-C genes of the b haplotype and the Igh-V genes derived from the BALB/c strain (13, 14). Specific precipitation and analysis by NaDodSO4 gel electrophoresis demonstrated that BAB/14 had the Igh-5b allotype and a high IgD1/IgDll ratio (Tables 4 and 5). Furthermore, the BxD-25 and SM/J strains both carried the Igh-5b allotype (data not shown). The Igh haplotypes of these strains represent additional examples of recombinant haplotypes in which a crossover event occurred between the Igh-Cb region loci and the Igh-V genes (13, 16). The Igh-5 locus therefore maps between the Igh-6 and Igh-V loci. DISCUSSION These studies have shown that the Igh-5 allotypes of a strain apparently determines the relative amounts of IgD1 and IgDj1 found on the cell surface. Mice bearing the Igh-5e allotype have a low IgD1/IgD11 ratio (0.4) and mice possessing the Igh-5a or Igh-5b allotype have high IgD1/IgD11 ratios ( ). F1 hy- Table 3. IgDI/IgDil ratios of F1 hybrid mice IgDI/IgD11 ratio Observed F1 hybrid Predicted (mean Lrange) C57BL/6 X A I 0.1 BALB/c X A ± 0.1 BALB/c X C57BL/ ± 0.9 BALB/c X DBA/ k 0.1 brids between an Igh-5e strain and an Igh-5a or Igh-5b strain have intermediate ratios (0.8). These intermediate ratios reflect the existence in F1 hybrids of two types of B-cell populations, each with its own IgDh/IgDEu ratio. Assuming that the IgDh/IgDII ratio is a characteristic of the 6 heavy chain structure, the Igh-5 locus may directly determine this ratio. An alternative explanation is that a distinct gene controls the IgD1/IgDn ratio and that this gene is closely linked to the Igh-5 locus. The IgDh/IgDll ratio provides a useful marker in that it uniquely defines the Igh-5e allotype. Known serological markers only define Igh-5e on the basis of its pattern of crossreactivity. When the IgDh/IgDug ratio was used to determine the Igh-5 allotypes of a number of strains, two examples of apparent crossing over between the Igh-5 gene and the other Igh-C genes were detected, in NZB and C.AL-20 mice. Serologic studies confirmed that these mice had unexpected IgD specificities. The NZB mouse had the Igh-54 allotype rather than the expected Igh-5e allotype, and the C.AL-20 line had the Igh-5e allotype instead of the predicted Igh-5a allotype. These findings suggested that recombinations occurred between the Igh-5 gene and the other Igh-C genes during the development of the NZB and AL/N strains. According to the classification scheme proposed by Lieberman (14) and Green (15), whica designates the NZB mouse as haplotype Ighn, the C.AL-20 line, and presumably the AL/N strain, should be tentatively designated as the 1gh haplotype. The heavy chain linkage group of the NZB mouse is identical to that of the A mouse for all the Igh-C genes except Igh-5. Because the Igh-V genes of NZB and A mice differ, the chain gene is probably at the end of the Igh-C locus closest to the Igh-V genes. Only one crossover would be required if one postulates this map position for Igh-5 (Table 5). The assumed recombination seen in the C.AL-20 line also places the 6 chain gene at the end of the Igh-C locus, closest to the Igh-V genes. Because the,u heavy chain allotypes of AKR and C.AL-20 mice are not known, one cannot determine whether the,a heavy chain is involved in the recombination (Table 5). The results obtained with BAB/14, BxD-25, and SM/J mice demonstrate that the Igh-5 locus maps to the right of the known Igh-V loci. Table 4. Correlation of IgD allotype and IgD1/IgDjj ratio IgD antigenic specificity* t Strain Igh-5 allotype IgDJIgD_ BALB/c a +t NZB a A e C.AL-20 e AKR a C57BL/6 b BAB/14 b NDt * Based on Oi et al. (11). -, antibody defining that specificity did not precipitate IgD from that particular strain; +, antibody did precipitate IgD from that particular strain. Not determined. Gels indicated that BAB/14 had a high IgDJfIgDIj ratio, although the bands were not quantitated.

