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Proc. Nadl. Acad. Sci. USAVol. 89, pp. 11688-11691, December 1992Cell Biology

A different sort of Mott cell(B/W mouse lyuphoa/ mUogOb nseo/Russell body)

HANS-MARTIN JACK*t, GABRIELE BECK-ENGESER*, BARBARA SLOANt, MEO LIE WONGt,AND MATrHIAS WABLt§*Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153; tDepartment of Microbiology and Immunology, Universityof California, San Francisco, CA 94143-0670; and tDepartment of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California,San Francisco, CA 94143

Communicated by Niels K. Jerne, August 4, 1992 (received for review February 18, 1992)

ABSTRACT NYC Is a B lymphoma cell line derived frovnB/W mice. Upon fusion of NYC cells with a _ ,which itself produces no Isunoglobulin, the r lg NYCHhybridoma cells are Mott cells; i.e., they contain large intra-cellular vesicles filled with Immuoglobulin, the so-called Rus-sell bodies. When NYCH.ac, a variant of NYCH that had lostthe ability to produce heavy chain, was transected with aheavy-chain construct, this concentration of iUn buinin the intracellular vesicles ourred only when the rn cimmunoglobulin heavy chain had the same variable region asNYC. Moreover, unlike conventional Mott cells, the hybridcells secrete immunoglobulin at a normal rate.

The rare but spectacular-looking Mott cells are found in cellsmears from lymphoid tissues (1). These cells are lympho-cytes that produce large amounts of immunoglobulin con-tained mainly in large vesicles, the so-called Russell bodies(2, 3). When viewed under the light microscope, Mott cellsoften appear to consist solely of an aggregation of Russellbodies. Mott cells are thought to represent a pathologicalstate of plasma cells, the final stage of B-lymphocyte devel-opment, when immunoglobulin is secreted at a high rate. If,for any reason, plasma cells produce immunoglobulin at arate significantly faster than it can be secreted, it will accu-mulate in the lumen of the endoplasmic reticulum to formlarge vesicles, which give the Mott cell phenotype (4-7).There is a strong selection for a particular antibody vari-

able (V) region in the B-cell tumors of (NZB x NZW)F1hybrid mice, conventionally designated as B/W (8). This factsuggests that these tumors are formed during the clonalexpansion that follows antigenic stimulation. As part of theprogram to elucidate the origin of the recurrent V region, weestablished a B-cell line, NYC, from a spontaneous B/Wlymphoma. Hybridomas generated from NYC cells have theMott cell phenotype. However, as we show below, thesehybridomas secrete immunoglobulin at a normal rate. There-fore, Russell bodies must form in these cells for completelydifferent reasons than they do in previously described Mottcells.

MATERIALS AND METHODSCell Culture. All cell lines were grown in RPMI 1640 or

Dulbecco's modified Eagle's medium supplemented with10% (vol/vol) fetal bovine serum (HyClone Laboratories), 4mM glutamine, 0.05 mM 2-mercaptoethanol, 0.1 mM sodiumpyruvate, 50 units of penicillin per ml, and 50 jig of strepto-mycin sulfate per ml. The B lymphoma NYC is derived froma spontaneous B-cell tumor that originated in a B/W mouse(9).

Cell Fun. To generate hybridoma NYCH, NYC lym-phoma cells and the hypoxanthine/aminopterin/thymidine(HAT)-sensitive plasmacytoma Ag8.653 (ratio, 2:1) werefused with polyethylene glycol 4000 (Boehringer Mannheim).Because NYC lymphoma cells are not HAT-sensitive, io-doacetamide selection (10) against unfused NYC cells wasused. Briefly, 4 x 107 NYC cells were resuspended in 20 mlof serum-free RPMI medium, and freshly prepared 0.2 Miodoacetamide (Sigma) in distilled water was added to give afinal concentration of 2 mM. After 25 min at 370C, serum wasadded to a final concentration of 50%1. The cells were washedtwice in medium with fetal bovine serum and once in serum-free medium prior to fusion and selection in HAT medium.

