ICANCER RESEARCH 53. 2147-2153, May 1. 1993]

Analysis of Ly-l+ B-Cell Populations and IgH Rearrangements in "Normal" Spleens

and in Lymphomas of AKR/J and AKR Fv-lb Mice1

A. Rosner, A. Peled, N. Haran-Ghera, and E. Canaani2

Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT

AKR mice are highly susceptible to development of spontaneous T-celllymphoma. Thymus removal at the age of 1-3 months greatly reducesT-cell lymphoma. Lymphomas that have the characteristics of T- and/orB-cells occur sporadically in peripheral lymphoid tissues of old thymec-

tomized AKR/J mice. These thymectomized mice were shown to carrydormant potential lymphoma cells. Transplantation of lymphoid cellsfrom 8-12-month-old AKR/J mice, thymectomized at the age of 6 to 8weeks, into intact or thymectomized young recipients yielded 80-100%I >-l ' pre-B or B-cell lymphomas. In the YKK-1A-I1' congenie strain the/•'>•-/"alíeleof AKR/J mice was substituted with the AV-/' alíele,thereby

limiting viral replication and spread of the endogenous N-tropic murineleukemia virus. As a result of this restriction in virus spread, AKR-Fv-lb

mice develop a low spontaneous incidence (7%) of T-cell lymphomas andabout 28% of !.>-!' B-cell lymphomas at old age. In spleens of 15-18-month-old thymectomized AKR/J mice and intact AKR-Fv-lb mice, 30-60% of the B-cells were of the Ly-1* B type. Analysis of the IgH locus inthese normal old spleens and Ly-1 ' B lymphomas indicated mono- or

oligoclonality. One particular IgH rearrangement was identified in manyindividual old spleens and tumors. A second specific IgH rearrangementwas found in some tumors. Possible mechanisms involved in the expansionof Ly-1 ' clones and their progression into tumors are discussed.

INTRODUCTION

AKR mice display a high incidence of spontaneous T-cell lymphomas that arise predominantly in the thymus of 6-12-month-old mice(1). The susceptibility of AKR mice to spontaneous T-cell lymphoma

development is dependent on the presence of an intact thymus and isassociated with two classes of endogenous retroviruses: the ecotropicvirus inherited in AKR mice at two chromosomal loci (2. 3): and therecombinant (MCF) virus class derived from recombination of ecotropic virus with endogenous xenotropic or MCF-like provirai gene

sequences (4, 5). These recombinant viruses are expressed in thethymus of AKR mice prior to the development of lymphoma (5, 6).Thymus removal at the age of 1-3 months reduces development ofT-cell lymphoma (7). Lymphomas occur in peripheral lymphoid tis

sues of old thymectomized mice (up to about 30% at a mean latencyof 600 days) and have the characteristics of T- and/or B-cell lympho

mas (8). Although removal of thymus from young AKR mice preventsthe development of spontaneous T-cell lymphoma, these thymectomized mice were shown to be carriers of PLC.1 Transplantation of

lymphoid cells from 8-12-month-old AKR/J mice, thymectomized at

the age of 6 to 8 weeks, into intact or thymectomized young recipients yielded 80-100% Ly-l+ pre-B or B-cell lymphomas of AKR or

igin (9). Thus, these PLC could be triggered to develop into overtlymphomas only after their removal from "restrictive host environ-

Received 11/6/92; accepted 2/24/93.The costs of publication of this article were defrayed in part hy the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the Leo and Julia Forschheimer Center for Molecular

Genetics at the Weizmann Institute and by the Israel Science Foundation (Grant 147/911.N. H. G. holds the Olin Sang Professorial Chair in Leukemia Research, and E. C. holdsthe Harry Kay Professorial Chair in Cancer Research.

2 Presen! address: Thomas Jefferson University. Jefferson Cancer Institute. Philadel

phia. PA 19107.' The abbreviations used are: PLC, potential lymphoma cells; FITC, tluorescein

isothiocyanate; FACS. fluorescence-activated cell sorting; kbp, kilobase pair.

ment" to appropriate compatible young recipients. Recently we dem

onstrated termination of the PLC dormant state in 12-month-old

thymectomized AKR mice by a variety of ways such as syngeneicthymus grafting, administration of the lymphokine IL-2, treatmentwith antibodies against CDS, etc.; 90-100% of such treated mice developed Ly-1 ' (CD5f) B-cell lymphomas (10).

