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Cytogenetic and Immunologic Identification of Clonal Expansion of Stem
Cells Into T and B Lymphocytes in One Atomic-Bomb Survivor
By Yoichiro Kusunoki, Yoshiaki Kodama, Yuko Hirai, Seishi Kyoizumi, Nori Nakamura, and Mitoshi Akiyama
Chromosome aberration frequency in peripheral blood lymphocytes is elevated in radiation-exposedpeople, and identical karyotypic changes are not infrequently encountered in
one blood sample as well as in separate samples from the
same donor. Suchclonal propagation originates either from
a single immature stem cell able t o expand anddifferentiate
into several cell types or from a single mature lymphocyte
able t o expand after antigen stimulation in vivo. In the present study, atotal 71 T-lymphocyte and 58 B-lymphocytecolonies were established from one atomic-bomb survivor,
who showed a persistent clonal aberration t(4;6), t(5;13) in
phytohemagglutinin culture of peripheral lymphocytes.
Nearly 10%of the colonies (6 T-lymphocyte and 7 B-lymphocyte colonies) showed the same chromosome abnormality.
Southern blot analyses of the T-cell-receptor or Ig heavychain gene showed all different rearrangement patterns
among T- or B-lymphocyte colonies, respectively. Thus, the
chromosome aberration occurred in a precursor cell before
differentiation into T and B lineages and was not derived
from monoclonal proliferation of mature T or Blymphocytes
in the periphery. To confirm the issue, cells from erythroid
burst-forming unit (BFU-E) colonies were examined by the
chromosome-paintingmethod. Two translocations, one between chromosomes 5 and13 and the other between chromosomes 4 and one of group C, perfectly consistent with
the t(4;6), t(5;13), were found in about 10% of the cells. The
results imply that a single stem cell of an adult is capable
ofgenerating long-lived myeloid and lymphoid progeny
amounting to several percent of the total population of circulating lymphocytes and hematopoietic progenitors.
0 1995 by The AmericanSociety of Hematology.
L
possible to explain the origin of such “monoclonal” derivatives. Either a mature lymphocyte bearing a chromosome
aberration has monoclonally expanded in the periphery, or
a precursor cell bearing the chromosome aberration has generated progeny cells through differentiation. In the former
case, all the clonal cells are expected to express the same
phenotype, whereas, in the latter case, lymphocytes in distinctively different lineages may be affected. In any event,
because such clonal cells cogstitute as much as several percent of the cells examined, the total number of major clones
contributing to lymphogenesis appears not to be enormously
large.
In the present study, lymphocytes from one A-bomb survivor were examined in detail. The examinee’s estimated radiation dose was 1.95 Gy, and PB phytohemagglutinin (PHA)
culture consistently showed stable chromosome aberrations
at a frequency of 30% to 45%. Characteristically, however,
clonal aberrations persisted over a period of 10 years in 3
to 8 cells per 100 metaphases. These clonal aberrations were
found to have been derived from a single stem cell in this
case.
YMPHOCYTES AND BLOOD cells originate from
common hematopoietic stem cells, and developmental
pathways from the stem cells have been extensively investigated in experimental ani mal^."^ Furthermore, analysis in
vivo of the fate of a single cell at a certain developmental
stage is possible by means of transplantation of cells carrying
specific markers such as allogeneic markers,’,2chromosome
aberration^,^ and transgenes.“’
However, human data for lymphopoiesis are limited except for some chronic myelogenous leukemia cases in which
Phl-bearing cells can differentiate into separate lineages of
mature blood cells; controversy remains as to whether such
cells can differentiate into T cells.6
Chromosome aberration frequencies in peripheral blood
lymphocytes (PBLs) increase with radiation dose in atomicbomb (A-bomb) survivor^^-^^ and in others exposed to radiation,” and stable aberrations (mainly translocations and inversions) persist for many years after such exposures.
Among the A-bomb survivors exposed toahigh dose of
radiation (more than 1 Gy), identical chromosome aberrations are occasionally encountered in 3 or more cells from
the same bloodsample.’ Two alternative mechanisms are
MATERIALS AND METHODS
From the Departments of Radiobiology and Genetics, Radiation
Effects Research Foundation, Minami-ku, Hiroshima, Japan.
Submitted October I I , 1994; accepted May 8, 1995.
MS
This publication is based on manuscripts (RP 2-66,3-87, 7-89,
23-93) resulting from research pegomed at the Radiation Effects
Research Foundation (RERF), Hiroshima and Nagasaki, Japan.
RERF is a privatefoundation funded equally by the Japanese Ministry of Health and Weware and the US Department of Energy through
the National Academy of Sciences.
Address reprint requests to Mitoshi Akiyama, MD, Department of
Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama
Park, Minami-ku, Hiroshima 732, Japan.
The publication costs of this article weredefrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 I995 by The American Society of Hematology.
