Scand. J. Immunol. 19, 447-^55. 1984

Different Fate of Antibodies to Surface IgM andIgD in Germinal Centre Cell-AssociatedLymphomas

E . R U U D . K . B E I S K E , A. DRACK, H . B . STEEN &T . GODALLaboratory for Immunology, Department of Pathology: and The Norwegian Cancer Society; andDepartment of Biophysics; Norsk Hydro's Institute for Cancer Research, The Norwegian RadiumHospital, Montebello. Oslo, Norway

Ruud, E., Beiske, K., Drack. A.. Slcen. H.B. & Godal, T. Different Faie of Amibodies loSurface IKM and IgD in Germinal Centre Cell-Associated Lymphomas. Scand. J. Immunol.19, 447-i'55. 1984.

In all of four human germinal centre cell-associated B lymphomas carrying s!gM + sIgD F(ab')2fragmenis of rabbit antibodies to human /(-chain vvero accumulated intraccMularly. whereas iheaccumulation of antibodies lo rt-chain was considerably less abundant. Thu accumulalion ofantibodies to lighi chain was intermediate between aniihodies to f- and (Vchain. These resultscould be explained by the following observations: (i) the difference in ihe level oi' reexpressionof sIgM and sIgD and (ii) the discovery thai antibodies to <S-chain bound to reexpressed sigDwere degraded at a higher rate ihan those bound lo primary slgD and at a higher rate thanantibodies to /i-chain, whether bound to primary or reexpressed sIgM.

Erik Ruud. Laboratory for Immunology. The Norwegian Radium Hospital. Montebello. Oslo3. Norway

A large proportion of B cells express bothsurface IgM (sIgM) and sIgD. sIgD has beenshown to appear after sIgM during B celldevelopment [27, 28], and sIgD-positive B cellsare involved in primary immune responses [2,6],but mature memory B cells lack sIgD [2, 13].Thus. sIgD appears to be present during alimited period of B ceil development andinvolved in the generation of memory B cells.

In neoplastic human B cells induction of DNAsynthesis with both antibodies to sIgM and sIgDwhen combined with 12-O-tetradecanoyl-phorboI-13-acetate (TPA) has been reported[10, 12]. In a recent report [20] we described aclear distinction between the functional prop-erties of sTgM and sIgD in a human germinalcentre celi lymphotna in which TPA-hanti-^i-chain antibodies induced DNA synthesis andincreased cytoplasmic Ig content, whereasTPA-i-anti-(%chain antibodies induced DNAsynthesis only. During these studies we observedthai antibodies to u-chain accumulated in-tracellularly, whereas very little accumulation ofantibodies to d-chain could be detected. In this

report we present data showing the samedifference in the accumulation of antibodies to/(- and d-chain in four human B-cell lymphomas.

MATERIALS AND METHODS

Lymphoma cell .suspensions. The present study wascarried oul on four different human B-lymphoma cellpopulations. Two of ihc lymphoma cell suspensions(242/80 and 209/78) were derived from lymph nodes asdescribed elsewhere [11], whereas two (214.''81 and90/80) were loukaemic cells separated from peripheralblood after centrifugation on it Ficoll-lsopaque gra-dient [4]. A significant proportion (iy-71'/f) of theneoplastic population in all four patients had germinalcentre cell morphology, indicating differentiationarresl analogous to a maturation siage of germinalcentre celts. For details about hislological classifiea-lion and surface phenotype, sec Table I.

Aliquots of ihe cell suspensions were stored frozenon liquid N^ with 10^/ dimeihyt sulphoxide (DMSO)and 25% heai-inactivated foetal bovine serum (FBS).The viability of thawed samples always exceeded 90%.as assayed by trypan blue dye exclusion. Cell cuilureswere set up in RPMI 1640 with penicillin andstreptomycin and supplemented with lOCf FBS.Cultures were kept at 37°C,5''f CO^ in humidified air.

