1. No category

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1996 87: 3429-3436
Molecular single cell analysis demonstrates the derivation of a
peripheral blood-derived cell line (L1236) from the Hodgkin/ReedSternberg cells of a Hodgkin's lymphoma patient
H Kanzler, ML Hansmann, U Kapp, J Wolf, V Diehl, K Rajewsky and R Kuppers
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Molecular Single Cell Analysis Demonstrates the Derivation of a Peripheral
Blood-Derived Cell Line (L1236) From the HodgkWReed-Sternberg Cells of
a Hodgkin’s Lymphoma Patient
By Holger Kanzler, Martin-Leo Hansmann, Ursula Kapp, Jurgen Wolf, Volker Diehl, Klaus Rajewsky,
and Ralf Kuppers
A novel cell line, L1236, was establishedfrom the peripheral
blood of a patient with Hodgkin’s disease (HD). Two Ig VH
and one V, gene rearrangementswere amplified by polymerase chain reaction (PCR) from the DNA of this cell line, demonstrating its derivation from the B-cell lineage. To test the
cell line for its clonal relationship to the Hodgkin/ReedSternberg (H-RS) cells in the patient, single H-RS cells were
micromanipulatedfrom tissue sections of a tumor-infiltrated
bone marrow specimen from that patient and analyzed by
PCR for Ig gene rearrangements. The same rearrangements
detected in the cell line were also found repeatedly in H-RS
cells of the biopsy material. Thus, L1236 is the first cell line
established from a case of HD for which a derivation from
the H-RS cells of the patient could be demonstrated. Furthermore, the selective isolation of identical V gene rearrangements from multiple single H-RS cells demonstrates that
these cells represented a clonal population in the patient.
0 1996 by The American Society of Hematology.
I
data concerning the case history are provided in the accompanying
article by Wolf et al.
PCR analysis of cell line LI236. DNA was isolated from cell
line L1236 by standard methods.” Using VH and V, framework
region I (FRI) family-specific primers together with J-segment
primers?’the cell line was analyzed for rearranged VHand V, region
genes as previously described.” In addition, VH family-specific
primers hybridizing to the leader region of VH genes were used. A
PCR product was obtained with a vH1 leader primer (5’TCACCATGGACTGGACCTGGAG 3’) and a vH3 leader primer (5’
ACCATGGAGTTTGGGCTGAGCTG3‘). PCR products were gelpurified and directly sequenced from both sides using the cyclesequencing system (Life Technologies, Eggenstein, Germany) and
the oligonucleotides used for amplification.
Micromanipulation and single cell PCR. Single cells were isolated from frozen sections of a bone marrow specimen of an HD
patient by micromanipulation as previously described.16.20 Single
CD30+ H-RS cells and CD3+ T cells were obtained from adjacent
sections of the same specimen. Single B cells were micromanipulated from the mantle zone of a tonsillar section of another individual.
That section was stained with Ki-67 antibody to identify germinal
centers and the surrounding mantle zone. Micromanipulated cells
were coded and analyzed in a blinded fashion.
Ten H-RS cells and seven T cells were analyzed in the first round
of amplification with a primer mix containing the VH1FRI primer,
FlOVHl (5’GCCTGGGGCCTCAGTGAAGGT3‘; Fig I), a primer
specific for a region in the intron between JH3 and JH4 (H3P1: 5’
CATCTCCCAGSTCCASGACAGA 3’; Fig 2), and the VH3, V,3,
and 3’ JH and J, primer mixes.” In the second round of amplification,
a 1-pL aliquot of the first round was reamplified in separate reactions
with the following primer combinations: FlOVHl/S‘ JHmix,’” VH3/
H3P2 (5‘ AGSTCCASGACAGAGGACGCTG3’, internal to H3P1;
Fig 2), and V,3/5‘ J, mix.