The AKR-Fv-lh congenie strain differs from the AKR/J mice at the

Fv-1 gene which encodes a product which inhibits viral replication byblocking integration of the DNA provirus (11). In the AKR-Fv-lhcongenie mice the Fv-1" alíeleof AKR/J mice was substituted with theFv-lh alíele.This substitution limits viral replication and spread of

the endogenous N-tropic murine leukemia virus in thymocytes, sple-nocytes, and bone marrow of AKR-Fv-lh mice (12, 13). As a resultof this viral restriction these AKR-Fv-lh mice develop a low incidence of spontaneous T-cell lymphomas and a similar Ly-1 ' B-cell

lymphoma incidence to that observed in old thymectomized AKR/Jmice.4

The development of Ly-1 ' B-cell lymphomas in AKR/J mice andAKR-Fv-lh mice and the identification of PLC in spleens of old"normal" mice raised the question whether the number of Ly-l +

B-cells was increased in the spleens of older mice, whether Ly-l +

B-cell populations were clonal, and whether the pattern of IgH rearrangements in those clones was similar to that observed in Ly-l +

B-cell lymphomas developing in these strains of mice. We find thatnormal spleens of old AKR/J and AKR-Fv-lh mice contain clonalpopulations of Ly-14 B-cells. A common IgH rearrangement is de

tected in these cells as well as in a substantial number of lymphomasoriginating in AKR/J mice. A second common IgH rearrangement isfound in some AKR lymphomas. This phenomenon is probably related to the unique properties of Ly-1 B-cells.

MATERIALS AND METHODS

Mice. AKR/J mice were obtained from The Jackson Laboratory (Bar Harbor. ME). Breeding pairs of AKR-Fv-lh congenie mice were kindly provided

by Dr. E. Boyse (Sloan Kettering Memorial Institute. New York. NY) and bredat the Weizmann Institute of Science. Thymectomy was performed on 6-8-week-old mice, as described previously (10).

Tumors and Cell Lines. Tumors were induced as described (9). Briefly,spleens, bone marrows, or lymph nodes were obtained from individual I -year-

old preleukemic AKR/J mice which were thymectomized at the age of 2months or from spleens of 16-24-month-old AKR-Fv-lh and transplanted

(from one donor to one recipient) into AKR/J x DBA/2 F, recipients. Lymphomas of AKR origin developing in the F, recipients, 30-180 days following

cell transfer, were characterized for their phenotype and genotype. The lymphomas of AKR origin identified as Ly-1 ' B were further analyzed for im-

munoglobulin rearrangement. Flow cytometry was performed on a FACS IIor on Coulter Profile, using methods described previously (9). Several celllines (24-182; 24-666; 24-674; 24-675; 24-683; 25-197) were establishedfrom primary AKR/J Ly-1 ' B lymphomas in collaboration with Dr. D. Zi-

pori. All primary lymphomas used for the establishment of cell lines expressed surface p chain as well as B220. la, and Ly-1. Three of the tumors

also expressed surface K light chain. During in vitro maintenance these lymphoma cell lines lost their surface u and K expression but retained cytoplas-mic u, B220, and Ly-1. Lymphomas obtained by retransplantation of the cells

from tissue culture back into animals regained their surface u and K.

4 N. Haran-Ghera. unpublished observations.

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Ly-r B-CELLS AND IgH REARRANGEMENTS IN MOUSE LYMPHOMA

Flow Cytometry Analysis and Cell Sorting. Single cell suspensions fromspleens were prepared in phosphate-buffered saline-0.05% sodium azide. Foranalysis, aliquots of 1-3 x IO6 cells were incubated with goat anti-FITC

(Sigma) or biotinylated anti-Ly-1 (CDS) (Becton-Dickinson), followed bystreptavidin-PE (Biomeda). Double staining of cells with FITC-labeled/

biotinylated antibodies was carried out in the combinations of IgM/CD5. Allincubations were done for 30 min at 4°C.The stained cells were examined by

FACScan (Becton-Dickinson) using a FITC/PE filter system. Lymphocyte

populations were gated by forward versus side scatter and fluorescence intensity was expressed by logarithmic amplification. Spleen cell populations usedfor Ly-l + B-cell sorting were treated with ammonium chloride to lyse RBC

with ami-Thy-1.1 monoclonal antibody (clone HO-22-1) and low toxic rabbitcomplement (Cederlane) to eliminate T-cells. Viable cells were collected

c•¿�TI

O£O.

