Proc. Nati. Acad. Sci. USA Vol. 85, pp. 875-879, February 1988 Immunology Precursor B cells transformed by Epstein-Barr virus undergo sterile plasma-cell differentiation: J-chain expression without immunoglobulin (hnmunoglobulin gene rearrangement/transormation/X chromosome-linked agammaglobulinemia) HIROMI KUBAGAWA*t, PETER D. BURROWSt, CARLO E. GRossI*, JIRI MESTECKYt, AND MAX D. COOPERO§ Departments of *Pathology, tMicrobiology and §Pediatrics, The Cellular Immunobiology Unit of the Tumor Institute, The Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294 Communicated by Marian E. Koshland, October 12, 1987

ABSTRACT Human bone marrow cells were depleted of polymeric immunoglobulin, is increased as a function of B lymphocytes to enrich for precursor B cells that could be plasma-cell maturation (4, 5). transformed with Epstein-Barr virus. Transformed immuno- The molecular details of this scheme of mammalian B-cell globulin-negative precursors either maintained their immuno- development have been deduced largely from studies of globulin genes in the germ-line configuration or had under- transformed mouse cell lines. To analyze changes in geno- gone DJ or abortive VDJ rearrangements (V, D, and J type and phenotype that occur during B-lineage develop- represent variable, diversity, and joining gene segments). All ment in humans, we have used Epstein-Barr virus (EBV) to cell lines and their derivative clones, even those with no rescue clones of early B-lineage cells. detectable immunoglobulin gene rearrangements, generated subpopulations of cells that produced high levels ofjoining (J) MATERIALS AND METHODS chain when analyzed by immunoprecipitation after biosyn- thetic labeling and by blot hybridization of cytoplasmic RNA. EBV Transformation of Early Pre-B Cells. Bone marrow Morphologic and immunofluorescence analyses revealed that specimens were obtained with parental consent from five J-chain production was confined to clonal progeny that had boys (ages 2-11 years) with X chromosome-linked agamma- exited the cell cycle to undergo plasma-cell differentiation. globulinemia (XLA) and from four fetuses (14-16 weeks of Analysis of cell surface antigens revealed expression of several gestation) aborted for reasons of maternal health. Mononu- B-cell maturational markers, including complement receptor clear cells from fetal marrow samples were depleted of sIg+ ype 2 (CR2) and plasma cell antigen 1 (PCA-1). Epstein-Barr cells by fluorescence-activated cell sorting (FACS) or by virus can thus transform B-cell progenitors, allowing them to "panning" over anti-Ig-coated plastic dishes (6, 7). The proliferate and undergo terminal B-cell differentiation coupled frequencies of pre-B and B cells were 5.2% and 4.0% before with J-chain expression. These events appear to occur inde- sorting and 7.0% and <0.1% after sorting; corresponding pendently of the immunoglobulin gene status of the trans- values were 8.0%o and 3.8% before panning and 8.3% and formed cells. 0.3% after panning. Wells containing p. + pre-B cells were infected with EBV produced by the B95.8 marmoset cell line During differentiation along the B-cell pathway, a lymphoid (8, 9). EBV-transformed cells were subcloned by limiting stem cell undergoes a series of somatic rearrangements of dilution (9). germ-line immunoglobulin gene segments to generate func- Immunofluorescence Analysis of Cells. Fixed smears of tional immunoglobulin heavy (H)- and light (L)-chain genes. EBV-transformed cells were examined for J-chain expres- The initial rearrangement brings together diversity (D) and sion by two-color immunofluorescence (10) using polyclonal joining (J) gene segments in the H-chain locus on both antibodies (11) and monoclonal antibodies (mAb). mAb chromosomes, and this is followed by joining of a variable NF-11 [IgGl(K) isotype], made against a V,, preparation of (V) gene segment to DJ. VL to JL joining may take place an