Another set of 10 H-RS cells, three ‘r cells, and 10 B cells were
N HODGKIN’S DISEASE (HD), the characteristic Hodgkin/Reed-Sternberg (H-RS) cells represent only a minor
population in the tumor-usually less than 1% of cells. Due
to the sparseness of these cells and the difficulties encountered in attempts to isolate them,’.’ clonality and lineage
derivation of H-RS cells is much
Furthermore,
little is known about gene expression and cellular differentiation of H-RS
To overcome the difficulties in studying
biopsy material and to establish an in vitro system for the
investigation of HD, numerous attempts have been made to
obtain cell lines from HD patients. So far, 14 such cell lines
have been described in the literature.”” However, since a
derivation from H-RS cells of the respective patients has not
been proven for any of these lines, their relevance for a
characterization of the H-RS cells in the patient remains
unc1ear.
Recently, we established a method to isolate single cells
from frozen tissue sections by micromanipulation and analyzed those cells for rearranged V region genes.I6 Since V
gene rearrangements are restricted to B-lineage cells and are
highly specific for a B-cell clone,I7 the detection and sequence analysis of such rearrangements can be used to study
the B-lineage origin and clonal relationship of the cells analyzed. The micromanipulation and polymerase chain reaction
(PCR) method was applied to study three cases of HD for
a possible clonality and B-lineage origin of the H-RS cells.”
Indeed, in all three cases, H-RS cells represented a clonal
population and appeared to be derived from B cells at various
stages of development.
Recently, a novel cell line, designated L1236, was established from the peripheral blood of a patient suffering from
HD of mixed cellularity subtype (see accompanying article
by Wolf et a l l g a ) . Since biopsy material of the HD patient
was available, we were in the position to address the question
of whether the H-RS cells of the patient and LIZ36 cells
carried Ig gene rearrangements, and if so, whether these
rearrangements were identical.
MATERIALS AND METHODS
Case history. In 1991, HD of mixed cellularity subtype was
diagnosed in a 31-year-old patient. Cell line L1236 was established
from the peripheral blood of this patient (see accompanying article
by Wolf et al). In April 1994, tumor infiltration of the bone marrow
was observed. The respective bone marrow specimen obtained for
monitoring tumor progression was the source of single H-RS cells
analyzed in this study. The patient died in May 1994. Additional
Blood, Vol 87, No 8 (April 15). 1996: pp 3429-3436
From the Institute for Genetics and the Departments of Pathology
and Internal Medicine, University of Cologne, Cologne, Germany.
Submitted June 20, 1995; accepted December 1, 1995.
Supported by grants from Deutsche Forschungsgemeinschafr
(Di184, Di1284/1 -5, and SFB243-).
Address reprint requests to Ralf Kiippers, PhD, University of
Cologne, LFI-Gebaude, E4 R706, Josef-Stelzmannstr. 9, 50931 Cologne, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
OOoS-4971/96/8708-O7$3.00/0
3429
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KANZLER ET AL
3430
Leader
Phe Cy6 Leu LeuAla Val AlaPro
