Rearrangement and Diversification of Immunoglobulin Light-Chain ...

Vol. 9, No. 11

MOLECULAR AND CELLULAR BIOLOGY, Nov. 1989, P. 4970-4976 0270-7306/89/114970-07$02.00/0 Copyright © 1989, American Society for Microbiology

Rearrangement and Diversification of Immunoglobulin Light-Chain Genes in Lymphoid Cells Transformed by Reticuloendotheliosis Virus JIAYOU

ZHANG,1 WILLIAM BARGMANN,1 AND HENRY R. BOSE, JR. 12*

Department of Microbiology,1 and The Cell Research Institute,2 The University of Texas at Austin, Austin, Texas 78712 Received 25 May 1989/Accepted 18 July 1989

Antibodies that recognize a wide variety of different antigens may be elicited. Antibody diversity in mammals results principally from a series of somatic recombinations that occur during B-cell differentiation to produce functional light- and heavy-chain immunoglobulin molecules. The lightchain loci in mice are composed of many different and potentially functional variable-region segments (V), several different joining regions (J), and four constant regions (C). Recombination between different V and J regions and the mechanism which joins the V and J segments produce antibody diversification. In heavy-chain genes, the diversity region (D) adds another element of diversification. After antigenic stimulation, mammals further diversify the sequences of active immunoglobulin genes by somatic mutation. Sequence heterogeneity is produced by a different mechanism in the V region of chickens. Chickens have a single X light-chain V region (17). In chicken B cells, this V region recombines with a single J region and a single C region to produce a functional A light-chain gene. In germ line DNA, 25 pseudo-V genes are clustered within a 20kilobase region located several kilobases upstream of the single functional V gene. Sequence heterogeneity observed in the functional V region corresponds to sequences in the V pseudogene library, suggesting that diversification is created by gene conversion events between the functional V segment and its pseudogenes. Rearrangement and diversification have been observed in both the bursa of Fabricius and the peripheral hematopoietic organs (17, 22). Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) may serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. REV-T induces invariably fatal lym*

Corresponding author.

phomatosis in experimentally infected birds with a latency period of 7 to 10 days (19). REV-T is replication defective and coreplicates with a helper virus, termed reticuloendotheliosis-associated virus (REV-A) (8). REV-A is a nontransforming virus which induces bursal and thymic atrophy (16, 26), transient immunosuppression (18, 27), and an acute runting syndrome (15). Injection of REV-T-transformed nonvirus-producing cells obtained by in vitro infection of splenic lymphoid cells induces fatal lymphomatosis (12). The principal target cells which are transformed by REV-T (REV-A), both in vivo and in vitro, are extremely immature lymphoid cells (3, 12, 20). The lymphoid cells transformed by REV-T weakly express B-cell surface antigens, contain low levels of terminal deoxynucleotidyltransferase activity, and generally fail to synthesize immunoglobulin molecules. Further evidence that REV-T (REV-A) transforms very immature lymphoid cells of the B-cell lineage was provided by defining the status of the immunoglobulin chain genes in REV-T-transformed cells (4). REV-T-transformed cells which express immunoglobulin M (IgM) have occasionally been identified, thus indicating that phenotypically different cells may be transformed by REV-T (REV-A) (9, 12). In addition to transforming and immortalizing very immature lymphoid cells, REV-T is capable of efficiently transforming mature B cells when pseudotyped with chicken syncytial virus (2). Cells transformed by REV-T (chicken syncytial virus) synthesize variable amounts of IgM and contain rearranged heavy- and light-chain immunoglobulin loci. The rearranged VA loci in any clone are either diversified or nondiversified, and further diversification did not occur during prolonged in vitro passage (2). In this report, we characterize the differentiation status of REV-T (REV-A)-transformed lymphoid cells obtained after in vitro infection of splenic lymphocytes. The extent of 4970

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Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

VOL. 9, 1989

LIGHT-CHAIN GENE IN REV-T-TRANSFORMED CELLS

immunoglobulin chain gene rearrangements observed in REV-T-transformed splenic lymphoid cells was directly related to the age of the host cells at the time they were obtained for transformation. REV-T did transform cells at several different stages of B-cell development. Furthermore, REV-T-transformed lymphocytes continued to undergo immunoglobulin chain gene rearrangements and diversification during prolonged in vitro propagation.