IgM(A) human myeloma (12) and subsequently shown by subsequently in the L-chain loci (1). immunoprecipitation analysis to have J-chain reactivity These initial gene rearrangements to generate H- and (data not shown), was used with rhodamine-labeled goat L-chain genes occur in bone marrow pre-B cells and result in antibodies specific for mouse immunoglobulin (Southern the emergence of the primary antigen-reactive cells of the B Biotechnology Associates, Birmingham, AL) to detect J- lineage, the surface-IgM-positive (sIgM+) B lymphocytes, chain expression. Cells were counterstained with fluoresce- which are then seeded to the peripheral lymphoid tissues (2). in-labeled goat antibodies specific for human immunoglobu- These cells are small and have a high nucleus/cytoplasm lin (10). Viable cells were similarly stained for surface ratio, and their scant cytoplasm is nearly devoid of organ- antigens and analyzed with a FACS IV (Becton Dickinson). elles. Stimulation with antigen can result in their activation Electron Microscopic Analysis. Cells fixed with 1.25% and differentiation into plasma cells with abundant cyto- glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5) were plasm containing a well-developed Golgi apparatus and washed, postfixed with 1% osmium tetraoxide, dehydrated rough endoplasmic reticulum. This morphological matura- with ethanol, and embedded in Spurr medium (13). Ultrathin tion is paralleled by a shift from production of the membrane form of immunoglobulin to the secreted form (3). Large Abbreviations: H, heavy; L, light; V, variable gene segment; D, amounts of immunoglobulin are then secreted by the termi- diversity gene segment; J, joining gene segment; C, constant gene nally differentiated plasma cells. The expression of joining segment; Am and A., H chains of membrane and secreted forms of (J) chain, a 15-kDa protein involved in the assembly of IgM, respectively; sIg, surface immunoglobulin; EBV, Epstein- Barr virus; XLA, X chromosome-linked agammaglobulinemia; mAb, monoclonal antibody(ies). The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: University of payment. This article must therefore be hereby marked "advertisement" Alabama at Birmingham, T263 Tumor Institute, University Station, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Birmingham, AL 35294. 875 Downloaded by guest on September 27, 2021 876 Immunology: Kubagawa et al. Proc. Natl. Acad. Sci. USA 85 (1988)

sections stained with uranyl acetate/lead citrate were exam- limited subpopulation (8-22%) of cells within each of the ined with a Philips EM 201 electron microscope. clones expressed cytoplasmic J chains (Table 1). Biosynthetic Analysis of Immunoglobulln and J-Chain Mol- The morphology of the cells in these lines and subclones ecules. Cells (5-10 x 106) were cultured for 8 hr at 370C in 1 differed from that characteristic of normal or leukemic pre-B ml of methionine- and cysteine-free RPMI 1640 medium cells; a spectrum of cell types ranging from lymphoblastoid containing 10%o dialyzed fetal bovine serum, [35S]methionine to plasmacytoid morphology was seen in each clone. J (100 MCi), and [35S]cysteine (100 MCi; 1 1ACi = 37 kBq). For chains were expressed strongly in the cytoplasm ofcells with inhibition of N-linked glycosylation, cells were incubated plasmacytoid morphology but weakly, if at all, by cells with with tunicamycin (2.5 ,ug/ml) for 1.5 hr at 370C, washed, and lymphoblastoid morphology within the clone (Fig. 1). More- resuspended in medium containing [35S]methionine and over, in two cell lines examined by electron microscopy and [35S]cysteine for an additional 8 hr in the presence of immunofluorescence, the proportion of J+ cells correlated tunicamycin. Radiolabeled cells were solubilized with 1% closely (± 3%) with the proportion of cells having fully Nonidet P-40 in 0.15 M NaCl/50 mM Tris HCl buffer (pH developed rough endoplasmic reticulum characteristic of 