L1236H1 TTC TGC TTG CTG GCT GTA GCT CCA GGTAGAGGGCCAACTGGTTCCAGGGCTG
HV1F10
_-- -__ ----_ - ---A-----------------------
___
___
___
___
GlY
__
L1236H1 AGGAAGGGATTTTTTCCAGTTTAGAAGACTGTCATTCTCTATTGTGTCCTCTCCGCAG GT
HVlF10 -------------------------G---------------C---------------Leader
Val His Ser GlnVal Gln Leu Val
Gln SerGly Ala Glu Ile Lys
Arg
CAA GTA CAA CTG GTG
C M TCT GGGGCT GAA ATT AAGA=
L1236H1 GTT CAC TCC
HV1FIO -C- --- --- --G --G --G _-- --- --G --- __-- _ - --G G-G
-A-
___
Pro Gly Ala Set Val
Lys Val His Cys Lys Thr Ser Gly
Val Tyr
Phe
L1236H1 CCT GGG W C TCA GTG AAGGTT CAC TGC AAG
ACA TCT GGA TAC GTC TTC
"- -"
_" "KLH 1
_-- -__ -_TC- _ _ ---- G"
--_ ACHVIF10
___
___
___ ___
___
___
"____
CDR I
Thr Ser Tyr Tyr Ile His Trp Val Arg Gln Pro Arg Gly Gln Gly Leu
L1236H1 ACG AGTTAT TATATT CAC TGG GTG CGA CAG CGT
CAA GGCCTT
KLH1
--- --- -_- --- ----- --- --- --- --- ---CCTGGA
--_ _-- --HV1F10 --C --C --C
--G
--- --_ _-- --- G-C -C- --_
--G
___
___
___
___
___
___
CDR I1
Glu TrpMet Gly GlyIle Gly Pro Gly Val Gly Ser
Met Thr
Cys Ala
L1236H1 GAG TGG ATG GGA GGA ATC GGC
GGT CCC
GTTGGC TCG ACA ATGTGC GCA
KLHl
--- --- -__--- --__HVIF10
-__ AT- _ _ -AA- --T A-- -G- --T A X _ _ _ -GC -A-
______
___
___
-_____ ___
___ ______
_________ ___
Glu LysPhe Gln Gly Arg Leu Thr Met Thr Arg Asn
Thr Ser Thr Thr
L1236H1 GAG AAGTTC CAG GGC AGA CTC ACC ATG ACC AGA AAT
ACG
ACG ACT
--- --- --- --- --- - --- --- --- - -- - --- --- TCC
--- --- --KLH 1
Fig 1. vH1 gene
rearrangeHV1FIO C-- --- -__
__- G" - _ _
--G G-C
-GC
ments from cell line L1236 and
___
__
___
______
__
______ ___
Leu Ser Arg Leu Arg Phe Glu Asp Thr Ala
Thr Val Tyr Met Glu
L1236H1 ACA GTT TATATG G M CTG AGC AGA CTG CGA
--- --- -__--- ----- -____---- TTT GAG
_-- GAC
--_ACG
_-- GCC
--- GTG
KLHf
--C
--C
--G
__--C
_-A--C----_
_---HVlFlO ---
___
___
___
___
___
___
CDR I11
Tyr Phe Cys Gly Arg Gly Gly Arg Trp Arg Ser Gly
Tyr Asn
Asn Gly
L1236H1 TAC TTC TGT GGG AGA GGT GGCAGG TGG CGC AGT GGG AAC TAC AAT
--- - _ _-_---KLH1
HVlFlO --T -A- --- -c- -" "- TT- -A-
___
-_____
___
"
His
Trp Gly Gln Gly
L1236H1 CAC TGG GGC CAG GGA
KLH 1
JH&
l'-- --------
---
analyzed in a blinded fashion with the standard VH and V, primer
mixes as previously described.'6.20
RESULTS
Amplification of rearranged V region genes from cell line
L1236. To analyze cell line L1236 for Ig gene rearrangements, three sets of oligonucleotides were used. For Ig H
gene rearrangements, either a set of six VH leader region
family-specific primers or a set of six primers that hybridize
to the VHFRI of the six human VHgene families were used
together with a J H gene primer mix. Analysis for V, gene
single H-RS cells. The vH1 gene
rearrangement obtained from
cell line-Ll236 (L1236H1) is comVal
pared with the vH1 sequences
amplified from single H-RS cells
(KLHl), the HVlFlO germline
gene.= and the JH4b gene segment." (---l Sequence identity.
Codons are numbered according
to the method of Kabat et al?'
GGC
Correspondingaminoacidsare
shownaboveeachcodon.The
leader region and CDRlto CDRlll
of the VH generearrangement
are indicated. No DH genesegment could be identified in
CDRIII. The sequence of primer
FlOVHl is underlined.
rearrangements was performed with a set of six V, familyspecific primers and a J, primer mix.