100

M

60-

40-

= 20 -

A 17/18(94%)

B 11/30(36%)

C 5/70 (7%)

D 20/70 (28%)

100 200 300 400 500Days

600 700 800

Fig. I. Cumulative incidence of spontaneous T- and B-cell lymphoma in intact andthymectomized AKR/J mice. A, T-cell lymphoma in intact AKR/J mice. B, B-cell lymphoma in thymectomized AKR/J mice. Thymectomy was performed at the age of 40-60days. C, T-cell lymphoma in AKRFv-lb mice. D. B-cell lymphoma in AKR-Fv-lb

from a Ficoll-Hypaque gradient and cells were stained as stated above. Sorting was performed on a FACStar* (Becton-Dickinson).

Probes. Immunoglobulin probes included the pJl 1 genomic fragment containing J3 and ¡4( 14), pm 173 1C»complementary DNA clone of the kappa chainlocus (15), Hopcl A20 complementary DNA derived from the A chain locus

(16), and a genomic Xbal fragment containing the u gene (17). All probes wereprovided by Drs. O. Bernard and J. Adams.

DNA Blotting, Cloning, and Sequencing. High molecular weight DNAwas extracted by standard procedures (18). Aliquots of 10 ug were digestedwith restriction enzymes, electrophoresed on 0.8% agarose gels, and blottedinto nitrocellulose filters. Hybridizations were conducted in 50% formamideat 42°Cas described (19). EcoRl DNA fragments were cloned into the Wes B

vector (18) following size enrichment. Sequencing was done by the chain ter

mination method (20) using the Sequenase kit of U.S.B. Primers were synthesized on the basis of sequence information obtained sequentially.

RESULTS

Spontaneous Lymphoma Development in AKR/J and AKR-Fv-lb Mice. AKR/J mice display a high incidence of spontaneous

T-cell lymphomas, 94% (17 of 18) at a mean average age of 250 days(Fig. 1). Thymus removal at the age of 1-3 months prevented T-cell

lymphoma incidence (2 of 30; 6% beyond the age of 1 year) andusually about 30% of these thymectomized mice developed Ly-l +

B-cell lymphomas at the mean latency of 600 days. In AKR-Fv-lb

mice spontaneous T-cell lymphoma was observed in 7% (5 of 70) ofmice at a mean latency of 470 days (Fig. 1). Ly-l+ B-cell lymphomas developed in 28% (20 of 70) of AKR-Fv-lb mice at a mean la

tency of 680 days. In vivo elimination of T-cell subsets by administration of monoclonal antibodies (anti-CD8) to 12-month-old AKR/Jmice, thymectomized when 2 months old, enhanced markedly Ly-1"1"

B-cell lymphoma development (up to 80-100%) (10). Similar in

B

Fig. 2. FACS analysis for the expression of u chain and Ly-1 (CD5)antigens on spleen cells from grossly normal 18-month-old (A, topiand 6-month-old (B, lop) intact AKR-Fv-lb mice, compared to 15-month-old M, bottami and 6-month-old (B, bottom) thymectomizedAKR/J mice. Cells were stained with biotinylated Ly-1 followed bystreptavidin-PE and goat anti-mouse FITC.

LUO.

J_I

-FITC

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Ly-r B-CELLS AND IgH REARRANGEMENTS IN MOUSE LYMPHOMA

Ba b e d e f

t -2.6

Fig. 3. Southern blot analysis of "normal" spleen DNA sam

ples digested by EcoRl. Blots were hybridized to the Jn probe.Each lane represents a different spleen. A, spleens from2-month-oldAKR-Fv-lb(Lonesa. ¿(andAKR/J (Lanec) mice.B. spleens from 12-month-old thymectomized AKR/J mice. C.spleens from 24-month-old AKR-Fv-1b female mice. D. spleensfrom 24-month-old male AKR-Fv-lh mice. gl. germ line.