unolu Analysis. The specificity of subclass-specific fluorescein- or Texas Red-labeled and unlabeledantisera (Southern Biotechnology Associates, Birmingham,AL) were tested on plasmacytomas or hybridomas producingvarious immunoglobulin classes and purified, ifnecessary, onan appropriate immunoglobulin column. To.detect membraneproteins, 2 x 106 cells in suspension were incubated withfluorescein- or Texas Red-labeled antibodies for 20 min onice. The cells were fixed onto a glass slide in absolute ethanoland covered with the mounting medium Cytoseal (VWRScientific). To detect cytoplasmic proteins, 5 x 104 cells werefixed onto a glass slide and incubated for 10 min at roomtemperature. The glass slides were washed in phosphate-buffered saline containing 1% bovine serum albumin and0.1% NaN3.Laeling and mnrepion of Cdhear Proteins.

Biosynthetic labeling of cultured cells was performed essen-tially as described by Burrows et al. (11). Cells (2-5 x 106 perml) were incubated for 2-6 hr in methionine-free RPMI 1640medium supplemented with I35S]methionine (1000 Ci/mmol,100 mCi/ml; Amersham 1 Ci = 37 GBq) and 10% dialyzedfetal bovine serum. Culture supernatants were filteredthrough 0.22-pum membrane filters and incubated with spe-cific antisera on ice overnight. Antigen-antibody complexeswere precipitated by the addition of heat-inactivated, forma-lin-fixed Staphylococcus aureus (12) and incubation for 15min. S. aureus pellets were washed as described (11). Solu-bilized protein samples were reduced with 2-mercaptoetha-nol and electrophoresed in SDS/10%o polyacrylamide gels.Labeled proteins were detected by fluorography.Transection ofDNA into Cultured Cell Lines. Some 5 X 106

cells were washed in protein-free RPMI 1640 medium, sus-pended in 500 ml of the same medium, and mixed with 5-20pAg of circular or linearized DNA. The mixture was electro-porated with a Cell Porator (BRL) at 330 IkF, 285 V, lowresistance. The cells were cultured in 10 ml RPMI 1640supplemented with 201% fetal bovine serum and supplementsas described above. After 2 days, the cells were suspended at104 per ml in RPMI 1640 containing 10% fetal bovine serum,

§To whom reprint requests should be addressed.

11688

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact. -

Proc. Natl. Acad. Sci. USA 89 (1992) 11689

FIG. 1. Fluorescence photomicrographs of NYCH hybridoma cells. The cells were fixed and incubated with a polyclonal, fluorescein-coupled, goat anti-c antiserum. (Upper) In addition to the uniformly distributed cytoplasmic IgM characteristic of hybridoma cells, IgM isconcentrated in vesicles that resemble the Russell bodies of Mott cells. (Lower) Evenly distributed oglobulin in two NYCH.Ktransfectomas, NYCH.K.ILT! and NYCH.K.icT2, derived from NYCH.K by stably transfecting it with a ,u gene using aV segment different fromthat used by the endogenous A gene in NYCH (ppgpt; see Fig. 3b). Staining was with fluorescein-labeled goat antibodies to mouse g chain.

1.25 Ag of mycophenolic acid (Calbiochem) per ml, and 250,ug ofxanthine per ml. One milliliterwas distributed in 24-wellCostar plates, screened for transfectants at day 7-10, and fedat day 14. We obtained an average transfection frequency of10-5 for plasmacytomas and hybridomas. The transfectantswere screened by cytoplasmic immunoflourescence andSDS/PAGE of immunoprecipitated radiolabeled proteins.

Construction of a Eukaryotic Expresion Vector. The pro-totype expression vector pp~gpt is a modification of pA (13),kindly provided by Rudi Grosschedl, University of Califor-nia, San Francisco; pA contains a complete genomic Au generearranged {VDJ}H17.2.25 (where V, D, and J representvariable, diversity, and joining gene segments and H indi-cates heavy chain), and A-chain constant region (C, genesegments of allotype a]. The Nae I restriction site wasconverted into a Cla I site allowing us to replace the Sal I-ClaI fragment. We then introduced the Escherichia coli gene gpt,encoding guanine-phosphoribosyltransferase, into the singleXho I site of pA&. Mammalian cells expressing gpt grow in thepresence of xanthine and mycophenolic acid. To makepgt.NYC, we replaced the Sal I-Cla I fragment by a fragmentthat contained the productively rearranged {VDJNh genesegment expressed in NYC cells. To generate this fiagmentwe inserted a4.3-kilobaseXba I fragment, which was isolatedfrom a A phage library and contained the productively rear-