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RESULTS Rearrangement in the light-chain locus during development in avian spleen cells. Initially, we analyzed the chicken light-chain gene rearrangements which occur in normal spleen cells obtained from embryos and chicks at various

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1 2 3 4 5 6 7 8 9 FIG. 1. Rearrangement of immunoglobulin light-chain genes in uninfected spleen cells from embryos and chicks at various times after hatching. Cellular DNAs (7 F±g) from spleen cells obtained at various times before and after hatching were digested with BamHI and Sall and hybridized with the nick-translated fragment containing the chicken light-chain constant region isolated from pXC (4). The 2.8-kbp fragment represents the germ line configuration (G), and the 6.5-kbp fragment represents the rearranged configuration in light-chain genes. The 20-kbp fragments represent the immunoglobulin light-chain genes methylated (M) in these restriction endonuclease sites (2). Molecular sizes are shown on the left. The sources of cellular DNA are indicated at the tops of the lanes.

times after hatching. Cellular DNA from these cells was digested with restriction endonucleases BamHI and Sall. The DNA fragments were separated by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized with the light-chain probe pXC (4). The pXC probe identified a 2.8-kilobase-pair (kbp) fragment in cells obtained from 5-day-old embryos (Fig. 1, lane 1). This fragment represents the germ line configuration of the light-chain genes. Lightchain immunoglobulin gene rearrangements were not detected by Southern analysis in cells from chicken spleens obtained from birds up to 1 week after hatching (Fig. 1, lanes 2 to 4). In spleen cells obtained from birds 2 weeks old or older, a 6.5-kbp DNA fragment hybridized with the pXC probe (Fig. 1, lane 5 to 9). This fragment represents the rearranged configuration (22), indicating that a significant percentage of the spleen cells from birds 2 weeks old or older had rearrangements in the light-chain locus. The high-molecular-weight fragment (20 kbp) detected in these Southern analyses is presumed to represent light-chain genes which were resistant to BamHI and/or SalI digestion because of methylation in these restriction endonuclease sites (2). Rearrangement of chicken immunoglobulin light-chain genes in lymphoid cell lines transformed by the v-rel oncogene. To define the immunoglobulin chain gene status in REVT-transformed lymphoid cells, chicken spleen cells from 14-day-old embryos, newly hatched chicks, and chickens at 1, 2, 6, 8, and 12 weeks after hatching were transformed in vitro by REV-T (REV-A). Individual colonies in soft agar were isolated, and 71 cell lines were established. DNA was isolated approximately 1 month after the cells were initially infected with REV-T (REV-A). Cellular DNA from each cell line was digested with BamHI and Sall and hybridized with the pXC probe (4). Southern analyses showing representative cell lines are shown in Fig. 2. Some of the REV-T-transformed cell lines isolated after in vitro infection of spleen cells obtained from embryos (Fig. 2, lanes 2 and 5) and newly hatched and 1-week-old birds (Fig. 2, lane 8) did not contain the 2.8-kbp fragment representing the germ line configura-


Virus and transformed cells. REV-T (REV-A) was harvested from cell line RECC-UTC4-1R (28). The procedure for the development of REV-T-transformed cell lines has been previously described (8). Single-cell suspensions used for in vitro transformations were prepared from spleens of Hyline SC embryos and chicks (Hyline International, Johnston, Iowa). The cell lines isolated from soft agar were grown as suspension cultures in RPMI 1640 medium supplemented with 3.3% fetal bovine serum and 6.6% calf serum. Clones were subsequently adapted to 5% calf serum with 2% Serum Plus (Hazleton, Lenexa, Kans.) as a supplement. Southern analysis. High-molecular-weight DNA was extracted from the various REV-T-transformed cell lines as previously described (28). DNA was digested with 35 to 70 U of the specified restriction enzyme (28). Southern transfers (21) and hybridization conditions were as previously described (28). RNA isolation and Northern (RNA) analysis. RNA was isolated from the cell lines by using a modified guanidine thiocyanate procedure (5, 11). Cells (1 x 108 to 3 x 108) were washed with phosphate-buffered saline and suspended in 5.3 ml of 4 M guanidine thiocyanate-25 mM sodium citrate (pH 7)-0.5% sodium N-lauryl sarcosine and vortexed for 10 s. Then, 1.7 ml of a 5.7 M CsCl solution was added to each preparation, mixed well, and layered over 4 ml of the 5.7 M CsCl solution in an SW41 tube (Beckman Instruments, Inc., Fullerton, Calif.). RNAs were centrifuged through the CsCl cushion at 100,000 x g for 22 h at 20°C. RNA pellets were suspended in 200 ,u of diethylpyrocarbonate-treated water. RNAs were precipitated by adding 0.1 volume of 3 M sodium acetate (pH 6) and 2.5 volumes of cold absolute ethanol. RNAs were pelleted by centrifugation in a microcentrifuge for 15 min, washed with 80% ethanol, dried in a vacuum, and suspended in diethylpyrocarbonate-treated water. RNA (10 ,ug) was loaded onto a formaldehyde-agarose gel and prestained by adding ethidium bromide to the loading buffer. After electrophoresis, the gels were rinsed in 10x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and the RNAs were transferred to a nitrocellulose filter by a standard Northern blot technique (13) with lOx SSC as the transfer solution. Immunoanalysis procedures and reagents. Immunoprecipitation and Western blotting (immunoblotting) have been previously described (24). The immunoreagents used to identify the light-chain proteins were rabbit anti-chicken IgG (heavy and light chains; Miles-Yeda) and goat anti-chicken IgG (heavy and light chains; Kirkegaard & Perry Laboratories).