bed

D

Kbp

•¿�gl

5.0

-2.6

a b e d e

-2.6

vivo treatment administered to 18-month-old intact AKR-Fv-lb mice Jn probe. This probe contained the genomic segments J, and J4 of theresulted in development of Ly-l+ B-cell lymphoma in 80% of the

animals.5Level of l,y-1" B-Cells and Pattern of IgH Rearrangements in

Normal Spleens of Old AKR-Kv-1" and Thymectomized AKR/JMice. The analysis of the Ly-1+ IgM+ B-cell population was done onspleens from 15-18-month-old and 6-month-old grossly "normal"mice. The percentage of Ly-1+ B-cells in each individual spleen was

determined by FACS analysis of cells double labeled with anti-Ly-1and anti-n antibodies. In spleens derived from 15-month-old thymectomized AKR/J mice, 30-60% of the B-cells were of the Ly-1 + B type

(Fig. 2A, bottom). In contrast, in 6-month-old thymectomized AKR/Jmice only 9% of the B-cells were Ly-l + (Fig. IB, bottom). Thisincrease in the Ly-1 + B population was encountered in a significant

proportion of animals and further increased with age. In healthyspleens of 18-month-old AKR-Fv-lb mice, 22-45% of the B-cells

were of the Ly-1 B-type versus 5% in spleens of 6-month-old mice

(Fig. 2, A, top; B, top, respectively). FACS analyses of such spleensamples are presented in Fig. 2. The results imply that the Ly-l +B-cell population in AKR/J mice and AKR-Fv-lb mice increases as

the animal becomes older.The IgH rearrangement pattern in spleens from AKR/J and AKR-

Fv-lb mice of different ages was studied. Spleen DNAs were digested

with £coRIand analyzed by Southern blotting for hybridization to the

•¿�'Unpublished observation.

heavy chain locus and could be used to detect all rearrangements ofthis locus. When normal spleens from 2-month-old mice were ana

lyzed, only the germ line EcoRI fragment of 6.2 kbp was detected(Fig. 3/1). Spleens from 6-month-old mice showed the germ line band

as well as many very faint bands corresponding to rearranged IgH(data not shown). Southern blot analysis of DNA samples derivedfrom individual spleens of 12-month-old thymectomized AKR/J miceor from 24-month-old old female AKR Fv-lb mice indicated IgH

EcoRI bands of 6.2 (germ line), 5.0, 2.6 kbp, and others (Fig. 3, fi andC, respectively). Spleens of 2-year-old Fv-lh males showed more

complex patterns (Fig. 3D). The results imply that the B-cell population in spleens of these mice is mono- or oligoclonal. It appears thatspleens of young mice (which have less Ly-1 B-cells) contain many

clones, each with a different IgH rearrangement, preventing detectionof individual rearranged bands. In contrast, the spleens of old mice arecomposed of only few clones easily detected by Southern analysis.Reduction to oligoclonality was detected in both old intact mice andthymectomized mice. The analysis of about 80 "normal" spleens of

old mice indicated several IgH EcoRI rearranged fragments of common size in different spleens. Fragments of 2.6, 2.9, or 5 kbp weremost common and were detected alone or in combinations in about50% of spleens analyzed.

To show that the clonal rearrangements observed occurred in Ly-1B-cells, we FACS sorted Ly-l+ B-cells from two spleens of 12-

month-old thymectomized AKR/J mice. The T-cells were eliminated

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Ly-r B-CELLS AND IgH REARRANGEMENTS IN MOUSE LYMPHOMA

kbp

—¿�9.4

—¿�6.5

—¿�4.3

—¿�2.3

—¿�2.0

Fig. 4. Rearranged IgH DNA in Ly-1 B-cells purified from two "normal" spleens of 12

months old thymectomized AKR/J mice (Lanes a, b).

(by anti-Thy-1.1 antibodies and complement) and the rescued cells(separated by Ficoll-Hypaque gradient) were double labeled withFITC-anti-u and PE-anti Ly-1 antibodies and sorted by FACS. DNAswere extracted from approximately 1-3 X IO6 sorted cells of each

spleen and analyzed by Southern blotting for hybridization to J,,probe. The Ly-1 + B-cells showed rearranged IgH DNA with or with

out a germ line band (Fig. 4).