ranged {VDJ}H gene segment, into the plasmid vector pblue-script KS(±) (Stratagene). The Xba I site upstream of thepromoter region was converted into a Sal I site, and the NaeI site downstream ofthe JH4 gene segment was converted intoa Cla I site. The Sal I-Cla I fragment was isolated and clonedinto the Sal I-Cla I vector fragment.

RESULTS AND DISCUSSION

NYCH Cels Contain IgM In Veicles, and They Abe SecreteIt. We fused cells of the B lymphoma NYC to the myelomaAg8.653, which by itself does not produce any immunoglob-ulin chain, and obtained the set ofNYCH hybridomas. Only22 of 32 hybridomas expressed A chain, perhaps because ofa growth disadvantage conferred by IgM synthesis. TheA-positive hybridomas accumulated pg-negative cells muchfaster than other hybridomas derived from spleen cells. Inanother fusion, assayed at a later stage of clonal develop-ment, only 9 of 20 hybridomas expressed AL chain.Under the fluorescence microscope, the A-positive cells

contained vesicles that were filled with IgM (Fig. 1 Upper)and were reminiscent ofthe immunoglobulin-containing Rus-sell bodies in Mott cells. For comparison, Fig. 1 Lower showstwo hybridoma cells without Russell bodies.

Cell Biology: Rick et al.

Proc. Nadl. Acad. Sci. USA 89 (1992)

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FIG. 2. Endogenous and transfected IgM secreted by NYCHhybridomas. V5S]Methionine-labeled immunoglobulin chains immu-noprecipitated from cell culture supernatants with an antiserum thatreacts with both A' and K chains were analyzed by SDS/PAGE underreducing conditions. NYCH (lane 1) is the original hybridomasecreting both I and K. NYCH.K (lane 2) is a variant of NYCH thathas lost the expression of IL. NYCH.K.IAT1 (lane 3) andNYCH.K.ILT2 (lane 4) are derived from NYCH.K by stably trans-fecting it with a , gene using a V segment different from that usedby the endogenous g gene in NYCH (p~&gpt; see Fig. 3b).

As mentioned in the Introduction, a Mott cell is thought tobe a plasma cell that, because of a defect in the particularimmunoglobulin produced by that cell, fails to secrete it.However, the NYCH hybridomas secrete immunoglobulincopiously. Complete IgM can be precipitated by a polyclonalrabbit anti-mouse IgM (Fig. 2, lane 1) or goat anti-mouse ,u(data aot shown) at a rate comparable to that of IgM mole-cules containing other C,, and VH regions-e.g., NYCH.KT1and NYCH.KT2 (lanes 3 and 4). Thus, although IgM is foundin large- quantities in vesicles, there is no defect in thismolecule that impedes its secretion.NYC 1gN Binds to the Intracellular Vesicles via Its Variable

Regipn. Although the immunoglobulin-containing vesiclesare reminiscent of the Russell bodies in Mott cells, the factthat the imnmunoglobulin is also secreted means that theNYCH Russell bodies must be of a very different origin. Ifthere is no defect in the ability ofthis molecule to be secreted,why is NYCH IgM found in the intracellular vesicles? It maybe targeted via its C region to this compartment, or it maybind to a component of the vesicles via its V region; i.e., thiscomponent could be the cognate antigen of the immunoglob-ulin receptor of the lymphoma, a possibility indicated by thefact that we found that NYC IgM binds to antigen of a virusthat is 'produced by the NYC cells (14). To distinguishbetween the two possibilities, we performed transfectionswith two I gene constructs: pjugpt (Fig. 3b) included the{VDJ}H 17.2.25 segment (13), which is different from the oneexpressed in the NYC line, and ppuNYC (Fig. 3c) included theVH exon from NYC; both constructs contained the C,, genesegments from pju (Fig. 3a and ref. 13)-i.e., not from theNYC line. When transfected into NYCH.K, a variant ofNYCH that produces K light chain without heavy chain, theg chain with {VDJ}H 17.2.25 (pugpt) was made in normalamoqnts, associated with the endogenous K chain, and wassecreted into the growth medium, where it could be precip-itated by a polyclonal goat anti-IgM (Fig. 2, lanes 3 and 4).When an antiserum specific for ju was used for precipitation,