7.4) containing 20 mM E-aminocaproic acid, 0.01% soybean cells with plasmacytoid features (Fig. 1). Cells within these trypsin inhibitor, 20 mM iodoacetamide, and 1 mM phenyl- null cell clones also expressed cell-surface differentiation methylsulfonyl fluoride. Solid-phase immunoisolation was antigens that are characteristically displayed by mature B performed in microtiter wells coated with a rat mAb to cells (e.g., HB2, ref. 21; CR2/EBV receptor, ref. 6; and mouse K chain and then with mouse mAbs to human J-chain HB7, ref. 22), activated B cells (BAC-1, ref. 23), or plasma or immunoglobulin-isotypic determinants (10). Antibody- cells (PCA-1, ref. 24) (Table 1). They also expressed HLA- bound molecules were dissociated by incubation with 2% DR determinants but did not express CALLA, interleukin-2 NaDodSO4/5% 2-mercaptoethanol for 30 min at 37°C, re- receptors, or T-cell antigens (CD1, CD3) in detectable solved by NaDodSO4/PAGE, and autoradiographed. amounts. DNA Blot Analysis. High molecular weight DNA from cell In contrast, J chain was not demonstrable by immunofluo- clones, placenta, and tonsillar T and B cells (12) was rescence in the spontaneously transformed pre-B leukemia digested to completion with restriction endonucleases, elec- cell lines 697 and 207, which are morphologically similar to trophoresed in 0.7% agarose, and transferred to Hybond normal pre-B cells (25), or in several established lines of nylon membranes (Amersham). The membranes were hy- T-cells (MOLT4, MOLT-3, HSB-2), myelomonocytic (HL- bridized at 65°C for 15 hr with nick-translated 32P-labeled 60, U937), or erythroid (K562) lineage. DNA probes: a 3.9-kilobase (kb) Bgl II JH probe (14, 15), a J-Chain Biosynthesis by Null Cell Clones. To examine the 1.3-kb EcoRI ,u constant-region (C,.) probe (14), a 2.0-kb Sac molecular nature of J chains produced by EBV-transformed I JK probe (16), a 3.6-kb EcoRI-HindIII CA2 (Ke-Oz-) null cells, the cells were metabolically labeled and analyzed probe (17), a 3.15-kb BamHI DH probe (a kind gift of S. by immunoprecipitation with a mAb to J chain. A polypep- Korsmeyer and U. Siebenlist), and a 1.6-kb Xba I J-chain tide of Mr 23,000 was immunoprecipitated from the cell probe (18). After hybridization, blots were washed first at lysates (Fig. 2) but not from the culture supernatants (data low stringency (3 x SSC, 65°C) and then at high stringency not shown). The same result was obtained using rabbit (0.2 x SSC, 650C). [Standard saline citrate (1 x SSC) is 0.15 anti-J-chain antibodies. Treatment of cells with tunicamycin M NaCl/0.015 M trisodium citrate, pH 7.0.] reduced the size of the anti-J-chain-reactive molecules to M, RNA Blot Analysis. Total cellular RNA prepared by the 18,000. These results are consistent with the behavior of guanidinium isothiocyanate method (19) was electropho- purified J-chain molecules when analyzed by NaDodSO4/ resed in 1.7% or 1.2% agarose/formaldehyde gels, blotted PAGE (26). In agreement with the immunofluorescence onto nylon membranes, and hybridized to nick-translated findings, immunoglobulin H and L chains could not be J-chain or C,, probes. precipitated from either cell lysates or culture supernatants. J-Chain Gene Transcription. J-chain mRNA of -1.6 kb RESULTS was detectable by blot analysis in all phenotypes of EBV- EBV-Transformed Cells Expressing J Chains Without Im- transformed clones but not in the pre-B-cell lines 697 and 207 munoglobulin Molecules. We examined samples of (i) normal Table 1. Characteristics of EBV-transformed null cell clones fetal bone marrow from which sIg+ B cells had been removed, either by immunofluorescence-activated cell sort- Phenotypic % positive cells ing or by panning, and (ii) bone marrow from boys with marker I-B37 III-1.54 III-3.65 III-11.57 S-22 XLA, whose marrow contained normal numbers of pre-B weeks after EBV J chain 15 22 15 10 8 cells but very few B cells (20). Four to six B-cell