447

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Antibodies to Immunoghhtdins. Rabbit antibodieslo human Ig heavy and liglil chains were purchasedfrom Dakopaus (Copenhagen. Denmark) with Ihefollowing specificities: anti-H-chain (code 10-091),anti-d-chain (code A-093), anti-A-chain (code 10-9L5).and anti-x-chain (code 10-9K.'i). F(iib')> fragments ofthese atilihodies were prepared by the method ofTurner et al, \2t\. Nortnai rabbit Ig (code X9I)3) wasalso from Dakopaits. Fluorescein isothiocyanate{FITC)-conjugated swine anti-rabbit immiinoglobuliti(code F2I90) was also from Dakopatts. Swineanti-rabbit immunoglobulin (code Z l%i was coupledto CNBr-aclivaied Sepharose 4B (Pharmacia) inaccordance with the dirccti<ins of the nianulaciurer.

F(ah')n fragmetils of rabbit anii-human Immunoglo-bulin were radioiodinated by using lodogen asdescribed elsewhere [2.^|. ''^I-Na. 4(H)() MBq/ml. wasobtained from the Radiochemical Centre. Amersham,England. Separation nf free ''^I-Na from protein-bound radioactivity after iodination was performed bygel fillralion on Bio Get P-ICR).

Measurement of accumulated antihodies in culturedcells. Cells were cultured for various intervals in thecontinuous presence of unlahelted F(ab'):; rabbitanti-human immunoglohulin and TPA (from Consoli-dated Midland Corp.. Brewster. N.Y.; final TPAconcentration in all experiments. 75 nM). Ceil-assoeiated rabbit anti-human immunoglobulin(surface+ CY[oplasmic) and DNA content were mea-sured simultaneously in single cells, using alaboratory-buill flow cytometer [24| as describedelsewhere [20]. Cultured eells were washed once inIce-eold phosphate-buffered saline (PBS) (Dulbccco)and subsequently resuspended in 1 ml ice-cold PBS.For fixation %*> ethanol was added lo the cellsuspension under constant pipetting during 5 min untilthe final elhano! concenlralion reached KV/r. The cellswere stained with FITC-conjugated swine antibodiesto rabbit immunoglobulin ( KH) ^i\ of a 1/80 dilution inminimal essential medium (MEM)) and ." 0 ugi'ml ofthe DNA stain propidium iodide (PI) after the sampleshad been treated with \% RNase (Sigma).

Cyloeentrifuge preparations of cultured cells werefixed in a formol sublimate solution and stained foraeeumulated rabbit anti-human immunoglobulins bythe peroxidase-anti-peroxidase (PAP) immunecomplex technique initially introduced by Sternbergeret ai |25]. as described elsewhere Il6| .

Measurement of slg ree.xpression. To remove sIgMor slgD. the cells were incubated lor I h at TiTC withTPA and 75 ug/ml unlabelled rahbii antibodies to /i- orrt-chains. respectively. Removal of one class of slgwith isotype-specific aniibodies did not significantlyalter the expression of the other SIg class. Alter 1 h ofincubation at .i7"C the cells were washed three times inice-cold RPMl 1640 and reeultured for variousintervals at a concentration of 10" cells/ml with TPAalone. Evidence of removal and internalization of slgafter 1-4 h of incubation at 37°C was ascertained withflow eytometry, as intact cells did not stain withFITC-eonjugated isotype-speeific antibodies orF219O. whereas fixed cells stained positively. Formeasurement of reexpressed slg. the cells were spundown, and O.d.S u% '" I-Na-labelled rabbii antibodies

Different Fate of Antibodies to sIgM and sIgD 449

to human /i- or fi-chain (specific activity, 7.7x10"cpm//ig)in^O/il PBS with O.IV FBS was added. Thesamples were incubated for 1 h on ice and washedthree times in ice-eold RPMl 1640 with 0.1 % FBS. andradioactivity counted in a 12-channcl LKB gammaspectrometer. Evidence of slg reexpression on asignifieant number of the cultured ceils was obtainedwith flow cytometry. The cultures were treated asdescribed above, except that the ceils were labelledwith FITC-conjugated rahhil aniibodies to human -or d-chain under saturating conditions 1111—foreMmpte, 100 /tl of a 1/40 dilution in MEM for 1 h onice.