Sequence of Rearranged Fragments of Common Size from Normal Spleens. Molecular cloning of the 2.6-kbp EcoRl fragment of theprominent rearranged species in normal spleens was performed. Several clones with identical restriction maps were obtained, thus ensuring that they represented the species observed by Southern blot analysis. EcoRl fragments (2.6 kbp) obtained from two different AKR-Fv-lh (2-year-old) spleens were sequenced. The sequences were found

to be identical (Fig. 5), suggesting germ line configuration as well asthe absence of an N region. The V region best fits the Vj gene whichis a member of the VHJ 558 family (21 ). The D region best resemblesDsp2.»(22). Surprisingly, VDJ joining in these molecules disrupts theopen reading frame. Three 5-kbp EcoRl fragments were cloned fromthree AKR-Fv-lh "normal" spleens (IgH patterns of these spleens are

shown in Fig. 3C); sequencing analysis indicated that the fragmentsrepresented DJ2 rearrangements, but the D segment was different ineach spleen (data not shown). Thus, while the 2.6-kbp EcoRl rear

ranged fragments detected in individual spleens appear to represent aunique DNA species, the 5-kbp EcoRl rearranged bands correspond

to at least three different types of rearrangements.Analysis of the Heavy and Light Chain Loci of Ly-1* Pre-B and

B-Cell Lymphomas of AKR/J Mouse Origin. Twenty-two Ly-1+ B

and pre-B-lymphomas derived from different individual donors (fromexperiments done during 1985-1989) were analyzed for rearrange

ment of the IgH and IgL loci. All lymphomas analyzed of AKR/Jorigin arose following transfer of lymphoid cells from old thymectomized mice into F, recipients. Aliquots of tumor DNAs were digestedwith the EcoRl enzyme, electrophoresed on agarose gels, blotted intonitrocellulose filters, and hybridized to the JM probe. Similar analysis

was performed to examine the loci coding for the light chains of theimmunoglobulins. None of the tumors showed rearrangement of the A

locus (not shown) but most tumors tested contained rearranged Kalíeles.An example of the Southern blot analysis of the IgH locus ofsix Ly-1 + B lymphomas is shown in Fig. 6. The germ line fragment

of 6.2 kbp is detected in three tumors (Lanes a, b, c). A rearrangedfragment of 5.5 kbp appeared in all tumors. In addition, a 2.6-kbprearranged DNA fragment appeared in three tumors (Lanes d-f), anda 3.0-kbp fragment was detected in one lymphoma (Lane a). Digestion of DNAs from tumors 24-182; 24-674; 24-675; 24-683 and25-197 with the Bglll and BamHl enzymes also showed rearranged

fragments of common size (not shown). The results of the entireanalysis for IgH and Igx rearrangements are summarized in Table 1.The tumors could be divided into 3 categories: group 1, tumors exhibiting rearranged fragments of 5.5 and 2.6 kbp; group 2, tumorsshowing a 5.5-kbp fragment; and group 3, consisting of 9 tumors with

IgH rearranged fragments of random size. Thus, in many tumors bothIgH alíeleswere rearranged; lymphomas showing more than two IgHfragments probably represented a mixture of two original tumors orwere contaminated with normal tissue. Rearranged IgH fragments ofcommon size were also observed in B-lymphomas from AKR Fv-lb

mice (not shown).Nucleotide Sequence of the Common IgH Rearrangements in

AKR B-Lymphomas. To further study rearranged fragments of common size, the 5.5- and 2.6-kbp species were molecularly cloned from

several independently derived lymphomas included in group 1. Comparison of the physical maps of these cloned fragments to the map ofthe cloned germ line DNA indicated deletion of sequences upstream ofJ2 and J3 in the 5.5- and 2.6-kbp fragments, respectively. The sequences of the 5.5-kbp fragments derived from the two Ly-l+ B

lymphomas 24-674 (which has a normal karyotype) and 24-683 [with

the (17;18) translocation marker (23)], were identical (Fig. 7). Thisindicates the absence of somatic mutations and no insertion of N-re-

gion nucleotides (24). Joining of VDJ segments retained the openreading frames in phase. Computer search indicated that the sequenceof the V region is nearly identical to that of A 8.1 (25). This VHgeneis a member of the VHQ52 family. The D region best fits the DFL16.2sequence (22). The sequences of the 2.6-kbp DNA fragments clonedfrom Ly-l+ B lymphomas 24-182, 24-674, and 24-675 were identical

to one another as well as to the sequence of the 2.6-kbp fragmentsobtained from normal AKR-Fv-lb spleens previously indicated in Fig.

