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Cellular Origin, Antigen Reactivity, and V, Segment Structure of IgM mAbs

from AIDS Lymphomas" PIERSANDRO RIBOLDI,b GIANLUCA GAIDANO,'

EDWARD W. SCHETTINO,b,d DANIEL M. KNOWLES,d

bDepartment of Pathology and Kaplan Comprehensive Cancer Center

New York University School of Medicine New York, New York 10016 'Department of Pathology

College of Physicians and Surgeons Columbia University

New York, New York 10032 dDepartment of Pathology

Cornell University Medical College New York, New York 10021

RICCARDO DALLA-FAVERA,' AND PAOLO CASALIb~d~e

INTRODUCTION

Stimulation of B lymphocytes by antigen (Ag) leads to cell activation, prolifera- tion, somatic hypermutation of the expressed Ig V genes, and, eventually, clonal selection of those B cells bearing a mutated surface receptor displaying the highest affinity for Ag. Evidence for persistent clonal B-cell expansion and proliferation by foreign and self Ags has been provided in chronic infectious diseases and autoimmune d i~orders . ' -~ In addition, it has been suggested that Ag stimulation also plays a role in the clonal B-cell selection associated with development of B-cell tumors, such as follicular Similarly, the generalized B-cell expansion and hyperactivity observed in HIV-infected patients may be at least in part triggered by Ag and might prepare the ground for the development of B-cell lymphomas, particularly those that arise in the absence of other putative lympho- magnetic cofactors, such as EBV.

a This work was supported by U.S. Public Health Service Grants CA-68541 to P.C., CA-37895 to R.D-F., and the Ministry of Health, I.S.S., AIDS Projects 1993, Grant 8206-07 (Italy) to P.R. G.G. was supported by a fellowship for AIDS research from the Ministry of Health, I.S.S. (Italy). This is publication #7 from The Division of Molecular Immunology.

P.R. current address: Dept. of Internal Medicine, Infectious Diseases, and Immunopathol- ogy, University of Milan, 20122 Milan, Italy.

Address for correspondence: Dr. Paolo Casali, Division of Molecular Immunology, Department of Pathology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021.

509

510 ANNALS NEW YORK ACADEMY OF SCIENCES

Because the assessment of a potential role for Ag in the clonal B-cell selection associated with B-cell malignant proliferation entails the definition of the specificity and of V, segment structure of the tumor-derived antibody, we established in uitro two cell lines from two patients with AIDS-associated Burkitt's lymphoma (BL) and analyzed the Ag specificity and the structure of the VH segment of the produced IgM mAbs.8 Here, we extend those findings to the analysis of a third AIDS-BL cell line and to the analysis of the B-cell origin of these three AIDS-BL clones. Two AIDS-derived BL cell lines produced anti4 IgM cold agglutinins and utilized somatically mutated VH4-21 genes.8 The third AIDS-BL cell line made an IgM antibody of undetermined specificity and used a putatively mutated VHIII gene. Two IgM-producing BLs originated from B-l cells and one from B-2 cells.

AIDS-BL CELL LINES

The features of the HBL-1, HBL-2, and HBL-3 cell lines and respective mAbs are summarized in TABLE 1. The cell lines were established from bioptic specimens of three AIDS patients with BL, diagnosed by histologic and immunophenotypic criteria. The monoclonality of the AIDS BL cell lines and the absolute relatedness to the respective tumors was formally established by the analysis of the Ig H and L chain gene rearrangements and that of the c-myc oncogene tran~locations.~ HBL-1 cells were positive for EBV and negative for HIV and HTLV-I DNA sequences. HBL-2 and HBL-3 were negative for genetic sequences of the three viruses.