5 Immunology: Pollock et al. Proc. Natl. Acad. Sci. USA 77 (1980) 4259 Table 5. Mapping the Lg)f5 locus within the Igh gene complex* Igh- V region Igh-C regiont Strain Igh-Ars Igh-Gtet Igh-Sa5 Igh-5 (Igdr) Igh-6 Igh-3 Igh-1 AKR a (h) d d C.AL e < (? - 1 ; d d A + + _ e(1) e e e NZB - - a(h) e e e BALB/c a (h) a a a BAB/ b (h)? b b C.B b (h) b b b * See refs t Sites of recombination are indicated by vertical bars, potential sites of crossing over are indicated by broken vertical bars. Arrows localize position of Igh-5 locus. S.-T. Ju and M. E. Dorf, unpublished results of the common idiotype on anti-gat antibodies expressed in mice bearing Igh-1b and Igh-1 e. Hypothetical locus controlling the ratio of IgDv'IgDiu. Symbols indicate high (h) or low (1) ratios. The murine Igh-C complex was previously considered to be a tightly linked cluster of structural genes because no recombinants were found in more than 5000 progeny from crosses designed to look for such recombinants (14, 15). However, recombination for the Igh-5 locus was not assessed in those studies. Table 5 maps the Igh-5 locus and the hypothetical locus which determines the IgD1/IgDi ratio (tentatively termed Igdr) within the Igh gene complex. The segregation of the Igh-V region marker loci is fully consistent with the interpretation that a single crossover event occurred between Igh-5 and the other Igh-C loci. The use of the NZB strain, which carries a "natural recombinant" haplotype, to map the Igh-5 gene is similar to the use of population studies to determine the Igh-C gene order in humans. Although there is little evidence for known crossovers among the Igh-C genes in human families, studies of different populations strongly suggest that crossovers have occurred within this complex (17). Because no allotypic markers have been discovered for human IgD, the IgD gene cannot be placed precisely within the human Igh-C map. By restriction mapping of the DNA containing the Igh-C genes, two laboratories have shown that the 6 chain gene is located on the 3' side of the,u chain gene, thus placing the Igh-5 gene to the right of the Igh-6 gene (F. Blattner, personal communication; L. Hood, personal communication). An apparent discrepancy therefore exists between the genetic data and the DNA restriction mapping data as to the exact location of the 6 chain gene within the Igh complex. One possible explanation for the genetic data is that multiple crossover events occurred during the development of the NZB strain. The occurrence of multiple recombination events within the Igh complex has been documented in other strains (13). Although the linkage of the genes controlling the IgDh/IgD11 ratio to the Igh-5 allotype further demonstrated that both IgD1 and IgDhi are native structures, the functional significance of the two forms is not known. Additional structural heterogeneity occurs because IgDI and IgDuj each contains both of the 6 heavy chains, 61 and 62. We have previously shown that 61 and 62 are native heavy chains that differ in their sialic acid content and in their susceptibility to papain cleavage (9). The cellular distribution of these IgD structures is not known. If different subpopulations express only IgDj or IgD11, there may be a functional basis for the structural heterogeneity of IgD. Note Added in Proof. IgD allotypic determinants of a large number of mouse strains have recently been examined by Woods et al. (18, 19). Their results also show that AL/N and C.AL-20 mice have the Igh-5e allele and they have also designated this recombinant haplotype Igh- CO. This work was supported by National Institute of Allergy and Infectious Diseases Grant 1-PO1-AI R.R.P. was supported by National Institutes of Health Training Grant CA Goding, J. W. (1979) Contemp. Top. Immunoblol. 8, Eidels, L. (1979) J. Immunol. 123, Pollock, R. R. & Mescher, M. F. (1980) J. Immunol. 124, Vitetta, E. S., Baur, S. & Uhr, J. W. (1971) J. Exp. Med. 134, Melcher, U., Vitetta, E. S., McWilliams, M., Lamm, M. E., Philips-Quagliata, J. M. & Uhr, J. W. (1974) J. Exp. Med. 140, Abney, E. R. & Parkhouse, R. M. E. (1974) Nature (London) 252, Lisowska-Bernstein, N. & Vassalli, P. (1975) in Membrane Receptors of Lymphocytes, eds. Seligmann, M., Preud'Homme, J. L. & Kourilsky, F. M. (North-Holland, Amsterdam), pp Corte, G., Viale, G., Cosulich, E., Bargellesi, A. & Ferrarini, M. (1979) Scand. J. Immunol. 10, Mescher, M. F. & Pollock, R. R. (1979) J. Immunol. 123, Goding, J. W., Warr, G. W. & Warner, N. L. (1976) Proc. Natl. Acad. Sci. USA 73, Oi, V. T., Jones, P. P., Goding, J. W., Herzenberg, L. A. & Herzenberg, L. A. (1978) Curr. Top. Microbiol. Immunol. 8, Pearson, T., Galfre, G., Ziegler, A. & Milstein, C. (1977) Eur. J. Immunol. 7, Weigert, M. & Riblet, R. (1978) Springer Sem. Immunopathol. 1, Lieberman, R. (1978) Springer Sem. Immunopathol. 1, Green, M. C. (1979) Immunogenetics 8, Kipps, T. J. & Dorf, M. E. (1979) Immunogenetics 9, Kunkel, H. G. & Kindt, T. (1975) in Immunogenetics and Immunodeficiency, ed. Benacerraf, B. (MTP Publisher, St. Leonard House, Lancaster, England), pp Woods, V. L., Finkelman, F. D., Scher, I. & Paul, W. E., J. Immunol., in press. 19. Woods, V. L., Scher, I. & Paul, W. E., J. Immunol., in press.