A vH1 gene rearrangement was obtained withthe vH1
leader primer, but not withthe FRI primer. Sequence analysis
of the PCR product revealed a potentially functional rearrangement of a vH1 gene with highest homology to the
HVlFlO vH1 germline gene2*(Fig 1, L1236Hl). There are
55 nucleotide differences between the VH gene segment of
L1236H1 and the HVlFlO gene (ie, 87% homology). A J H ~
gene was used in the rearrangement, but no DH gene could
be identified (Fig 1). The J H gene carried four point mutations
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
HRS CELL DERIVATION OF CELL LINE L1236
8#88
40
Ll236H3
KLH3
v341
25
TCT
FR 1
343 1
FR 11
8888*888888888-
##**88~88
29
CAA ATC ACC TTC A
"_ T__
"_ ___ "___
_ -_GT AN
___ a_
-
50
"_
"_ CGC
"_ CAG GCT CCA
"_ GGG AAG
"_ GGG
"_ CTG
"_ W
"_ TCA
"_GTCTCATGTGTCAAT"_TATTGT"_ "_ "_"_
TAT AGC __G __c
______ ______ ___ ___ ___ ___ ___ ___ -4 -GG ___ ___ -CC A-T _G_ AG#**U******
FR
70
L1236H3
KLH3
v 3 4
*Pe*Pe88*888**"---------'
35
ATT AAG
TGG
GTC
111
***88****#
****PO***# FR 111
A__
88*#*88#8*888
60
TTT TTT CAT GCA GAC
GAC
AAT
GA CGA TTC AAC
ATC
TCC
AGA
GAC
AGC
ACC
___ AG___ ___
___ ______ ______ TNA4- ?TG
___ GAC
___GTC___ CCA
___ TGC
___ATC
___ATNTCCACA
--C ___ ___ ___ ___ __ ___ ___ _______________
___
___
-4 A-A _AC T-C ___ ___ ___ G__ AG4 _G_ _T_ _c_ ___ ___
_G
___
-4
4
___
___
_c_
___
___
___
___
_A_
G__
-
CTT CCTTAT
AG
[--------- dupljc=&.ion
-----------]
[--"""-
duplication
""""--l
#**LI**#*###~**pM4*****LL.***t***
90
80
L1236H3
KLH3
v341
94
___
______ _________ _______________-4
___ ___ G-NNN-- -________ ___ ___ ___ N-N __-__-__-____
___ ___
_AC ___ ___ T__ ___ ___ A__ ______
--G ___ ___ G" G& -A- -4 ___ ___ _G__
-TG"""""""-
AAG AGG TCACTGCATCTG
CAA CTG AAC AGCCTAAGA
Ca: GAC GAC ACGACTCTTTCTTATTGT
GCG ACA G A G A C A T C G T C C A ~ T C C T T
"
intron JH3-JH4
g-
L1
236H3
JH-lms
36 bp
to end of JH3
G T C G T ~ ~ ~ G A T ~ T G T ~ ~ G G T C T C T T C T ~ T G T ~ ~
__~____G________A___-_____~-~-------~---__-_A_---_---__---_
212bp to JH4 ->
Fig 2. V d gene rearrangements from cell line L1236 and single H-RS cells. Sequence format is the same as in Fig 1. VH3 rearrangement
detected in cell line L1236 iL1236H31is comparedwith VH3 sequences amplifiedfrom single H-RS cells (KLHI), the V3-21 germline gene:6 and
part of the intron between JH3 and Jn4." FRI to FRlll are indicated. A tetranucleotidemotif (5'CCAG3'/5'CTGG3') often found near nonhomologous recombination breakpointsin Ig genes*' is underlined nearthe deletion in CDRI, within the duplication in FRIII, and at the recombination
breakpoint in the Jn intron. A duplication of codons66 to 73 is indicated.N. nucleotides not clearly readableon the sequence gels. The location
of primers H3Pl and H3P2 is indicated below and above the JH3/JH4intron sequence, respectively.
as compared with the J ~ 4 bgermline ~equence.'~
The failure
to amplify this rearrangement with the vH1 F R I primer was
most likely due to the fact that there were three mismatches
in the rearranged VH gene versus the vH1 FRI primer, one
close to the 3' end of the primer.
A vH3 gene rearrangement of unexpected length was amplified with both the vH3 FRI and leader primer. The PCR
product was about 250 bp longer than would be expected
for a typical VH gene rearrangement. Sequence analysis of
the product obtained with the FRI primer showed several
unusual features. The V gene segment was rearranged into
the intron between JH3 and JH4, and there was a deletion
of codons 30 to 33 in complementarity-determining region
(CDR) I and a duplication of codons 66 to 73 in FRIII (Fig
2, L1236H3). No DH gene segment could be identified. The
VH gene segment of L1236H3 showed 77% homology to
the V3-21 vH3 germline gene." Interestingly, close to the
rearrangement breakpoint in the JH intron, a tetranucleotide
sequence motif (5'CTGG3'/5'CCAG3') commonly found
near nonhomologous recombinations involving Ig gene sequence~~'
was found twice (Fig 2). The same motif was also
seen near the deletion in CDRI and within the duplication
in FRIII (Fig 2). Rearrangement of the VHgene into the JH
intron about 250 bp upstream of the JH4 gene segment explains the unusual length of the PCR product.