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tion. The BamHI and SalI sites of both alleles are presumed to be methylated and, therefore, resistant to enzyme digestion. Cell lines with a single methylated allele would not have been detected by this Southern analysis. In REV-T transformants established from spleen cells obtained from birds 2 weeks old or older, methylation of both alleles in the germ line fragment was not detected. In cell lines transformed from embryonic spleen cells at day 14 (seven of seven cell lines) and day 18 (six of six cell lines) (data not shown), rearrangements were not detected (Fig. 2, lanes 1 to 5). In cell lines which contained rearrangements, some of the cell lines established from spleen cells obtained from newly hatched (one of two cell lines) and 1-week-old (seven of nine cell lines) chicks had one (Fig. 2, lanes 7 and 9) or two (Fig. 2, lanes 6 and 10 to 12) light-chain alleles rearranged. All of the REV-T-transformed cell lines obtained from spleen cells of chickens at 2 weeks or later after hatching had one (Fig. 2, lanes 13 to 17) or two (Fig. 2, lane 18) rearranged light-chain alleles. Two cell lines from this group (RECC-UT6W51 and RECC-UT12W23) appeared to have both alleles in germ line configuration. However, when the DNAs from these cell lines were digested with Scal and SaI, these cell lines had at least one rearranged allele (see Fig. 5, lanes 15 and 19). The failure to detect rearrangements in these two cell lines by BamHI and SalI digestion may be due to restriction enzyme polymorphism. Essentially all of the REV-T transformants derived from spleens obtained from chickens 2 weeks old or older had at least one rearranged light-chain allele. Therefore, cells transformed by REV-T appear to be blocked at various stages in B-lymphoid cell differentiation. Functional light-chain immunoglobulin rearrangements in lymphoid cells transformed by REV-T. To determine whether the rearrangements in light-chain genes are functional in cells transformed by REV-T, the presence of mRNA for light-chain immunoglobulin was detected by cytoplasmic blots using the pXC light-chain probe. Light-chain immunoglobulin transcripts were not detected in cell lines in which both light-chain genes remained in germ line configuration or in MSB-1 cells (data not shown). MSB-1 cells are avian lymphoid cells transformed by Marek's disease virus (14). All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain immunoglobulin transcripts, although mRNA levels varied among the cell lines. Northern analysis indicated that the light-chain gene transcript was approximately 1.2 kilobases long (Fig. 3, lanes 2

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genes have begun rearrangements, subsequent rearrangement can take place. The availability of permanent REV-T-transformed cell lines representing different stages of differentiation provides a system to study the mechanism of avian immunoglobulin gene rearrangement and diversification.

ACKNOWLEDGMENTS

LITERATURE CITED 1. Alt, F., N. Rosenberg, S. Lewis, E. Thomas, and D. Baltimore. 1981. Organization and reorganization of immunoglobulin in genes in A-MnLV-transformed cells: rearrangement of heavy but not light chain genes. Cell 27:381-389. 2. Barth, C. F., and E. H. Humphries. 1988. A nonimmunosuppressive helper virus allows high efficiency induction of B cell lymphomas by reticuloendotheliosis virus strain T. J. Exp. Med. 167:89-108. 3. Beug, H., H. Muller, S. Grieser, G. Doederlein, and T. Graf. 1981. Hematopoietic cells transformed in vitro by REV-T avian reticuloendotheliosis virus express characteristics of very immature lymphoid cells. Virology 115:295-309. 4. Chen, L., M. Y. Lim, H. Bose, Jr., and J. M. Bishop. 1988. Rearrangements of chicken immunoglobulin genes in lymphoid cells transformed by the avian retroviral oncogene v-rel. Proc. Natl. Acad. Sci. USA 85:549-553. 5. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:52945299. 6. Hagiya, H., D. D. Davis, T. Takahashi, K. Okuda, W. C. Raschke, and H. Sakano. 1986. Two types of immunoglobulinnegative Abelson murine leukemia virus-transformed cells, implication for P-lymphocyte differentiation. Proc. Natl. Acad. Sci. USA 83:145-149. 7. Hoelzer, J. D., R. B. Franklin, and H. R. Bose. 1979. Transformation by reticuloendotheliosis virus: development of a focus assay and isolation of a non-transforming virus. Virology 93: 20-30. 8. Hoelzer, J. D., R. G. Lewis, C. R. Wasmuth, and H. R. Bose. 1980. Hematopoietic cell transformation by REV: characterization of the genetic defect. Virology 100:462-467. 9. Keller, L. H., R. Rutner, and M. Sevoian. 1979. Isolation and development of a reticuloendotheliosis virus-transformed lymphoblastoid cell line from chicken spleen cells. Infect. Immun. 25:694-704. 10. Keshet, E., and H. M. Temin. 1979. Cell killing by spleen necrosis virus is correlated with a transient accumulation of spleen necrosis virus DNA. J. Virol. 31:376-388. 11. Krawetz, S. A., and R. A. Anwar. 1984. Optimization of the isolation of biologically active mRNA from chick embryo aorta.