K light chain was coprecipitated from medium ofNYCH.KT1and NYCH.KT2 cells but not from medium ofNYCH.K (datanot shown). Yet the IgM from this construct was not trappedin the vesicles. The cytoplasm ofthe transfected cells stainedevenly with anti-pu antibody (Fig. 1 Lower).The construct pjuNYC allowed us to formally rule out the

possibility of a mutation in the C,,gene segment. The Augenesegment encoding NYC VH restored the targeting to thevesicles (results not shown). As in the original NYCH IgM,the molecular weight and the rate of secretion of the resultingIgM molecules were normal. Therefore, we conclude fromthe transfection experiments that the endogenous IgM spe-cifically (i.e., via its V region) binds to antigen present in thevesicles.We also selected two "switch" variants from the NYCH

line that seemed to contradict the conclusion reached above:one variant synthesized IgGl, the other IgG3. Neither ofthese isotypes was concentrated in the vesicles, and thisresult could be taken as evidence for retention of the NYCIgM via the C region. However, the behavior of these twovariants is also explicable by the low affinity ofthe combiningsite, which we previously noted when discussing the effect ofTriton X-100 in blocking the precipitation of a 31-kDa proteinby NYC IgM (14). Although the V region of the IgM ispresumably the same as that of both IgG isotypes, the IgMmolecules are assembled to pentamers in the endoplasmicreticulum. Thus intracellular IgM will have a much higheravidity than IgG. On the basis of the latter interpretation,there would be some IgG binding to the vesicles, but therewould not be enough to be seen above the background levelof IgG uniformly distributed throughout the cytoplasm.

Possible Origin of the Russell Body-Like Structures inNYCH Mott Cells. When we stained the cytoplasm of cells ofthe NYC lymphoma with fluoresceine-labeled anti-A anti-bodies, there were many bright spots that were much largerthan is usual in B lymphocytes. Under the electron micro-scope, there were vesicles filled with a loose, irregularlyspaced membranous material or with a denser material,which was sometimes enclosed by membranous whorls. Wethink that these vesicles, which often contained virus-sizedspheroidal particles, correspond to the bright spots seen influorescence microscopy.We have recently shown that NYC IgM is specific for an

antigen of a retrovirus carried by NYC. Moreover, it wasimpossible to obtain variants of NYC that had lost thecapacity to produce IgM. Thus the basis for maintaining thetumor state of NYC seems to be the binding of the antibodyproduced by these cells with its cognate antigen produced bythe same cells (14). Interestingly, Russell (2) interpreted theintracellular bodies named after him, which he found inmarginal areas of tumors, as the etiological agent of cancer.We speculate that the vesicles in the hybridomas are com-posed ofabnormally polymerized viral proteins, analogous tothe polyhead structures of phages (reviewed in ref. 15).Further ultrastructural studies of these vesicles are clearlywarranted.Coding Remarks. In classical Mott cells, the Russell

bodies are presumably formed by distortion of the endoplas-mic reticulum because the structure of a particular immuno-globulin does not allow it to be efficiently secreted (7). If thatimmunoglobulin could be efficiently secreted, the classicalRussell body would not exist. In NYCH Mott cells, thevesicles are presumably formed by a defective retrovirusindependently of immunoglobulin production. Part of theNYC immunoglobulin, which otherwise is as easily secretedas any other immunoglobulin, is trapped in preformed vesi-cles because of its specificity for a component ofthe vesicles.NYCH Mott cells are hybridomas and, as such, are ex-

perimental artifacts. But there is no reason to believe thatNYCH Russell bodies would not arise in vivo. Imagine that