antigens* infection, transformation occurred in 60-90% of the culture 12 1-2 the trans- HB-2 41 40 19 12 wells. After expansion for another weeks, CD21 (CR2) 95 11 32 65 77 formed cells were analyzed for immunoglobulin and J-chain In both fetal CD38 99 36 68 57 65 expression by two-color immunofluorescence. BAC-1 11 62 51 26 26 and XLA marrow cultures, almost every well contained cells PCA-1 ND 33 37 62 52 H or L chains. expressing J chains without immunoglobulin CD10 (CALLA) <1 2 1 3 2 1% to The frequency of this J+Ig- phenotype varied from HLA-DR 99 96 97 97 97 50o (mean + SEM: 25.4 + 3.4% for 64 fetal cultures; 15.9 3.5% for 57 XLA cultures). By immunofluorescence, the CD25 (IL-2R) ND 3 3 3 3 intensity of J-chain staining in these Ig- cells was much The expression of surface antigens and cytoplasmic J chain was brighter than that observed for EBV-transformed cells ex- determined by automated flow cytometry (FACS IV) and immuno- only ,u chains or both ,u and L chains. fluorescent microscopy, respectively. The monoclonal antibodies pressing used to detect the CD antigens were as follows: CD21, HB-5; CD38, Five Ig- "null" cell clones were obtained from 6- to HB-7; CD10, W8E6; CD25, Tac-1. No cells expressing surface or 12-month-old bulk cultures of two fetal marrow samples cytoplasmic immunoglobulin were detected in any clones. ND, not (I-B37, 111-1.54, III-3.65, and III-11.57 clones) and one XLA done. marrow sample (S-22 clone) and were\ analyzed further. The *CR2, complement receptor type 2; BAC-1, B-cell activation anti- doubling time of the null cell clones ranged from 40 to 60 hr, gen 1; PCA-1, plasma-cell antigen 1; CALLA, common acute a relatively slow growth rate. Even after subcloning, a lymphoblastic leukemia antigen; IL-2R, interleukin-2 receptor. Downloaded by guest on September 27, 2021 Immunology: Kubagawa et al. Proc. Natl. Acad. Sci. USA 85 (1988) 877

FIG. 1. Immunofluorescence micrograph of EBV-transformed null cells expressing cytoplasmic J chain (Left, x 400) and transmis- sion electron micrograph of cell

I. P A with mature plasma-cell morphol- ogy (Right, x 4000). (Fig. 3). Failure to detect J-chain mRNA in the two leukemic containing fragments, of approximately 1.4, 0.7 and 0.4 kb, pre-B-cell lines was not due to RNA degradation, because were demonstrated in both the I-B37 clone DNA and the A-chain mRNA of -2.7 kb was detected. placental DNA; specifically, fragments of A:1 kb, which Immunoglobulin Gene Rearrangements in Null Cell Clones. would be generated by DQ52-JH, rearrangement, were not To determine the relationship between J-chain expression observed in the I-B37 DNA. These results strongly sug- and immunoglobulin-gene configuration in the null cell gested that the immunoglobulin H-chain genes in this clone clones, we performed DNA blot hybridization analysis. were retained in their germ-line configuration. Since the DNA from the fetal marrow-derived clone I-B37 yielded L-chain genes in the I-B37 clone were also in the germ-line only one JH-containing fragment, identical in size to that context (see below), these results were consistent with the found in placental DNA digested with HindIll, BamHI, or report (27) that EBV could transform precursor B cells in EcoRI (Fig. 4) or with Bgl II (data not shown). These results fetal liver prior to immunoglobulin gene rearrangement. suggested that the I-B37 clone had not undergone JH re- In the other null cell clones, JH gene rearrangements arrangements. In humans, however, the most 3' D gene occurred at either one allele (clone III-1.54) or both (clones segment, DQ52, is located within the JH gene cluster, -90 III-3.65, III-11.57 and S-22) as determined by HindIII (Fig. base pairs to the 5' side of the first functional J gene 4) or BamHI digestion (data not shown). Both JH and Cl, segment, JHl (14). Thus, a DQ52-JH, recombination would probes gave the same hybridization results in BamHI di- not alter the