5 (not shown).

DISCUSSION

Spleens from old "normal" intact AKR-Fv-1b mice or from thymec

tomized AKR/J mice were found to contain large clones (mono- oroligoclonal) of Ly-l+ B-cells. The size and number of these popula

tions varied between individual mice and between batches of animals.Similar findings have been described for peritoneal cells of NZB andNZB X NZW F, females (26, 27) and CB.20 mice (28). BecauseLy-l+ B-cells are long lived and have self-renewal properties, they

have an increased tendency to become malignant (29). The largeLy-l+ B population in the "normal" spleens might already represent

the preleukemic stage. The preleukemic phase seems to be establishedbefore the pro-B or the pre-B stages since the large Ly-l+ B populations detected in "normal" spleens had germ line configuration of

the IgL loci. We speculate that the different experimental manipulations used by us to enhance progression of the preleukemic cells toovert lymphomas involve breakdown of immunological surveillancewhich normally arrests the proliferation of the preleukemic clones.

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Ly-l* B-CELLS AND IgH REARRANGEMENTS IN MOUSE LYMPHOMA

V3 GOT GTC CAC TCC CAG CTC CAG CTG CAG CAG CCT GGGGCT GAA CTG CTG AAG CCT GGGGCT TCA102-28102-29

V3 GTG AAC CTG TCC TGC AAG GCT TCT GCA TAC ACC TTC ACT AGC TAC TCC ATG CAC TGG GTG AAG102-28102-29

V3 CAG ACG CCT GGA CAA GGC CTT GAG TGG ATC GQA GAG ATT GAT CCT TCT GAT AGT TAT ACT TAC102-28

Fig. 5. Nucleotidc sequence of the VDJ rear- 102-29

rangement within 2.6-kbp DNAs cloned from twonormal spleens (102-28, 102-29) of AKRFv-I"

mice compared to the sequence of V,, D>p2K,andJ, V3 TAC AAT CAA AAG TTC AAG GGC AAG GCC ACA TTG ACT GTA GAC AAA TCC TCC AGC ACA GCC TAC(see text). , nucleotide identities. 102-28

102-29

V3 ATG CAA CTC AGC AGC CTG ACA TCT GAG GAC TCT GCG GTC TAT TAC TGT GCA AGA102-28 -

102-29

I D.p2.8 I ->3

»AGGGG AGT ATG GG GCC TGG TTT GCT TAC TGG GGC CAA GGG ACTCTG GTC ACT GTC TCT GCA102-28102-29

f

KbR_

-5.5

-2.6

IBI f.Fig. 6. Southern blotting analysis of the IgH locus in AKR Ly-l * B-lymphomas. DNA

samples were digested with Ecofu, and following electrophoresis and blotting werehybridized to an IgH-specific probe. Lymphomas of AKR/J mice were: 57-450, 95-56,95-219, 24-182. 25-197. 24-674 (Lanes a-f. respectively).

This enhanced proliferation would increase the risk for additionalgenetic alterations culminating in the malignant transition to autonomous tumors.

Analysis of individual "normal" spleens of old mice and individualprimary Ly-l ' B-cell lymphomas indicated common utilization of

two specific sets of IgH VDJ segments. A 2.6-kbp EcoRl fragmentdetected in many "normal" spleens and in 11 tumors was analyzed on

the sequence level in two spleens and three tumors and found identicalin all cases. A 5.5-kbp £c«RIDNA detected in 13 tumors was se-

quenced in 2 tumors and found identical. The latter species showed acommon restriction pattern with three enzymes in several tumors andtherefore represented a unique set of VDJ segments. An EcoRl fragment of 5-kbp detected in many normal spleens represented several or

many different DNA species as evidenced by sequence analysis of thisfragment cloned from three individual spleens. While in the common5.5-kbp rearranged DNA detected in tumors the open reading frames

of the VDJ elements were fused in phase, this was not the case in the2.6-kbp DNA species. It is possible that the latter rearrangement