11690 Cell Biology: Rick et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 11691

aNa. I

Sail `

Ampr

b cClal Cla I

Sail

1

M1,2

Immunoglobulinintrons, promotorand enhancersequences

Ig exons A sm Pog(A)

FIG. 3. Schematic representation of the plasmids transfected into the su-negative variant NYCH.K. (a) Plasmid pL (13) contains a ," geneencoding both the secreted and the membrane form of the s heavy chain; the rearranged {VDJ}H segment VH17.2.25 is separated from the C.segments 1-4 by the major intron. Transcription of this gene is driven by the immunoglobulin heavy-chain promoter. The ampicillin-resistance(Ampr) gene allows selection in bacteria in medium containing ampicillin. A, and Am indicate the polyadenylylation signals for the mRNAsencoding the secreted and the membrane form of A chain, respectively. M1,2 indicates the two membrane-form-specific exons. (b) In ppugpt,the gpt gene, which permits the selection oftransfected mouse cells in medium containing mycophenolic acid and xanthine, has been introduced.(c) pNYC is similar to pjt, but the VH17.2.25 segment has been replaced by VHNYC, the {VDJ}H segment of the NYC .& chain.

aB/W lymphoma cell in vivo were to receive a differentiativesignal. Now that cell would develop into a clone ofMott cellscontaining NYCH-type Russell bodies. Indeed, large num-bers of Mott cells were seen in the mesenteric lymph nodesof a 17-month-old B/W mouse (5). Moreover, differentiationof a clone of B lymphocytes into Mott cells, albeit conven-tional ones, has been described in a patient with a lymphopro-liferative disorder (16).

We thank Helen Engh, Mindy McDowell, and Nancy Chiang fortechnical help; Michael Bishop, Geoffrey Davis, Leon Levintow,and Charley Steinberg for discussions and extensive editing; andRudi Grosschedl for plasmids. This work was supported by NationalInstitutes of Health Grant lRO1 GM37699, by University of Cali-fornia, San Francisco, Grant MSC no. 03 Simon Fund, and by fundsfrom the Markey trust to M.W., as well as by Potts Foundation GrantRCC/OI, LU no. 4004, Loyola University, Chicago, to H.-M.J.

1. Mott, F. W. (1905) Proc. R. Soc. London 76, 235-242.2. Russell, W. (1890) Brit. Med. J. 2, 1356-1360.3. Bessis, M. (1977) Blood Smears Reinterpreted (Springer, Ber-

lin).

4. Weiss, S., Burrows, P. D., Meyer, J. & Wabl, M. R. (1984)Eur. J. Immunol. 14, 744-748.

5. Alanen, A., Pira, U., Lassila, O., Roth, J. & Franklin, R. M.(1985) Eur. J. Immunol. 15, 235-242.

6. Schweitzer, P. A., Taylor, S. E. & Shultz, L. D. (1991) J. CellBiol. 114, 35-43.

7. Valetti, C., Grossi, C. E., Milstein, C. & Sitia, R. (1991) J. CellBiol. 115, 983-994.

8. Tarlinton, D., Stall, A. M. & Herzenberg, L. A. (1988) EMBOJ. 7, 3705-3710.

9. Wofsy, D. & Chiang, N. Y. (1987) Eur. J. Immunol. 17,809-814.

10. Wright, W. E. & Hayflick, L. (1975) Nature (London) 256,495-497.

11. Burrows, P. D., Beck, G. B. & Wabl, M. R. (1981) Proc. Natl.Acad. Sci. USA 78, 564-568.

12. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624.13. Grosschedl, R., Weaver, D., Baltimore, D. & Costantini, F.

(1984) Cell 38, 647-658.14. Jack, H.-M., Beck-Engeser, G., Lee, G. & Wabl, M. (1992)

Proc. Natl. Acad. Sci. USA 89, 8482-8486.15. Kellenberger, E. (1990) Eur. J. Biochem. 190, 233-248.16. Posnett, D. N., Mouradian, J., Mangraviti, D. J. & Wolf, D. J.

(1984) J. Clin. Invest. 77, 125-130.

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