size of the HindIII, BamHI, and EcoRI germ- gests, indicating that no deletion ofthe C, gene had occurred line fragments sufficiently to be detected. Double digests in any of the null cell clones. with Sma I, which cuts To determine whether these JH gene rearrangements were between DQ52 and JH, (14), and Bgl due to DJ or VDJ recombination, we used a human genomic II were therefore performed, as the resultant JH-containing D probe, D3.15 (28). In BamHI-digested placental DNA, fragments are small enough to allow DQ52-JHl rearrange- three major fragments (6.0, 3.7, and 3.2 kb) and a minor ments to be detected. In such a digestion, three JH- fragment of 10.5 kb were detected with the D3.15 probe (Fig. 4). The same was true for the I-B37 clone, which had no .1_, Y,- -, immunoglobulin gene rearrangements. Among the other four C C CC 19g /i +LK' LC- r 'm ml CM r-_>,rCV 66 - .- '-0 ' T CNI N0 sN MCO I II II(a< 0) oI - 66 El El )Ico coEl 45 - probe: *N -2- 45; .. J chain ,wg00 ft*,~ - 1.6 kb - _.. - 25 25 _0..f

10% PAGE 5-15% PAGE FIG. 2. Biosynthetic analysis of J chain and immunoglobulin molecules in EBV-transformed null cell clone. Metabolically labeled YL 2.7 kb proteins immunoprecipitated from Nonidet P-40-solubilized I-B37 C/I :1* 2.4 kb cells by mouse mAbs were analyzed by NaDodSO4/PAGE under I reducing conditions. Asterisk indicates the material from tunicamy- cin-treated cells. The -46-kDa coprecipitating band may be actin. Positions of standard proteins (M, 66,000, 45,000, and 25,000) run in FIG. 3. RNA blot analysis ofJ-chain and C,, gene transcription. parallel are shown. LC, L chain. Downloaded by guest on September 27, 2021 878 Immunology: Kubagawa et al. Proc. Natl. Acad. Sci. USA 85 (1988)

Hm CZ CZ CO r Lo IoP-co.- CZ CZ St LO c C s cLoIO CM .- = (1 a) CY) ) CY) 1) CO ) C- C v ) = Ct n CM m 0 m ' m C m 0 0 c c CZ CZ rC CZ N 0 CZ I 0 0 _ nI m _ sC: H- CL CLH-- co (I -4 3o ~i~-~CD i F- aC M dOm CL _L __ _= _~ __ c,

18 -10.5 kb kb, *4.~~: 17 kb- _,W * -10 kb 10 kb- - aa.' *UPs_ *41Jp.-6.0 -T.. FIG. 4. DNA blot analyses of EBV-transformed null cell clones. *EInmi - 37 Genomic DNA samples were di- DH3.1 proeBa gested with the indicated restric- 4.a tion endonucleases. Hybridization probes corresponded to human JH and DH gene segments. The sizes of germ-line JH and D3.15 fragments t Hind m Bam HI EcoRl DH 3.15 probe, Barm HI were estimated on the basis of the Hind m sizes of HindIII-digested phage A JH probe DNA.

null cell clones with JH rearrangements, three (III-1.54, transcripts were also observed when poly(A) + RNA, rather 111-3.65, and 111-11.57) were also found to contain all of the than total RNA, was analyzed (data not shown). The III- germ-line D gene fragments, although the 10.5-kb minor 11.57 clone contained a transcript of mobility intermediate to band was not easily photographed. In contrast, the S-22 those of Aum and ,u mRNAs. clone exhibited complete deletions of the 3.7- and 3.2-kb fragments accompanied by the appearance of a minor, DISCUSSION non-germ-line fragment of =16 kb; the 6.0-kb genomic fragment was not deleted. Similar deletion patterns were The results indicate that EBV can be used to transform the observed in an EBV-transformed, ,UK' B-cell clone (JB- earliest cells in human marrow that have made the commit- 11.3) that had JH rearrangements of both alleles (Fig. 4). ment to differentiate along the B-cell lineage. The trans- Although the orientation of D3.15 gene segments has not yet formed pre-B cells give rise to Ig- clones that either have been established, these findings suggest that the JH gene not yet undergone any immunoglobulin gene rearrangements rearrangements found in III-3.65, III-11.57, and perhaps or have initiated the earliest rearrangements juxtaposition III-1.54 are likely due to DJ recombination rather than to ofD and J) in the H-chain locus. A similar EBV-transformed VDJ rearrangements. null cell line without apparent