Measurement of anli-lg rclfa.se from cultured celts.Both the initial and the reexpressed slg were taggedwith '• "'I-Na rabbit aniibodies to human Ig for 1 b onice. Tlie eells were washed three times in ice-coldRPMl with 0.1% FBS and subsequently reculturedwith TPA alone for various intervals, after which Ihecells were spun down, and radioactivity in ihesupernatant was measured. Cell-associated radioactiv-ity was measured after one wash in ice-cold RPMl1640. Supernatants were absorbed for 2 h at 20°C on arocker with 2.5rf (v.'v) swine antibodies to rabbit Igeoupfed to Sepharose heads. ITie beads were spundown, and the remaining radioactivity in the super-natant (degraded anti-Ig) was measured. Radioactivityassociated with the Sepharose beads (antigenically

intact anti-Ig) was measured after ihrcc washes iniee-cold RPMl 1640 with 0.1% FBS.

RESULTS

Accumtilation of anti-Ig in cultured cells

During a study of the induction of prolifera-tion and cyloplastnic Ig expression by antibodiesto sIgM or sIgD and TPA in human B celllymphonijts. we observed ihut rabbii antibodiesto IgM accumulated Jntraccilularly. whereasvery little such accumulation was observed withantibodies to IgD. The basic observations areshown in Figs I and 2. Fig. I shows cytocentri-fuge preparations of cells from btopsy 214/81,cultured for 72 h with TPA and rabbit antibodiesto human fi- or (^chain. After fixation the cellswere stained for the presence of rabbit im-munoglobulins by the PAP technique. Anti-bodies toii-chain (Fig. la-d) were found partlybound to the cell surface and partly accumulatedin vesicle- or tubule-like structures correspond-ing to the Golgi zone, even in morphologically

FIG. 1. Micrographs of eytocentrifuge preparations of ceils from biopsy 214/81cultured with TPA and rabbit antibodies to human /j- or (Vehain for 72 h. The cellswere fixed and stained for the presenee of rabbit immunoglobulins hy thePAP-technique. la-d. Cells cultured wiih anti-/(-t-TPA show accumulation ofrabbit immunoglobulins in vesicle- and tubule-like structures in lhe Golgi-zone(arrows) irrespective of either immunoblastic (Fig. I a and h) or ptasmablastic (Fig.Ic and d) morphology. The cell in Fig. la additionally presents hinding of rabbitimmunoglobulins to the cell surfaee (arrowheads). Note weaker staining ofaeeumulated than surface-bound immunuglobulins (objective. 1{H)/1.2?). le. Cellscultured with anii-*') + TPA show no significant surface-bound or accumulated rabbitimmunoglobulin (objeclive. AM).90).

450 E. Ruud et al.

diffcrctit cells. The same fixation and stainingprocedure, when applied to celis cultured withantibodies to < -chain, did not reveal significantaniount,s of rabbit immunoglobulins either onthe surface or in subccllular structures (Fig. le).

The results of the flow cytofluorometricanalysis of anti-Ig accumulation (Fig. 3) showedthat cells from all four lymphomas accumulatedantibodies to /(-chain, whereas very little accu-mulation of antibodies to (Vchain could bedetected. A flow cytofluorometric study ofaccumulation kinetics in lymphoma 214/81showed that the bulk of anti-n-chain was ac-cumulated between 24 and 72 h of incubation.Moreover, only minute amounts of additionalanti-/j-chain was accumulated between 72 and 96h of incubation (data not shown). The accumula-tion of anti-light chain was between that ofanti-/i- and anti-d-chain (Fig. 2). However, nostraight correlation was obtained between anti-

light-chain accumulation (Fig. 2) and the initialratioofsIgD to slgM (Table I). Both this lack ofcorrelation and lhe difference between anti-bodies to sIgM and slgD could at least in part bedue to differences in (he level of reexpression ofsIgM and sIgD. Among the lymphomas. 214/81showed both the highest slgD/slgM ratio (TableI) and the highest level of reexpression. Thislymphoma was therefore selected for furtherstudies.