CD5 mRNA EXPRESSION

To determine the lymphocyte origin of the cell lines, we analyzed its CD5 mRNA expression, using a PCR amplification method we recently devised ad h o ~ . ~ As we previously showed, CD5 mRNA is characteristically expressed by cells of the B-1 population, be they surface CDS' (B-la cells) or surface CD5- (B-lb cells).I0 HBL-I and HBL-2 cells expressed detectable amounts of CD5 mRNA suggesting a B1 cell origin, while HBL-3 cells did not (FIG. 1B). The specificity of the PCR method was detailed in our original report lo and was further supported by the proportionality between amount of input mRNA and amount of CD5 cDNA amplified (FIG. 1A and C).

ANTIGEN-SPECIFICITY OF THE IGM MABS

The Ag-specificity of the HBL-1, HBL-2, and HBL-3 IgM mAbs was deter- mined using specific ELISA4.'0s'l and self and exogenous Ags among the most commonly recognized by natural antibodies and autoantibodies, including IgG Fc fragment, human thyroglobulin, ssDNA, phosphorylcholine, insulin, human recombinant TNF-a, TNF-P, and IL-IP, i/I blood group determinants, myelin- associated glycoprotein, P-galactosidase and LPS from E . coli, tetanus toxoid, HIV-1, recombinant glycoproteins of CMV and parvovirus B19, Haemophilus injluenzae type b capsular polysaccharide, and Pneumococcus polysaccharides. The HBL-1 IgM mAb did not bind any of the Ags tested. The HBL-2 and HBL-3

TA

BL

E I

. Fe

atur

es o

f th

e A

IDS-

BL

Cel

l Lin

es a

nd R

elat

ed m

Abs

mA

b Tu

mor

Ag

Cha

ins

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l EB

V

HIV

H

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-I

Line

So

urce

H

isto

logy

D

NA

D

NA

D

NA

c-

myc

H

L

Spec

ifici

ty

HB

L-1

Peri

pher

al

SNC

C

Yes

N

O

NO

t(8

;14)

p

K

ND

' bl

ood

(L3)

H

BL-

2 Pl

eura

l SN

CC

N

o N

o N

O

t(8;1

4)

p

A 1

fluid

4f

H

BL-

3 Li

ver

SNC

C

NO

N

O

NO

5(

8;22

) p

A

1 m

ass

0.6f

VH

Seg

men

t N

ucle

otid

e N

ucle

otid

e (A

min

o D

iffe

renc

es in

:

VH

Iden

titya

~

D

JH

FR

Aci

d)

CD

R

Gen

e %

R

S

R

S G

eneb

G

ene

Hum

5igv

hd

94.6

5

2 4

5 D

LR3

JH

~

(V,II

I)d

(91.

0)

(2.9

)' (9

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vH

4-2 I

93

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7 6

20P3

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6

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d 84

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22

DX

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(88.

6)

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(80.

4)

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6.2Y

(I

nver

ted)

" C

ompa

red

with

the

gen

omic

ger

mlin

e se

quen

ces.

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and

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s ha

ve b

een

repo

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by

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ne 2

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has

been

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orte

d by

Sch

roed

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Not

det

erm

ined

. T

he c

ompl

ete

sequ

ence

of

the

geno

mic

ger

mlin

e H

um5i

gvh

gene

is n

ot p

ublis

hed

and

is a

vaila

ble

from

Gen

Ban

k un

der

acce

ssio

n nu

mbe

r M99401.

Expe

cted

num

bers

of

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utat

ions

cal

cula

ted

as re

port

ed in

Cha

ng a

nd C

asal

i.2"

The

sequ

ence

of

the

geno

mic

ger

mlin

e V

d-21

gen

e ha

s be

en r

epor

ted

by S

anz

er

f Sm

alle

st a

mou

nt (

ng) o

f Ig

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ain-

treat

ed c

ord

RB

Cs

in a

10O

-pl r

eact

ion

volu

me.

512 ANNALS NEW YORK ACADEMY OF SCIENCES

B

,T Lymphocytes

c

FIGURE 1. Expression of CD5 mRNA by AIDS-BL cells. mRNA from AIDS-BL cells (1 .O and 0.5 pg) and T lymphocytes (1 .O to 0.001 wg) were reverse transcribed. cDNAs were amplified by PCR using p-actin or CDS-specific primers. (A) Ethidium bromide-stained gel of amplified p-actin DNA (-0.6 kb). Panels B and C, hybridization of the 32P-labeled “inter- nal” CD5-specific oligonucleotide probe with the fractionated CD5 DNA amplified from cDNA independently reverse-transcribed from different amounts of purified mRNA from AIDS-BL cells and T lymphocytes, respectively.