A potentially functional V,3 gene rearrangement amplified from genomic DNA of L1236 was composed of the L2
V,3 germline gene2*rearranged to J,lZ9 (Fig 3, L1236K3).
The V, gene segment of L1236K3 carried 41 nucleotide
differences versus L2 (84% homology). One mutation was
found in the J,l gene segment (Fig 3).
Since in a Southern blot analysis for rearranged Ig genes
in L1236, two rearrangements at the Ig heavy-chain locus
and one rearrangement at the K locus were detected (see
accompanying article by Wolf et al), all V gene rearrangements carried by the cell line were successfully amplified
by PCR.
For the three V gene rearrangements amplified from
L1236, an analysis of the mutation pattern was performed.
The relation of amino acid replacement (R) to silent (S)
mutations (IUS value) can be taken as an indication for a
possible selection of the antibody molecule for high-affinity
binding to an antigen: R mutations should be underrepresented in the F R S to preserve the structure of the antibody
V domain, whereas they are often overrepresented in CDRs,
where R mutations can lead to an increased affinity to the
respective antigen. IUS values of the three V gene rearrangements were compared with the expected values, assuming
random mutagenesis (Table 2). It is evident that R mutations
are underrepresented in the FRS of the two potentially functional V region genes (L1236H1 and L1236K3). In the nonfunctional vH3 gene rearrangement-which cannot be selected, since no protein is expressed-IUS values of F R S
and CDRs are as expected for random mutagenesis (see
Table 2).
Amplijication of rearranged V region genes from single,
micromanipulated H-RS cells. The availability of an H-RS
cell-infiltrated bone marrow specimen of the HD patient
offered the unique chance to analyze the cell line for its
clonal relationship to H-RS cells in the patient.
Using a recently established method,16 single H-RS cells
were micromanipulated from a frozen tissue section of a
bone marrow specimen from the HD patient (Fig 4). Twenty
CD30+ H-RS cells and 10 T cells micromanipulated from a
CD3-stained adjacent section of the same biopsy material
and 10 B cells micromanipulated from the mantle zone of a
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KANZLER ET AL
3432
L1236K3
KLK3
L2
Leu Ser Leu Ser ProGly Glu Thr Ala Ile Leu TyrCys Arg
CTGTCT CTG TCT CCC GGA GAA ACC
GCC ATC CTC TAC
TGC AGG
_ _ _ ___
"- "-
G"
___
"_--A
"- ----G
---
"_
-" "- "-GA --- -C- ---
-"
---
-C- ---
"_
Ala Ser
GCC AGT
"- "--_ _ _ ----
CDR I
Asp Ser Ile Gly T h r Asn Leu Ala
Trp Tyr Gln Gln Arg Pro Gly Gln
L1236K3 GAC AGC ATT GGC ACC AAC
GCC TGG
TTA TAT CAA CAG AGG
CCT GGC CAG
KLK3
_-- --- -_- --_ --- --- --- --- --- --- _-- --- --- --_
C-G --T G" A-- -G- --- --- --- --- --C --G -__-AA --_ _-- _-L2
___ ___
CDR I1
Phe
Ser Pro Arg Leu Val Ile Phe Ala Ala AlaThr Arg Ala Ser Ala
L1236K3 TCT CCC CGC CTC GTC TTT
ATC GCT GCA GCC ACC AGG GCG
TCT GCT TTC
--- -_- --- --- --- --- --- --- --- --- --- --- --- --_ _-_-__
KLK3
G"
A-G __-C--A- -G- __-T--__--C A-- -G- A-L2
___
___
___
Pro Pro Arg Phe Ser Gly Gly Gly
Ser Gly Thr Glu Phe Thr Leu Ser
L1236K3 CCA CCC AGGTTC AGT GGC GGT
GGG TCT GGG ACT GAGTTC ACA CTC TCC
_-- --- __---- --- --- --- --- --- --- --- --- --- --_
Km3
G"
-__ A-----A ___ _ _ ---T
A-L2
___
______
___
___
___
___ ___
___
Ile Thr Ser Leu Gln Pro Asp
AlaVal Ala ValTyr Tyr CY8 Gln Gln