sIg reexpression

A kinetic study of sIg reexpression in lym-phoma 214/81 is shown in Fig. 3. The cellsreexpressed sIgM at about the original valueafter 24 h of incubation, whereas sIgD stabilizedat a level of about lO'-v of the original value. At72 h of incubation sIgM had increased to aboutn^'^f of the original value, wbereas no increase

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Different Fate of Antibodies to sIgM and sIgD 451

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FIG. 3. Reexpression of (•) slgM and (•) slgD on cellsfrom lymphoma 214/81 at MX' as measured by' *I-anti-;j or '''l-anti-d labelling, respectively. Toremove the initial sIg. the cells were cultured with 75nM TPA and 75 /ig'ml unlabellcd antibodies to u- orA-ehain for I h at MX'. Al zero hours the cells werewashed and recultured with 75 nM IPA alone. Noreexpression of either (O) slgM or (0) sIgD could bedelected at J'C.

in sIgD was detected, sIg reexpression could notbe detected when the cultures were kept at 4°C.Concomitant removal of the initial sIgM andsIgD in lymphoma 214/81 with antibodies to Alight chain resulted in the same pattern of sIgreexpression as ihat depicted in Fig. } (data noishown). The studies of lymphoma 214/81 (Fig. 3)showed that, although sIgD became reexpressedin relative terms at a lower level, the totalamount of sIgD as compared with sIgM duringthe whole period appeared to be of the samemagnitude. The negligible eytoplasmic accu*mutation of sIgD could therefore not beexplained by differences in the level of reexpres-sion. The degradation of antibodies to sIgM andsIgD was therefore studied.

Release of radioactivity from cultured cellsTo determine the fate of the initially bound

antibodies, sIg were tagged with '"^I-Na-29

labelled antibodies to sIgM or sIgD. The resultsof the analysis are shown in Fig. 4a. As shown inFig. 4a, the radioactivity of antibodies to ft and dheavy chains was released from the cells atsimilar rales when bound to the initial sIg. Whenbound to reexpressed sIg (at 24 h). however, lheradioactivity of anti-(^-chain aniibodies dis-appeared from the cells at a considerably higherrate than that of anti-//-chain antibodies, and theradioactivity was recovered in the supernatant(Fig. 4b). Thus, antibodies to 6 heavy chainbound to initial and reexpressed sIgD wereprocessed differently, whereas radioactivity ofanti-//-chain aniibodies was released from thecells at a similar rate wbether bound to initial orreexpressed slgM (Fig. 4a nnd b).

Stimulation of cells carrying initial sIg withantibodies lo /(-chain. TPA. or medium alonedid not induce the expression of slgD with theability to clear the cells rapidly of aniibody to5-chain (Fig. 5). As shown in Fig. 5, theexpression of functionally changed sIgD de-pended on the interaction of the initial sIgD withantibodies tu d-chain.

Absorption of supernatanls

To determine the degree of degradation of theradioactive material released from the cells, thesupernatants were absorbed with swine anti-rabbit lg coupled to Sepharose beads. Theresults are shown in Fig. 6. About the samerelative amount nf radioactivity was removed bysuch beads from supernatants of cultures car-rying primary slgM or slgD. respectively (Fig.6a). In supernatants from cells carrying reex-pressed sIg. considerably less antigenicallyintact antibody lo A-chain than to /i-chain wasdetected (Fig. 6b). Thus, antibodies to tVchainbound to reexpressed slgD were degraded at ahigher rale than aniibodies to //-chain bound toeither primary or reexpressed sIgM (Fig. 6a andb).