IgM mAbs strongly and uniquely bound to the i determinant on human erythro- cytes, as shown by their agglutination of cord (smallest agglutinating dose: 4 and 0.6 ng/107 RBC, respectively), but not adult (no agglutination by HBL-2; HBL-3 mAb smallest agglutinating dose, 38.8 ng/ lo7 RBC) papain-treated human RBCs.

IcM MAB V, GENES

To determine the structure of the V, segments of the HBL-1, HBL-2, and HBL-3 IgM mAbs, we cloned and sequenced the respective cell line Cp cDNA.~

RIBOLDI el al.: IGM MABS FROM AIDS LYMPHOMAS 513

FIGURE 2 depicts the nucleotide (A) and deduced amino acid (B) sequences of the HBL-1, HBL-2, and HBL-3 IgM mAb V, genes and those of the closest reported germline VH genes. The nucleotide differences are summarized in TABLE 1. The HBL-1 mAb V, gene sequence was 94.6% identical to that of HumSigvh gene, a member of the V,III family. Nine of the 16 nucleotide differ- ences displayed by the HBL-I gene sequence resulted in putative amino acid replacements yielding replacement to silent (R: S) mutation ratios of 2.5 in the CDRs and 0.8 in the framework regions (FRs). The HBL-2 and HBL-3 mAb V, gene sequences were 93.5% and 84.2% identical, respectively, to that of VH4.21 gene, a member of the VHIV family." Twelve of the 19 nucleotide differences displayed by the HBL-2 VH gene sequence resulted in putative amino acid replace- ments yielding R : S mutation ratios of 5.0 in the CDRs and 1.1 in the FRs. Nineteen of the 46 nucleotide differences displayed by the HBL-3 V, sequence resulted in putative amino acid replacements yielding R : S mutation ratios of 2.2 in the CDRs and 0.3 in the FRs.

The structure of the CDR3s of the HBL-1, HBL-2, and HBL-3 IgM mAbs is depicted in FIGURE 2 (panel C, nucleotide sequence compared to the closest germline D and JH genes; panel D deduced amino acid sequences). The HBL-I, HBL-2, and HBL-3 mAb D gene sequences displayed some identity with those of the DLR3, the expressed fetal 20P3 and the DXP'I D gene^,''.'^ respectively, and were juxtaposed with the untruncated and mutated form of the J,3 gene (HBL-I), and truncated and mutated forms of JH6 and JHS genes (HBL-2 and HBL-3, respectively).

SOMATIC MUTATIONS IN THE HBL-3 IGM MAB V, GENE

The high number of nucleotide differences displayed by the HBL-3 VH gene sequence when compared with that of the VH4-21 germline gene and the presence of mutations in the HBL-3 mAb JH5 segment, in conjunction with the conservation of the VH4-7,1 gene in humans, strongly suggested that the HBL-3 mAb V, segment consisted of a somatically mutated form of the VH4-21 gene. Thus, we amplified by PCR DNA from the HBL-3 cell line or autologous fibroblasts using the sense VH4-21 FR1 primer in conjunction with the antisense VH4-21 FR3 primer, both encompassing sequences shared by the VH4-21 segment and the expressed HBL-3 VH gene (FIG. 3). The two combined primers amplified DNA from both fibroblasts and HBL-3 cells, yielding products of appropriate molecular size.* The same antisense VH4-21 FR3 primer was also used to amplify fibroblast DNA in conjunction with the sense HBL-3 leader primer, encompassing a stretch of the leader sequence of HBL-3 V, gene and differing in only one nucleotide from that of the corresponding area of the V58 gene, the VHIV family member displaying the highest degree of identity with the vH4-21 gene. The amplified product of appropriate molecular size (-400 bp) was obtained.* The three DNA amplification products were analyzed for their ability to hybridize the [y-32P]-labeled HBL-3 CDR 1 oligonucleotide, encompassing a stretch of the HBL-3 VH gene FR1-CDRl sequence that displayed seven putative mutations when compared with corresponding germline VH4-21 gene sequence. The oligonucleotide strongly hybridizes with the DNA amplification product from the HBL-3 cell line but not with the -260 or -400 bp DNA amplified from the autologous fibroblasts.*