L1236K3 ATA ACC AGT CTG CAG CCT GCA GAT GTT
GTT TAT
GCA TAC TGT CAG cAG
--- --- _-- --- --- --- --- _-- --KLK3
-" "--"
""- "- -"
L2
--C -G- --C ------ T-- -A- __-T-- --- --- -"
___ ___
CDR I11
Tyr AspLys Trp Pro Pro Val Thr
Phe Gly Gln
GGC CAG
L1236K3 TAT GAT AAG TGG CCT CCG GTGTTCACG
=K3
--- ---_
-_- A-- --C
-__
L2
"- "JKI
--A
______
___
__
_"
tonsillar germinal center were analyzed by single cell PCR
for rearranged VH and V, region genes. In the first experiment, 10 H-RS cells and seven T cells were analyzed in a
blinded fashion with a set of primers that allowed amplification of the three V gene rearrangements (one vH1, one vH3,
and one V,3) detected in the cell line. Seven of 10 H-RS
cells gave rise to at least one PCR product, with 15 products
altogether (Table 1). Sequence analysis of the three vH1,
seven vH3, and five V,3 PCR products revealed sequences
identical to the ones carried by cell line L1236 (Fig 1 to 3).
No PCR product was obtained for the seven T cells analyzed
as negative controls.
In a second experiment, 10 H-RS cells, three T cells, and
10 mantle zone B cells were analyzed in a blinded fashion
with a set of family-specific primers for the six VH and
six V, gene families as previously described.'6,20Six PCR
products-three vH3 and three VK3-were obtained from
four of 10 H-RS cells (Table 1). Three of 10 B cells gave
rise to a total of five PCR products; three T cells were
negative in the analysis. Sequence analysis of amplificates
obtained from the H-RS and B cells showed sequences identical to the vH3 and V,3 rearrangements of cell line L1236
for H-RS cells, whereas B cells harbored clonally unrelated
V region genes (Table 1). One of the PCR products obtained
from a B cell represented a contamination from a previous
experiment.
Taken together, the same V gene rearrangements carried
by cell line L1236 were repeatedly amplified from single H-
"_
"_
Fig 3. V 2 gene
rearrangements derived from cell line
L1236 and single H-RS cells. Sequence format isthe same as in
Fig 1. The V 3 gene rearrangement
obtained
from
cell
line
L1236 (L1236K3) is
compared
with V 2 sequencesamplified
fromsingle H-RS cells (KLW),
the L2 germline gene,= and the
J,1 gene segment.FJ
RS cells isolated from a bone marrow specimen of the HD
patient from which the cell line was established. These V
gene sequences could not be amplified from any of 10 T
cells and 10 B cells micromanipulated on the same day and
analyzed coded together with the H-RS cells, confirming the
reliability of the single cell approach.
DISCUSSION
A novel cell line (L1236) established from the peripheral
blood of a patient with HD (see accompanying article by
Wolf et al) was analyzed for a possible B-cell lineage origin
and its clonal relationship to H-RS cells in the HD patient.
One V, and two VHregion genes were amplified from this
line. To investigate cell line L1236 for its clonal relationship
to tumor cells in that patient, single H-RS cells were micromanipulated from a bone marrow specimen and analyzed by
PCR. Using two different primer sets, we showed that the
H-RS cells harbored the same V gene rearrangements as the
cell line, thus proving its derivation from the tumor cells in
the patient. L1236 is the first cell line derived from an HD
patient for which anorigin from the H-RS cells of the respective patient could be demonstrated.