DISCUSSION

Two mechanisms eould contribute to theobserved difference in intracytoplasmic accu-mulation of F(ab')i anti-//- and anti-< -chainantibodies; (i) differences in the level ofexpression, reexpression. and turnover of slgMand sIgD. and (ii) differences in the degradationof specific antibodies bound to slgM or sIgD.

452 E. Ruud et al.

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FIG. 4. Release of radioactivity from cultured cells of lymphoma 214/81 carrying (a) primary sIg or(b) reexpresstd sIg. The cells were labelled with iodinated antibodies to ft- or (%chain al zero hours,washed, ;ind consequently cultured wilh 75 nM TPA. {•) Cell-associated radioactivity ofantibodies to i-chain: (•) cell-associated radioaciivity of antibodies to<!)-chaini (O) radioactivity inthe supcrnatants of cultures labelled with '-^l-anti-/i; and (D) radioactivity in the supernatants ofcultures labelled with '-^I-anti-d. The bars represent ± SE of (a) two parallel and (b) three parallelexperiments.

With regard to the first possibility, largedifferences in iti t racy topi asmic accumulationbetween anti-^-chain and anti-5-chain wasobserved irrespective of the initial amounts ofslgM and sIgD as determined by flow cyto-fluorometry. Earlier studies have shown that Ihefluorescent antibodies used were comparablewith regard to sensitivity for sIg detection [11].Auit & Unanue [ 11 have reported that, althoughsIg was poorly reexpressed on human peripheralblood lymphocytes after interaction with specificantibody, sIgD had a tendency to be reexpressedto a higher extent than sIgM. Our findingsrevealed an opposite tendency. This may bedifferentiation-related, since our lymphomaswere germinal centre cell-related. The turnoverof sIgM and sIgD molecules in lymphoma 214/81was studied by using lactoperoxidase-catalysedmembrane iodination. In good accordance withFerrarini et al. [7], we found that both initial

sIgM and sIgD had a similar turnover, with ahalf-life of permanence in the membrane ofabout 3 h. The sensitivity of the method did notallow any conclusions to be drawn with regardto the half-life of reexpressed sIgM and sIgD.Thus, differences in expression, reexpression.and turnover of sIgM and sIgD could not explainthe large differences in intracellular accu-mulation. This was most apparent inlymphoma 214/81. where sIgD showed a 2.6-foIdhigher initial expression than sIgM and a similarlevel of reexpression during the first 24 h. Therate of degradation of antibodies to /i- and5-chain was therefore studied in this lymphoma,

Whereas the degradation of initially boundanti-^ and anti-// was closely similar, significantdifferences were found with regard to anti-dantibodies bound to reexpressed sIgD. whichwere found to be degraded at a higher rate than

bound to reexpressed sIgM. This was

Different Fate of Antibodies to slgM and slgD 453

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shown both by a higher speed of release of'' I-anti-fS from the cells (Fig. 4) and by anincreased proportion of degraded '"^I-anti-fi(Fig. 6). We consider it most unlikely that the

two rabbit anti-human Ig reagents should havedifferent susceptibility to degradation. Usinglactoperoxidase-catalysed membrane iodina-tion, we found that the binding and internatiza-tion of specific antibody involves the degrada-tion of slgM and slgD along with the antibodies.This was in accordance with the findings ofSidman & Farr [21]. We could not providedefinite evidence that anti-<') antibodies bound toreexpressed sIgD were internalized. Internaliza-tion could only be ascertained by flow cyto-fluorometric analysis of initially bound anti-/iand anti-(>. but specific fluorescence was toolow on cells carrying reexpressed slg (only twicethe background) to allow definite conclu,sions.Nevertheless, we consider it most unlikely thatsuch an extensive degradation would take placeat the surface of the cells. Thus, our studiessuggest a differential intracetlular fate of anti-bodies to n- and tVchain.