becomes productive through posttranscriptional or translational modifications such as those found in other systems (30, 31). An alternative possibility is that the 2.6-kbp DNA rearrangement is giving rise

to a truncated immunoglobulin, containing predominantly the V region but not Cp. The reproducible emergence and properties ofclones with particular IgH rearrangements is most likely due to thecharacteristics of the cell population from which they derive, theLy-l B-cells. These cells vary from the general population ofB-lymphocytes by their establishment early in ontogeny, their self-

renewal capacity, and their propagation in adults as mature IgM-positive B-cells. The normal Ly-l B-cells isolated from the perito

neal cavity display a unique repertoire to certain antigens andthereby differ from conventional B-cells (32). Ly-l cells (and theirLeu-1 human counterparts) were shown to be predominant in the fe

tus, rare in the adult, and consisting of a finite number of clones (33,34). They produce antibodies directed against self-antigens and bacterial antigens (35, 36) and as such are directly involved in autoim-

munity and in natural defense (32, 34).Several recent publications have shown repetitive utilization of

certain V,, and/or D segments in hybridomas or Ly-l B-lymphomas(27, 28, 32, 37-39). For example, Tarlinton et al. (27) showed thatmany Ly-l B-cells isolated from peritoneal cavities of different (NZB

X NZW)F1 mice had the same size rearrangements at both IgH andIgk loci. In one mouse, the productive rearrangements had identical

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Ly-r B.CELLSANDigHREARRANGEMENTSINMOUSELYMPHOMA

Table I IgH and IgK rearrangements in Ly-1^ B lymphomas nfAKR/J origin

No. ofexperiment

No. ofmouse

Cellstransferred"

Latency*

(days)IgH EcoRI

fragment (kbp)IgKEcoRI

fragment (kbp)

Group 1242424242424242425

Group 257579595

Group 3242828575757959598

1825176666676746756826X3197

450566

56219

IXI)94

20432

4X253023

682109

BMBMBMBMSpSpLNLNBM

SpBMSpSp

BMBMSpBMLNSpSpSpSp

142152445030504055209

22065250270

150180120130160100110150120

5.5; 2.6gl; 5.5; 2.65.5; 2.6gl: 5.5; 2.65.5; 2.65.5; 2.6gl; 5.5; 2.65.5; 2.65.5; 2.6

5.5;3.0gl; 5.5;2.9gl; 5.5gl; 5.5

gl;2.93.1; 2.4gl; 8; 2.9; 3.35.6; 4.512; 2.96.9; 4.3; 2.6gl;ll;9.26.8; 5.86.8; 4.9

glNDgl;21gl;21gl; 18.5; 13; 9.4; 7.5gl; 9.0glglgl;8.4

gl; 14gl; 14NDND

NDNDNDNDNDNDNDNDND

old).

' Mice were thymectomized at the age of 40-60 days. Lymphoid cell suspensions (~4 x IO7cells) were transferred from one donor to one recipient AKR/J x DBA/2 F, (2 month

I.' Latency in development of B-cell lymphoma in the F, recipients. BM, bone marrow; Sp. spleen; LN, lymph nodes; gl, germline; ND. not done.

unmutated VH and D elements joined to different JH elements. Forsteret al. (28) described repetitive utilization of VH segments in hybrido-

mas derived from unrelated clones. Two extreme examples, antibodyproduced in cell lines obtained as spontaneous outgrowth cultures ofspleen cells (39) and autoantibodies against bromelain-treated mouse

RBC (37), exhibit the same properties of IgH molecules described in

our work: (a) usage of identical V, D and J elements; (b) lack of somatic mutations; (c) absence of an N-region. The sequence withinthe 5.5-kbp EcoRI fragment shows 88% homology to the IgH expressed in two Ly-lB hybridomas (28). Moreover, the sequencewithin the 2.6-kbp DNA species exhibits 94% homology to that ofIgH expressed in the CH 10 and CH 13 tumors (38) and in 12 hybri-