The vH1 and the V,3 gene rearrangements carried by
the cell line and the H-RS cells were potentially functional,whereasthe
vH3 gene rearrangementharbored
several unusual features that rendered it nonfunctional:
there was a deletion of 1 1 bp between codons 30 and 33
and a duplication of codons 65 to 73, and the VH gene
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
HRS CELL DERIVATION OF CELL LINE L1236
Fig 4. Micromanipulation of a single RS cell. (AI
Frozen section of an HD-infiltrated bone marrow
specimen with an RS cell in the middle. (B) Frozen
section after picking, showing a hole where the RS
cell was located. The section was stained with antiCD30 antibody.
Table 1. Summary of the Single Cell Analysis of Micromanipulated
H-RS Cells for Ig Gene Rearrangements
Rearrangements
Experiment
Cell
Cells
No.
Type
Positive
PCR
Products
1
H-RS
7/10
15
3 VH1
7 Vn3
5 vx3
T
OD
H-RS
4/10
6
3 VH3
3 v,3
T
0/3
3/10
4
2
B
Repeated
Unique
3 VH3
1 v,2
In experiment 1, only primers for the 3 V gene rearrangements
detected in cell line L1236 were used; in experiment 2, a mixture of
primers for all VH and V, gene families was used. The latter primer
set does not allow amplification of the VH1 rearrangement of L1236.
One further V,4 PCR product obtained from a B cell represented a
contamination from a previous experiment.
was rearranged to the intron between JH3 and JH4. A
tetranucleotide motif (5'CCAG3'/5'CTGG3' ) commonly
found near nonhomologous recombination breakpoints in
Ig genes" may be involved in these recombination
events, since it was found near the deletion in CDRI,
within the duplication in FRIII, and exactly at the recombination breakpoint in the JHintron (Fig 2). Interestingly,
such unusual V gene rearrangements had also been detected in three of five V H gene rearrangements described
in our previous analysis of three cases of HD." Although
more cases need to be analyzed, the frequent detection
of deletionhsertion events in H-RS cells may indicate
some specific genetic alteration of these cells, the nature
of which remains to be explored.
All three V region genes carried by the cell line and the
H-RS cells seemed to be heavily mutated, since multiple
nucleotide differences were seen in the sequence comparison
to the most homologous germline genes (Figs 1 to 3). That
(at least most of) these differences in fact represent somatic
point mutations and not V gene polymorphism or usage
of hitherto undescribed V genes is based on the following
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
3434
KANZLER ET AL
Table 2. RIS Ratio Mutation Analvsis of L1236
V Gene
L1236H1
L1236~3
L1236H3
Region
FR
CDR
FR
CDR
FR
CDR
Random
Found
2.9
4.4
3.0
3.6
3.0
4.4
1.2
3.4
1.5
5.0
3.3
4.0
The random R/S ratio was calculated taking the codon composition
of the respective germline genes into account.
arguments. (1) The human V, locus has been extensively
characterized-most likely, all V, genes and their alleles
have been identified.30 Therefore, the V,3 gene sequence
L1236K3 can be reliably assigned to the L2 germline gene.
(2) There is one mutation in the J, segment of L1236K3 and
there are four mutations in the JH segment of L1236H1 as
compared with the corresponding germline genes. Since no
polymorphism has been described for human J, genes29 and
only three polymorphic forms of JH4 have been found in
numerous VH gene rearrangements,23 the sequence differences in these J gene segments are likely to represent somatic
point mutations.
The detection of VH and V, gene rearrangements clearly
shows that H-RS cells in the patient are derived from a Blineage cell. Cross-lineage Ig gene rearrangements have been
described in non-B-cell leukemias3'; however, they appear
to be restricted to DHJH rearrangement^.^^ VHDHJH and lightchain gene rearrangements have only been detected in B
Furthermore, based on the detection of a high load
of somatic mutations in each of three V region genes, the
progenitor of the H-RS cells appears to be a mature B cell,
since the mechanism of somatic hypermutation is activated
in antigen-stimulated human B cells proliferating in the microenvironment of the germinal center of lymphoid tissues."