It is well established that T cells [19] andmacrophages [3] can produce factors thatregulate the growth and differentiation of Bcells. It was recently reported by Fogelmann etrt/.[9] that lymphokines from a lymphocyte cellline modulated receptor-mediated endocytosisin human monocyte-derived macrophages. Suchf;;Ctors could influence the fate of anti-Ig in Bcells. In our cultures, accumulation of anti-Igwas only observed in the presence of TPA.Apart from having a positive effect on cellviability. TPA could provide the stimulus for theproduction of such factors by T cells ormacrophages present in the cultures. Antibodiesto fi' or d-chain in combination with TPA couldstimulate the production of factors that promoteor suppress anti-Ig accumulation, respectively.However, the simultaneous presence of anti-bodies to ft- and (5-chain did not reduce theamount of accumulated anti-Ig as comparedwith cultures with antibodies to /j-chain alone.This result does not favour anti-('j stimulatedproduction of factors that suppressed anti-Igaccumulation. It has been reported [15] thatphorbol myristate acetate, a compound closelyrelated to TPA. could directly influencereceptor-mediated endocytosis in mouse mac-rophages by stimulating the fusion of phago-somes with lysosomes. However, such amechanism could not easily explain the observeddifferences between antibodies to /t- and 6-chain.

The reexpressed sIgD was functionally diffe-

454 E. Ruud et al.

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FIG. 6. Absorption of supernatants with swine antibodies to rabbii Ig coupled to Sepharose beads.Cells carrying (a) initial or (b) reexpressed slg were labelled with iodinated antibodies to ft- orA-chain at zero hours, washed, and reculiured with 75 nM TPA. (O) Radioactivity absorbedfrom supernatants of cultures labelled with '-''I anii-/i: and (D) radioactivity absorbed fromsupernatanis of cultures labelled with '-'i-anti-A. The bars represent ±SE of three parallelexperimenu.

rent from the initial slgD. Antibodies to i -chainbound to reexpressed sIgD were degraded at ahigher rate than antibodies bound to initial sIgDand to both initial and reexpressed sIgM (Figs. 4and 6). The reexpression of functionally diffe-rent sIgD depended on the interaction of theinitial sIgD with antibodies to d-chain and couldnot be induced by initially treating the cells withantibodies to fi-chain. TPA. or medium alone(Fig. 5). Two explanations of the alteredfunctional properties of reexpressed slgD mustbe considered: (i) an alteration in the structureof slgD itself, and (ii) a change in the cellmembrane-associated function of a surfacecomponent associated with sIgD.

Mouse spleen cells contain multiple mRNAsencoding for the membrane form of d heavychains [8]. It has been suggested that differentexons in the gene sequence encoding thedifferent d membrane forms may be translatedand expressed as alternative or additional3'-polypeptide termini. Thus, such different dKeavy chains might provide multiple cytoplasmictermini aviiilable for different effector functionsof slgD. Differences in the glycosylation ofinitial and reexpressed slgD could also interfere

with the rate of intemalization and degradation.In the case of sIgM a non-giycosylated abnormal//-chain in a mutant cell line has been reported tobe degraded at a rate of less than 1/10 of theglycosylated form [22].

The functional significance of the presentfindings in relation to antigen triggering has tobe considered. Our findings suggest that anti-bodies to/i- ( -chain reach different intracellularcompartments. Our findings with antibodies tolight chain suggest that the fate of an antigen willbe determined by the ratio between sIgD andslgM at any time—that is, for both initial andreexpressed slg. Thus slgM. in addition lofunctions related to triggering, may have anantigen storage function. This stored antigencould act as a depot for prolonged stimulation ingerminal centres. Is it possible that the antigenspresent on dendritic cells for extended periods oftime {see Ref. 14) are derived from such depots?

SIgD. on the other hand, apparently leads to amore complete degradation. Such smaller anti-genic fragments could be suitable for antigenpresentation to T cells, which appear moreprone to 'see' primary structures of antigen thanantibody [5].