Biotechniques 2:342-347. 12. Lewis, R. G., J. McClure, B. Rup, D. W. Niesel, R. F. Garry, J. D. Hoeizer, K. Nazerian, and H. R. Bose. 1981. Avian reticuloendotheliosis virus: identification of the hemotopoietic target cell for transformation. Cell 25:421-431. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual, p. 202-203. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Matsuda, H., K. Ikuta, and S. Kato. 1976. Detection of T-cell surface determinants in three Marek's disease lymphoid cell lines. Biken J. 19:29-32. 15. Mussman, H. C., and M. J. Twiehaus. 1971. Pathogenesis of reticuloendotheliosis virus disease in chicks. An acute runting syndrome. Avian Dis. 15:483-502. 16. Purchase, H. G., C. Ludford, K. Nazerian, and H. W. Cox. 1973. A new group of oncogenic viruses, reticuloendotheliosis, chick syncytial, duck infectious anemia and spleen necrosis viruses. J. Natl. Cancer Inst. 51:489-497. 17. Reynaud, C.-A., V. Anguez, H. Grimal, and J.-C. Weill. 1987. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48:379-388. 18. Rup, B. J., J. D. Hoelzer, and H. R. Bose. 1982. Helper viruses associated with avian acute leukemia viruses inhibit the cellular immune response. Virology 116:61-71. 19. Sevoian, M., R. N. Larose, and D. M. Chamberlain. 1964. Avian lymphomatosis-VI. A virus of unusual potency and pathogenicity. Avian Dis. 8:336-347. 20. Shibuya, T., I. Chen, A. Howatson, and T. Mak. 1982. Morphological, immunological, and biochemical analysis of chicken spleen cells transformed in vitro by reticuloendotheliosis virus strain T. Cancer Res. 42:2722-2728. 21. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-507. 22. Thompson, C. B., and P. E. Neiman. 1987. Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell 48:369-378. 23. Tosafo, G., G. E. Mazti, R. Yazcoan, C. A. Heilman, F. Wang, S. E. Pike, S. J. Korsmeyer, and K. Siminovitch. 1986. EpsteinBarr virus immortalization of normal cells of B cell lineage with nonproductive rearranged immunoglobulin genes. J. Immunol. 137:2037-2042. 24. Walro, D. S., N. K. Herzog, J. Zhang, M. Y. Lim, and H. R. Bose, Jr. 1987. The transforming protein of avian reticuloendotheliosis virus is a soluble cytoplasmic protein which is associated with a protein kinase activity. Virology 160:433 444. 25. Weill, J.-G., C. A. Reynaud, 0. Lassila, and J. R. L. Pink. 1986. Rearrangement of chicken immunoglobulin genes is not an ongoing process in the embryonic bursa of Fabricius. Proc. Natl. Acad. Sci. USA 831:3336-3340. 26. Witter, R. L., and L. B. Crittenden. 1979. Lymphomas resembling lymphoid leukosis in chickens inoculated with reticuloendotheliosis virus. Int. J. Cancer 23:673-678. 27. Witter, R. L., L. F. Lee, L. D. Bacon, and E. J. Smith. 1979. Depression of vaccinal immunity to Marek's disease by infection with reticuloendotheliosis virus. Infect. Immun. 26:90-98. 28. Zhang, J., and H. R. Bose, Jr. 1989. Acquisition of new proviral copies in avian lymphoid cells transformed by reticuloendotheliosis virus. J. Virol. 63:1107-1115.


We thank M. Bishop and L. Chen for providing the plasmids containing chicken light-chain sequences and Jean-Marie Buerstedde for defining the nucleotide sequence of the variable lightchain region from two cell lines. This research was supported by Public Health Service grants CA 26169 and CA 33192 from the National Cancer Institute.

MOL. CELL. BIOL.