Thus, somatically mutated V region genes appear to be confined to germinal center B cells and their descendants, ie,
memory B cells. H-RS cells of the present case may be
derived from an antigen-selected (memory?) B cell. This is
supported by the mutation analysis of the V gene rearrangements: the underrepresentation of replacement mutations in
FRs of the two potentially functional V gene rearrangements
(L1236H1 and L1236K3) but not in the nonfunctional VH
region gene (L1236H3; Table 2) indicates selection for preservation of a functional antibody structure. However, stimulation and selection by an unknown antigen appears to be
no longer needed in the cell line, since no surface Ig was
detectable (see accompanying article by Wolf et al).
Alternatively, malignant transformation may have occurred in a germinal center B cell in which (after leaving
the germinal center) further somatic hypermutation ceased.
The lack of detectable intraclonal diversity due to ongoing
somatic mutation suggests that the differentiation stage of
H-RS cells is distinct from that of tumor cells in follicular
lymphomas. In the latter lymphoma, a germinal center B-cell
derivation is indicated by the detection of ongoing somatic
mutation.3bInterestingly, ongoing somatic mutation was also
detected in one case of lymphocyte-predominant HD in our
previous analysis. "
Since V gene rearrangements lead to permanent changes
at the DNA level, their detection in a cell allows identification of this cell as originating from the B-cell lineage even
if Igs are not expressed. Thus, based on the results of V
gene analysis, the B-cell lineage origin of L1236 is evident,
although the line expresses neither Igs nor B-lineage markers
like CD19 and CD20 (see accompanying article by Wolf et
allsa). Further studies are needed to reveal whether the block
of Ig expression occurs at the level of transcription or translation. The lack of expression of B-cell surface antigens in the
cell line (and in H-RS cells in the patient (see accompanying
article by Wolf et all8") shows that expression of such antigens can be deregulated in H-RS cells derived from the Bcell lineage.
The successful amplification of at least one of three V
gene rearrangements carried by the cell line from 1 I of 20
H-RS cells and the lack of other detectable V gene rearrangements in those cells demonstrate that H-RS cells in this
patient represent a clonal population. It is most likely due
to technical reasons that PCR products were not obtained
for all H-RS cells, eg, the fact that these cells were picked
from about 10-pm tissue sections, so for most of the large
H-RS cells, part of the nucleus should be missing."," The
finding of a clonal population of H-RS cells in this patient
confirms an earlier study of three cases of HD for which
clonality of H-RS cells could also be demonstrated." Clonality of H-RS cells has also been shown using chromosome
probes and interphase cytogenetics in each of seven cases
of HD analyzed by Inghirami et
On the other hand,
Delabie et al" provided evidence that there might also be
cases of HD in which H-RS cells represent a polyclonal
population. Considering the hypothesis that HD may begin
as an infectious process that, only after acquisition of further
mutations in the H-RS progenitor, leads to the outgrowth of
a fully malignant clone,39the discrepancy between the analyses by Delabie et a13' and the studies of Inghirami et al" and
ourselves may reflect different stages of disease progression.
Indeed, two of four patients studied by us (the present case
and a case of lymphocyte-predominant HD in our previous
investigation'') were in a late stage of the disease.
The method of micromanipulation and single cell analysis
for rearranged V region genes is suitable not only to study
H-RS cells for a potential B-cell lineage origin and clonality,
but also for the investigation of various aspects of the pathogenesis of this disease.40 Besides the study of cell lines derived from HD tissues for their derivation from H-RS cells
of the patient-as demonstrated in the present analysis-V
gene rearrangements detected in H-RS cells can be used as
molecular markers to search for members of the tumor clone
in various parts of the body. Using tumor clone-specific
primers (designed from the V gene sequences), stem cell
preparations can be tested for a contamination by tumor cells
in autologous bone marrow transplantation of HD patients,
residual tumor cells may be detected after therapy, and small
lymphocytes in the tumor tissue may be investigated for
potential precursors of large H-RS cells.
Taken together, using a recently established micromanipulation method, we show that a cell line established from the
From www.bloodjournal.org by guest on November 24, 2014. For personal use only.
HRS CELL DERIVATION OF CELL LINE L1236
peripheral blood of an HD patient is derived from that patient’s H-RS cells. This cell line should therefore represent
a valuable in vitro system for molecular and cellular characterization of H-RS cells.
ACKNOWLEDGMENT
We thank Giinter Simons for technical help.
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