0006-4971/p5/8606-02$3.00/0
2106
Blood donor. Venous PB was obtained from a female A-bomb
survivor (65 years old at the time of examination) exposed to the
A-bomb in Hiroshima when she was 20 years old (referred to as the
“study subject”). Her estimated dose was 1.95 Gy by the Dosimetry
System 1986.14 She has no complicated disease history except for
surgery for uterine myoma before 1983.
Lymphocyte colonies. PB mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density-gradient centrifugation after defibrination with glass beads.” The PBMCs were washed twice with
Earle’s balanced salt solution (Nikken, Kyoto, Japan) supplemented
with 2.5% fetal calf serum (FCS; Flow Laboratories, McLem, VA).
To form T-lymphocyte colonies, PBMCs were distributed to 96well round-bottom microtest plates (Costar, Cambridge, MA) at a
mean frequency of 0.5 or 1 celUwell each in 200 pL of GIT medium
(a serum-free medium specified for hybridoma cells; Wako Pure
Chemical Industry, Osaka, Japan) containing 10%FCS, 1:6400 PHA
(Difco Laboratories, Detroit, MI), 2 ng/mL human recombinant interleukin-2 (rIL-2; provided by Takeda Pharmacy, Osaka, Japan),
and feeder cells (5 X lo4 allogeneic PBMCs and 104 lymphoblastoid
Blood, Vol 86, No 6 (September 15), 1995: pp 2106-2112
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2107
CLONAL EXPANSION OF STEM CELL INTO T AND B CELLS
101
102
103
1,a4
FITC-CD34
Fig 1. A flow cytogram of PB mononuclear cells stained with antiCD34 and anti-CD38 MoAbs. The window for isolation of CD34+ cells
is shown by the square.
cells, OKIB, irradiated with 50 and 100 Gy of x-rays, respectively).
After a 2- to 3-week incubation, each growing colony was transferred
into the well of a 24-well plate (Costar) with the same culture medium containing 5 X 105 and 10s irradiated allogeneic PBMCs and
OKIB cells, respectively. The T-lymphocyte colonies were propagated by weekly feeding of the feeder cells in 24-well plates and
were used for further analyses at least 1 week after the last feeding.
Feeder cell contamination was negligible for cytogenetic and immunologic analyses in the present study when the cells growing from
colonies were tested 1 week after the feeding.
B-lymphocyte colonies were obtained by infecting PBMCs with
Epstein-Barr virus. The PBMCs were incubated with the supernatant
of a virus-producing B95-8 cell line for 2 hours at 37”C, were washed
and resuspended in GIT medium supplemented with 20% FCS, and
distributed into the wells of 96-well round-bottom microtest plates
at a mean frequency of 1 0 0 cells/well with irradiated allogeneic
PBMCs (104/well). The cells were cultured in 200 pL of medium
per well, with half of the medium replaced each week. After 4 to 5
weeks of culture, each growing colony was transferred into the well
of a 24-well plate and propagated in the same medium without feeder
cells.
Stem cell sorting and culture. PBMCs were treated withfluorescein-labeled anti-HPCA-2 (CD34) and phycoerythrin-labeled
anti-Leu17 (CD38) monoclonal antibodies (MoAbs; Becton Dickinson Immunocytometry Systems [BD], San Jose, CA) for 45 minutes
on ice. After washing with phosphate-buffered saline containing 2%
FCS, the cells were subjected to a cell sorter, FACStar (BD). The
flow-cytometric pattern is shown in Fig 1, and the fraction of CD34+
cells was 0.062%. About 2,000 CD34+ cells were sorted, and the
purity was nearly 99% by reanalysis of the sorted cells (not shown).
Colony formation of the sorted CD34’ cells was conducted by methylcellulose culture, as described previously.’6 Briefly, CD34’ cells
were plated in 24-well plate at a concentration of about 650 cells/
mL with culture medium containing 1% methylcellulose, 2 U/mL
recombinant human erythropoietin (Sankyo, Tokyo, Japan), and 100
ng/mL human rIL-3 (Genzyme, Cambridge, MA). Colcemid was
added to the culture at a final concentration of 0.1 pg/mL after 7
days of culture, except for several wells that were left to determine
the number of colonies on day 14. After 15 hours of treatment with
colcemid, cells were harvested for chromosome analysis. Colonyforming units (CFUs) on day 14 were characterized as having 40 or
more cells. Cloning efficiency of the CD34+ cells was about 20%.
Nearly 80% of thecolonies consisted of multiple clusters of erythroid
cells (BFU-E), and the remaining colonies were of granulocytes a n d
or macrophages (CFU-G and CFU-GM).
When nonsorted PBMCs were cultured in the same methylcellulose medium, BFU-E and CFU-GM colonies were obtained.