immunoglobulin gene re- When the DNAs of tonsillar B and T cells from a single arrangements has been derived from a fetal liver sample (27). normal individual were analyzed with the D3.15 probe, sim- The advantage of the method described here is that by ilar, but incomplete, deletions of 3.7- and 3.2-kb fragments depletion of B cells, one can rescue these progenitors with were observed in the B-cell population (Fig. 4). Again, the relative ease, although their growth rates are slower than 6.0-kb genomic fragment hybridizing with the D3.15 probe those of conventional Ig' B-cell lines. was not deleted in the tonsillar B cells, suggesting that it Transformation of the early pre-B cells results in a popu- might not be linked to the immunoglobulin H-chain locus. lation of proliferating cells responsible for the persistent All null cell clones in BamHI digestions exhibited only one growth of the clone. In addition, a significant fraction of the 12-kb JK-containing fragment identical in size to that in cells exit the cell cycle and differentiate into plasma cells as placental DNA samples (data not shown). HindIII digests defined by classical morphological features and expression also failed to reveal any J'K rearrangements. No rearrange- of a plasma-cell-specific antigen (PCA-1, ref. 24). This ments of the A L-chain locus were detected in the null cell morphological and physiological maturation indicates that clones, although EcoRI restriction fragment length poly- EBV-transformed pre-B cells can undergo all of the genetic morphisms were observed as previously described (17). In events required for terminal plasma-cell differentiation and agreement with previous findings (18), hybridization of a that this differentiation is independent of the rearrangement J-chain probe with EcoRI-digested DNAs from placenta and status of the immunoglobulin genes. Thus the signals pro- all null cell clones revealed one major fragment of 8.0 kb and vided by antigen, antigen-presenting cells, and T cells that one minor fragment of 4.0 kb, suggesting no J-chain gene normally trigger terminal B-cell differentiation can be by- rearrangements in these clones. passed by EBV infection of cells from the earliest stages in C,, Gene Transcription. Blot hybridization analysis of total the B-cell pathway. cellular RNA was used to examine the u H-chain mRNA A most interesting finding is the expression of J chain content of the various cell lines (Fig. 3). EBV-transformed observed in the "sterile" (Ig-) plasma cells. Based on clones with pre-B-cell (III-3.37) or B-cell phenotype (JB- intensity of immunofluorescent staining, the level of J-chain 11.3, LAC-I-32) appeared to synthesize predominantly a expression in the sterile plasma cells is equivalent to that secretory form (.,u, 2.4 kb) of 1 message, whereas the 697 seen in normal plasma cells. The sterile plasma cells appear and 207 pre-B leukemia lines contained message mainly for terminally differentiated and destined to die since, even after the membrane form (Aum, 2.7 kb). All of the Ig- clones subcloning, the frequency of J+ cells did not increase examined were found to contain RNA that hybridized with significantly. Interestingly, the sterile plasma cells failed to the C, probe. The 111-1.54 clone contained a u-chain tran- secrete J chain despite its abundance in cytoplasm. script that was smaller than normal. The III-3.65 and S-22 Studies in both mice and humans indicate that when a lines also contained such transcripts, as well as a species of resting B cell is activated and induced to undergo differen- RNA slightly larger than authentic ILm mRNA. These larger tiation to a plasma cell, the expression of J chain increases Downloaded by guest on September 27, 2021 Immunology: Kubagawa et al. Proc. Natl. Acad. Sci. USA 85 (1988) 879 dramatically (4, 5, 29-32). This is true not only in plasma "pre-B cell" phenotype (40). The EBV-transformed B-cell cells