A 5 ig

vh

HBL-

1

5igv

h HB

L-1

5igv

h HB

L-1

5igv

h HB

L-1

..

. FR

3 .

.3'

AGGC

AGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCTGC~TGAACAGCCTG-CCGAGGACACAGCCGTGTATTACTGTACTAGA

300

b 3 8 b

4

B Sigv

h HBL-1

VH4.21

HBL-3

HBL-2

C DLR3

HBL-1

20P3

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. FR1

. . CD

Rl

. FR2

. CDRZ .

. FR3

. QV

QLPP

WGAG

LLKP

SETL

SLTC

AVYG

GSFS

GYYW

SWIR

QPPG

KGLE

WIGE

INHS

GSTN

YNPS

LKSR

VTIS

VDTS

KNQF

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SSVT

AADT

AVYY

C~ 97

5'

. A

GC

AT

AT

TG

TG

GT

GG

TG

AT

CT

AT

TC

C

GGccCT------ AACAC-T--C-C-

ACGTGGGAGCTACT

G-CCTT--G---G

DXP'l(1NV)

GTTATAATAACTCCCCGAACCATAGTAA

TAC

ACAACTGGTTCGACTCCTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA

HBL-3

--AGAGG-A--TTC-GA-----ACAGTGGAAGACTT

-----G--------G----A-T-----------------

JH3

HBL-1

JH6

HBL-2

JH5

HBL-3

FIGURE 2.

Nuc

leot

ide

and

dedu

ced

amin

o ac

id s

eque

nces

of t

he V

H (A

and

B),

D, a

nd J

H (C

and

D) g

enes

util

ized

by

the

HB

L-1

, HB

L-2

, and

H

BL

-3 I

gM m

Abs

. The

top

sequ

ence

is g

iven

for

com

pari

son

and

repr

esen

ts th

e pu

blis

hed

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line

VH

, D, a

nd J

H ge

nes

disp

layi

ng th

e hi

ghes

t de

gree

of

iden

tity

to th

e ex

pres

sed

VH

-D-J

, gen

es. D

ashe

s in

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te id

entit

y. S

olid

line

s on

the

top

of e

ach

clus

ter

depi

ct C

DR

s.

516 ANNALS NEW YORK ACADEMY OF SCIENCES

HBL-3 GL I I 1

FIGURE 3. Evidence that the expressed HBL3 V, segment is somatically mutated. Contin- uous lines depict sequence identities. Boxes depict CDRs or primer sequences. Using the VH4-21 FRl and VH4-21 FR3 oligonucleotide primers appropriate products were amplified by priming genomic DNA from fibroblasts (HBL-3 GL) and HBL-3 cells (HBL-3 FRI/FR3). Analysis of these products showed that HBL-3 GL sequence was identical to that of the V,4-21 gene, and the HBL-3 FRl/FR3 sequence was identical to that of the expressed HBL-3 VH segment.

To identify the autologous germline V, gene that putatively gave rise to the expressed HBL-3 V, gene, the product amplified from fibroblast DNA using the sense VH4-21 FR1 and the antisense VH4-21 FR3 primers was cloned and se- quenced. The sequences of eight independent clones were all identical to each other and to that of the VH4-21 germline gene throughout the overlapping area (FIG. 3; HBL-3GL sequence). DNA amplified from the HBL-3 cell line using the sense vH4-21 FRl and the antisense V,4-21 FR3 primers was also cloned and sequenced. The sequences of three independent clones were identical to each other and to that of the expressed HBL-3 V, gene throughout the overlapping area (FIG. 3; HBL-3 FRl/FR3 sequence). These experiments proved that the expressed HBL-3 VH segment was somatically mutated and suggested that VH4-21 is the germline gene that gave rise to it.