Different Fate of Antibodies to slgM and slgD 455

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lymphocytes in human blood to regenerate surfaceimmunogiobulin after its removal by antibody. J.Immunol. 119. 327. 1977.

2 Black. S.J., Loo, W. van der. Loken. M.R. &Ilerzenberg. L.A. Expression of IgD by murinelymphocytes. Loss of surface IgD indicatesmaturation of memory B cells. J. exp. Med. 148,984, 1978.

3 Booth. R.J.. Prestidge. R.L. & Watson, J.D.Constitutive production by ihe WEHl-3 cell line ofB cell growth and differentiation factor Ihatco-purifies with interleukin 1. J. Imtnunot. 131,1289. 1983.

4 Boyum, A. Separation of leucocytes from bloodand bone marrow. Scand. J. clin. Lab. Invest. 21.Suppi. 97. 1968.

5 Chesnul, R.W.. Endres. R.O. & Grey. H.M.Antigen recognition by T cells and B eells:recognition of eross-reactivity between native anddenatured forms of globular antigens. Clin. Im-munol. Immunopathot. 15. 397. 1980.

6 Dresser. D.W.& Parkhouse. R.M.E. The effect ofparenteral administryiion of a rabbit anti-(mouse)-IgD serum on lhe immune response of mice tosheep erythrocytes. Immunology 35, 1027, 1978.

7 Ferrarini, M.. Corte. G., Viale, G.. Durante.M.L. & Bargellesi, A. Membrane Ig on humanlymphoeyies: rale of turnover of IgD and IgM onthe surface of human tonsil cells. Eur.J. Immunol.6, 372. 1976.

8 Fitzmaurice, L.. Owens, J., Blattner. F.R..Cheng. H.. Tucker. P.W. & Mushinski. J.F.Mouse spleen and IgD-secreting plasmacytomascontain multiple IgD 6 chain RNAs. Nature 296,459. 1982.

9 Fogetmann, A.M., Seager. J.. Groopman, J.E.,Berliner, J,A.. Haberland. M.E., Edwards. P.A.& Golde, D.W. Lymphokines secreted by anestablished lymphocyte line modulate receptor-mediated endocytosis in macrophages derivedfrom human monocytes. J. Immutioi. 131, 2368.1983.

10 Godal. T.. Henriksen, Aa.. Ruud, E. &Michaelsen, T. Monoclonal human B lymphomacells respond by DNA synthesis to anti-immunoglobulins in the presence of the tumourpromotorTPA. Scund. J. Immunol. 15, 267. 1982.

U Godal, T.. Lindmo. T.. Marton, P.F.. Landaas.T.0.. Langhotm, R.. Hoie. J. & Abrahamsen.A.F. Immunological subsets in human B celllymphomas. Scand. J. Itnmunot. 14, 481. 1981.

12 Godal. T.. Lindmo, T., Ruud, E.. Heikkilii, R.,Henriksen, Aa.. Steen. H.B. & Marton. P.F.Iramunologic subsets in human B-ceil lymphomasin relation to normal B-cell development. Haema-tologv and Blood Transfusion. Modern Trends inHuman Leukaemia IV. 26, 314. 19«1.

13 Herzenberg, L.A.. Black, S.J.. Tokuhisa. T. &Herzenberg, L.A. Memory B cells at successivestages of differentiation. Affinity maturation andthe role of IgD receptors. J. e.xp. Med. 151, 1071,1980.

14 Humphrey. J.H. & White. R.G. Immutiology forstudettts of medicine, p. 238. 3rd edn. BlackwellScientific Publications. Oxford, 1970.

15 Kielian. M.C. & Cohn, Z.A. Phorbol myristateacetate stimulates phagosome-lysosome fusion inmouse maerophages. J. exp. Med. 154. 101. 1981.

16 Landaas, T.0., Godal, T. & Halvorsen, T.B.Characterization of immunoglobulins in Hodgkincells. Im. J. Cancer 20, 717, 1977.

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Received 14 September 1983Received in revised form 18 January 1984