synthesizing IgM and IgA, where J chain may be precursors should also be useful for the analysis of normal required for polymerization of immunoglobulin, but also in development compared to that in patients with defective cells making monomeric IgG, where J chain has no obvious B-cell differentiation. function. A for the of J chain possible explanation presence We thank Dr. M. G. Weigert for his suggestions and support; Drs. in plasma cells synthesizing monomeric IgG and in the sterile S. J. Korsmeyer, U. Siebenlist, and E. E. Max for probes; Drs. plasma cells noted here comes from analysis of the cloned C. G. Carmack, S. A. Camper, M. J. Schlomchik, and G. V. Bor- gene (33). Upstream of the mouse J-chain gene, penta- and zillo for helpful advice; Dr. G. L. Gartland for FACS analysis; and decanucleotide sequences exist that are similar to sequences N. C. Martin, A. Hicks, R. Chow, J. B. Dashoff, and E. A. Brook- upstream of human and mouse VK genes and mouse VA shire for technical assistance. This work was supported by Grants genes. This region has been shown to be important in CA16673, CA13148, A110854, A123694, A118745, and GM20964-14 establishing tissue-specific transcription of VK genes (see awarded by the National Institutes of Health. review in ref. 4). An inverted sequence complementary to 1. Yancopoulos, G. D. & Alt, F. W. (1986) Annu. Rev. Immunol. 4, the decanucleotide is also found upstream of the VH genes. 339-368. 2. Kincade, P. W. (1981) Adv. Immunol. 31, 177-245. If a trans-acting factor that recognizes these conserved 3. Wall, R. & Kuehl, M. (1983) Annu. Rev. Immunol. 1, 393-422. sequences and induces increased transcription of immuno- 4. Koshland, M. E. (1985) Annu. Rev. Immunol. 3, 425-453. globulin genes is produced as a function of plasma-cell 5. Mestecky, J. & McGhee, J. R. (1987) Adv. Immunol. 40, 153-245. 6. Fingeroth, J. D., Tedder, T. F., Strominger, J. L., Barbosa, J. A. & differentiation, J-chain transcription could easily increase, Fearon, D. T. (1984) Proc. Natl. Acad. Sci. USA 81, 4510-4514. irrespective of the status of the immunoglobulin loci. 7. Kuritani, T. & Cooper, M. D. (1982) J. Exp. Med. 155, 839-851. Studies with mice indicate that J chain is expressed only at 8. Miller, G. & Lipman, M. (1973) J. Exp. Med. 138, 1398-1412. 9. Webb, C. F., Cooper, M. D., Burrows, P. D. & Griffin, J. A. (1985) the later stages in the B-cell differentiation pathway (4), Proc. Natl. Acad. Sci. USA 82, 5495-5499. whereas with humans, biosynthesis of J chain has been 10. Kiyotaki, M., Cooper, M. D., Bertoli, L. F., Kearney, J. F. & Kuba- reported in pre-B and Ig- lines derived by EBV transforma- gawa, H. (1987) J. Immunol. 138, 4150-4158. tion of XLA bone 11. Kutteh, W. H., Moldoveanu, Z., Prince, S. J., Kulhavy, R., Alonso, F. marrow (34). J chain has also been & Mestecky, J. (1983) Mol. Immunol. 20, 967-976. detected by immunohistologic or RNA blot analysis in some 12. Kubagawa, H., Mayumi, M., Kearney, J. F. & Cooper, M. D. (1982) J. pre-B leukemias and B-cell lymphomas (18, 35, 36). Expres- Exp. Med. 156,1010-1024. sion of J chain could 13. Spurr, A. T. (1969) J. Ultrastruct. Res. 26, 31-43. throughout B-lineage development 14. Ravetch, J. V., Siebenlist, U., Korsmeyer, S., Waldmann, T. & Leder, indicate that it has functions other than polymerization of P. (1981) Cell 27, 583-591. immunoglobulin and binding of polymeric IgA and IgM to 15. Borzillo, G. V., Cooper, M. D., Kubagawa, H., Landay, A. & Burrows, secretory component (4, 5). Our results suggest the following P. D. (1987) J. Immunol. 139, 1326-1335. 16. Hieter, P. A., Maizel, J. V. & Leder, P. (1982) J. Biol. Chem. 257, interpretation. Sterile plasma cells and Api pre-B "plasma 1516-1522. cells" differentiating within the EBV-transformed lines 17. Hieter, P. A., Hollis, G. F., Korsmeyer, S. J., Waldmann, T. A. & would be the source of J chain. Similarly, some members of Leder, P. (1981) Nature (London) 294, 536-540. clones of and B cells may exit the cell 18. Max, E. E. & Korsmeyer, S. J. (1985) J. Exp. Med. 161, 832-849. neoplastic pre-B cycle 19. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. and undergo plasma-cell differentiation with the concomitant (1979) Biochemistry 18, 5294-5299. and perhaps obligatory expression of J chain. 20. Pearl, E. R., Vogler, L. B., Okos, A. J., Crist, W. M., Lawton, A. R. & All of the Ig- clones contained C,,-hybridizable RNA, but Cooper, M. D. (1978) J. Immunol. 120, 1169-1175. the sizes 21. Abo, T., Landay, A., Balch, C. M. & Cooper, M. D. (1985) Hum. of these differed from authentic Hum and As mRNAs. Immunol. 13, 253-264. The smaller A-chain transcripts in three Ig- lines (III-1.54, 22. Tedder, T. F., Clement, L. T. & Cooper, M. D. (1984) Tissue Antigens 111-3.65, S-22) are likely DJ-CLL transcripts as have been 24, 140-149. observed in murine pre-B-cell lines (37). The larger A RNA 23. Suzuki, T., Sanders, S. K., Butler, J. L., Gartland, G. L., Komiyama, in the and S-22 clones K. & Cooper, M. D. (1986) J. Immunol. 137, 1208-1213. species III-3.65 may be sterile ,A RNA 24. Anderson, K. C., Park, E. K., Bates, M. P., Leonard, R. C. F., Hardy, components (IAs) that initiate within the immunoglobulin R., Schlossman, S. F. & Nadler, L. M. (1983) J. Immunol. 130, H-chain enhancer in the murine JH-C,, intron and that are 1132-1138. especially abundant in B-cell precursors (38, 39). None of 25. Findley, H. W., Cooper, M. D., Kim, J. H., Alvarodo, C. & Ragab, the RNA in the lines A. H. (1982) Blood 60, 1305-1309. components Ig- hybridized with VHI or 26. Koshland, M. E. (1974) Adv. Immunol. 20, 41-69. VHII gene probes (unpublished data), but further analysis 27. Katamine, S., Otsu, M., Tada, K., Tsuchiya, S., Sato, T., Ishida, N., using other VH and JHFC,. intron probes will be required to Honjo, T. & Ono, Y. (1984) Nature (London) 309, 369-372. determine whether they are all sterile transcripts. 28. Siebenlist, U., Ravetch, J. V., Korsmeyer, S., Waldmann, T. & Leder, The molecular P. (1981) Nature (London) 294, 631-635. analysis of human B-cell precursors has 29. Lamson, G. & Koshland, M. E. (1984) J. Exp. Med. 160, 877-892. been difficult, given their low frequency and the lack of 30. Mestecky, J., Winchester, R. J., Hoffman, T. & Kunkel, H. G. (1977) J. known differentiation-stage-specific surface antigens and of Exp. Med. 145, 760-765. techniques for long-term pre-B-cell cultures. EBV transfor- 31. Kaji, H. & Parkhouse, R. M. E. (1974) Nature (London) 249, 45-47. mation of bone marrow B cells 32. Brandtzaeg, P. (1974) Nature (London) 252, 418-419. depleted of should be useful 33. Matsuuchi, L., Cann, G. M. & Koshland, M. E. (1986) Proc. Natl. for this type of analysis, as Abelson leukemia virus has been Acad. Sci. USA 83, 456-460. in the mouse (1). One important difference between EBV 34. McCune, J. M., Fu, S. M. & Kunkel, H. G. (1981) J. Exp. Med. 154, and Abelson transformants is that the former maintain stable 138-145. 35. Hajdu, I., Moldoveanu, Z., Cooper, M. D. & Mestecky, J. (1983) J. Exp. immunoglobulin gene-rearrangement status during in vitro Med. 158, 1993-2006. growth (9), whereas many of the latter can undergo immu- 36. Mason, D. Y. & Stein, H. (1981) Clin. Exp. Immunol. 46, 305-312. noglobulin gene rearrangements involved in V gene assem- 37. Reth, M. G. & Alt, F. W. (1984) Nature (London) 312, 418-420. bly as well as isotype switching in vitro (1). We have already 38. Nelson, K. J., Haimovich, J. & Perry, R. P. (1983) Mol. Cell. Biol. 3, the EBV to 1317-1332. exploited transformation system identify primi- 39. Lennon, G. G. & Perry, R. P. (1985) Nature (London) 318, 475-478. tive B-cell precursors that produce immunoglobulin L chains 40. Kubagawa, H., Burrows, P. D. & Cooper, M. D. (1988) FASEB J., in in the absence of H-chain synthesis, a totally unexpected press. Downloaded by guest on September 27, 2021