SUMMARY AND CONCLUSIONS

In the present studies we analyzed the Ag specificity, VH gene structure, and cellular origin of three IgM mAb-producing cell lines established in uitro from bioptic specimens of three AIDS patients with BL. We found that (i) both HBL-2 and HBL-3 IgM mAbs were cold agglutinins highly specific for the i blood group determinant, a self Ag the expression of which is dominant in the early stages of life, and both mAbs used somatically point-mutated VH4-21 segments; (ii) HBL-I IgM mAb, the Ag-specificity of which has not been determined, used a putatively mutated member of the VHIII family; and (iii) both HBL-1 and HBL-2, but not HBL-3, cells expressed CD5 mRNA, suggesting a B-1 cell origin.

The utilization of vH4-21 by the HBL-2 and HBL-3 cold agglutinins is consistent with the usage of this gene segment by all the reported pathogenic except the naturally occurring cold agglutinins. 15-18 This restricted V, gene usage may reflect an inherent affinity of the germline vH4-21 gene product for the i/I carbohydrate structure, and, perhaps, an overrepresentation of VH4-21

RIBOLDI et al.: IGM MABS FROM AIDS LYMPHOMAS 517

in the human early and late B-cell r e p e r t ~ i r e . ' ~ . ' ~ Consistent with both an early and late developmental expression of the VH4-21 gene is the B-1 and B-2 cellular origin of the two VH4-21+ cold agglutinins reported here. Thus, the two cold agglutinin autoantibodies possibly emerged at different stages of the natural history of the B-cell repertoires of these patients and might display a different temporal relationship, as discussed below, to the time of emergence of the respective tumoral cells.

The somatically mutated status of the HBL-2 and HBL-3 mAb V, segments was suggested by the monomorphism of the human VH4-21 gene, the extension of the nucleotide differences to the, in general, highly conserved J H segment; and it was formally proved in HBL-3 mAb. Positive selection by Ag of the R mutations in the HBL-2 and HBL-3 mAb VH segments was suggested by the differential R : S mutation ratios in the CDRs and FRs (HBL-2 mAb, 5.0 and 1.1, respectively; HBL-3 mAb, 2.2 and 0.3, respectively) but not substantiated by appropriate statistical analysis according to the binomial distribution model.?" In fact, changes in the structure of the VH segment CDRs may be irrelevant to a self Ag selection of the HBL-1 and HBL-3 mAbs, as it has been recently postulated that the structural correlate for i/I Ag binding is provided by an unmutated sequence of the VH4-21 FRl (Capra et al., unpublished findings). Consistent with the suggested primary role of structurally conserved VH4-21 in binding the i Ag, both HBL-2 and HBL-3 mAb V, segments displayed significant lower numbers of R mutations in the FRs than theoretically expected ( p = 0.03 and p = 0.00000002, respectively).

In these AIDS-associated BLs, it is unclear whether the initiation of the anti-i/I autoantibody response constituted a crucial event in the neoplastic transformation. The B-1 cell origin of the HBL-1 and HBL-2 mAbs is consistent with an early (fetal or neonatal, when B-1 cells are predominant in the B-cell repertoire) emergence of the respective B-cell clonotype, perhaps preceding the occurrence of the genetic accident, c-mvc translocation; the B-2 origin of the HBL-3 mAb would be equally consistent with a similar and relatively early emergence of the respective B-cell clonotype or with a later (adult) origin, when B-2 cells are predominant. In the latter case, c-myc translocation might have preceded the rearrangement and expression of the Ig genes in this B-cell clone. Knowledge of the sequential order of activating, proliferating, and transforming events is crucial for a better understanding of the molecular pathogenesis of AIDS BL.

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