ATGTGAACTCCAGACA TG CAGAAAATCATTCCTTATGTTCCTCTCCAGGTGCTCCAACAAGCACAGTGTAAATTTCTGTCACC

A8.124-674 CA T X 33

A8.1 ATAAAAGCTCACACTAACCTGAGAAGCTCCATCCTCTTCTCATACAGCCTCCATCAGAGCATG GCT GTC TIG GGG CTG24-674 C C T 121

A8.1....CTC TTC TOC CTG GTG ACÕTTC CCA AGC TGTAAGTGTTTCAGGGTTTCAGAAGAGGGACTAAGACATGTCAGCTAG24-674..— —¿� X 186

A8.1 GAGATGTGTGACTAATGTTGATGTTGCTTGTCCCCAGGTGTC CTA TCC CAG GTC CAG CTG AAG CAO TCA CCA24-674 G T— 258

A8.1 CCI CGC CTA GTG CAG CCC TCA CAG AGC CTG TCC ATC ACC TGC ACA GTC TCT GOT TTC TCA TTA24-674 321

Fig. 7. Nucleotide sequence analysis of the VDJsegments,within the rearranged 5.5-kbp DNA frag u^ ^ ^ MI GGTGTACA£.TB(. ,.„ coc CM TCT CCAGOAAAGBGI CTOOAGTBO „¿�„BOA„¿�,.ment. Fragments were cloned from the 24-674, and 2 __ __ .__24-683 AKR/J tumors. , nucleotide identities;

24-683 —¿�———¿�———¿�———¿�———¿�——___ —¿�-_ ___ —¿�— ___ —¿�— ___ ___ ___ ___ ___ ___ ___ ___

X. sequence uncertainties. Published sequenceswith the closes, similarity are shown for compar,- OOA ^ ^ ^ uc ^ ^son(A8.1,andD,.L|62).

24-683

A8.1 GAC AAT TCC AAG AGC CAA GTT TTC TTT AAA ATG AAC ACT CTG CAÕGCT AAT GAC ACÕGCC ATA24-674 C G 51024-683 —¿�C G

I »FL16.2 I J2

A8.1 TAT TAC TGT GCG AQA TT CAT TAC TAC GGT TAC TAC TTT GAC TAC TGG24-674 —¿�C CAÕT-C —¿�C GTA GG C TAC TGG 65524-683 —¿�C GAA T-C —¿�C GTA GG C TAC TGG

J2 CGC CAA CGC ACC ACT CTC ACA GTC TCC TCA GGTCAGTCC24-674

24-683

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Ly-l* B-CELLS AND IgH REARRANGEMENTS IN MOUSE LYMPHOMA

domas (28) and 89% homology to the sequence of IgH expressed inLy-1 B-cells found in B/W mice (27).

Repetitive utilization of immunoglobulin gene segments could bedue to favorable rearrangements during the recombination process orto selection of the cells through their antibody receptors. The firstpossibility is usually based on the assumption that the VDJ elementsproximal to each other are easiest to recombine. The VH region withinthe 5.5-kbp fragment most commonly used in our tumors is a member

of the VHQ52 family, which is indeed positioned as the second mostproximal to the D region (40). In contrast, the VH region within the2.6-kbp species belongs to the VHJ 558 family positioned away fromthe D-J-C segment. Another possibility is that the prominent Ly-1 Boligoclones that eventually give rise to Ly-1 B lymphomas in AKRmice are antigen driven. The antigen could be an AKR self-antigen, anendogenous virus such as 24-666 isolated from a B-cell lymphoma(41 ), or an anti-idiotypic antibody. Interestingly, the sequence of the Vregion within the 2.6-kbp AKR rearranged DNA fragment is 90%homologous to VH sequences expressed in lupus-prone mice (42).Thus, it is possible that the AKR Ly-1 B clones studied here, by virtue

of specific IgH molecules which they express, are stimulated to proliferate by repeated exposure and interaction with a self-antigen. Con

tinuous proliferation of these clones would increase their risk foradditional genetic alterations leading to malignant transition.

ACKNOWLEDGMENTS

We are indebted to Dr. J. Haimovich for his interest and useful suggestionsand to Dr. O. Bernard for valuable discussions and probes.

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1993;53:2147-2153. Cancer Res   A. Rosner, A. Peled, N. Haran-Ghera, et al.  

MicebFv-1in ''Normal'' Spleens and in Lymphomas of AKR/J and AKR

B-Cell Populations and IgH Rearrangements+Analysis of Ly-1

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