Chromosome analysis. Metaphase spreads were prepared by the
conventional air-drying method.’ The G-band preparations were obtained by a minor modification of the trypsin technique of Seabright”; slides were treated with a 0.1% trypsin solution for 10 to
20 seconds at 30°C and then stained with a 2% Giemsa solution for
20 minutes. Two to six metaphases per colony were analyzed. As
for karyotypes of whole blood leukocytes, 100 metaphases were
analyzed after 48 hours of whole-blood culture in the presence of
PHA.~~
For detection of the clonal aberration t(4;6), t(5; 13) in stem cell
cultures, the fluorescence in situ hybridization (FISH) method was
used with composite probes for chromosomes 4 and 13. The details
have been described previously.’*
Cell surface phenotypes. Cell surface phenotypes of T-lymphocyte colonies were analyzed byflow cytometry using a FACScan
(BD) as described previously.” Fluorescein-labeled anti-Leu-3a
(CD4) and phycoerythrin-labeled anti-Leu-2a (CD8) MoAbs (BD)
were used.
Measurement oflL-2 production. Cells from each T-lymphocyte
colony (0.5 X lo6) were inoculated into a well of a 24-well plate
with 500 pL of medium (RPM1 1640 plus 10% FCS) and 2 X lo5
OKIB, with or without stimulation by 1 pg/mL anti-Leu-4 (CD3)
MoAb (BD). The culture Supernatants were harvested after a 24hour incubation. The quantity of IL-2 in the supernatants was estimated using a modified method of Gillis et al.’’ Murine CTLL-2
cells dependent on E - 2 (4 X 103/well) were exposed to doubling
dilutions (tested at 1:4 to 1:128 dilutions with RPM1 1640 medium
containing 10% FCS) of the supernatant and cultured for 24 hours
in 96-well flat-bottom microtest plates (Costar). Six hours before
termination of the culture, 1.85 X lo4Bq (0.5 pCi) of [’H] thymidine
(New England Nuclear, Boston, MA) was added to each well. These
cultures were aspirated onto a glass-fiber filter, and the incorporated
radioactivity was measured with a liquid scintillation counter (Aloka,
Tokyo, Japan).” A titration curve was generated for each supernatant, and the titer that gave 50% of the radioactivity in a culture
containing 1 ng/mL rIL-2 was defined as 1 U/mL of IL-2 activity.
Cytotoxicity assay. Cytotoxic activity against the K562 leukemia
cell line was measured in the presence or absence of PHA (1: 1600)
at an effector-to-target ratio of lO:l, according to a 4-hour 51Cr
release assay method described elsewhere.”
Southern blot analysis. Southern blot analyses of genomic DNA
from lymphocyte colonies were performed according to the method
described previously.z2Rearrangements of the T-cell receptor (TCR)
Table 1. Frequencies of Cells With Identical Chromosome
Aberrations in Whole Blood Sampled at Various Timer
Date
July 1980
July 1988
July 1989
AgeatFrequency
of Cells With
Examination
Aberrations:
(yr)
tl4;6),tl5;13) (%)*
55
3
63
64
6
8
* t(4;6)(qll;qll),t(5;13)(p13;q12).
Frequency of Cells With
the Other Independent
Aberrations (%)
30
34
37
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KUSUNOKI ET AL
2108
Table 2. Chromosome Analysis of T- and B-Lymphocyte Coloniesand Whole Blood From One Atomic-Bomb Survivor
No. of Colonies
or Cells Analyzed
Cell Origin
No. of Colonies or Cells With Aberrations
No. of Colonies or Cells
With the Other Structural
t(4;6),t(5;13)*
t(1;12)i
t(1;2).t(2;15)$
Chromosome Aberrations
4
2
0
1
0
0
33
0
0
2
2
0
2
2
2
1
20
43
T-lymphocyte colonies
CD4'
CD8'
CD4-8-
62
8
1
58
100
B-lymphocyte colonies
Whole blood
7
8
* t(4;6)(qll;qll),t(5;13)(p13;q12).
t t(1;12)(q42;q15).
t(1;2)(q23;q37),t(2;15)(p23;q15).
*
and Ig genes were studied with a 770-bp cDNA probe of TCR Cp
anda 4.5-kb EcoRI-Hind111fragmentfromagermline
IgCp gene
clone, kindly provided by Dr T.W. Mak (Ontario Cancer Institute,
Toronto, Canada) andDr T. Honjo (Kyoto University, Kyoto, Japan),
respectively.*'.''
RESULTS
Previous cytogenetic studies showed the persistence of
clonal cells bearing t(4;6) (41 1;ql l), t(5; 13)(p13;q12), in
the subject's PBLs for thepast 9 years (Table 1). In the
present study, a total of 71 T-lymphocyte colonies were
established and analyzed for karyotypes and cell surface
phenotypes (Table 2). A total of 43 T-lymphocyte colonies
carried stable chromosome aberrations, and among them 4
CD4' and 2 CD8' T-lymphocyte colonies shared the identical chromosome aberration, t(4;6), t(5; 13). The same chromosome aberration was further observed in 7 of 58 B-lymphocyte colonies examined andin
a whole-blood PHA
culture, as well (8 of 100 metaphases). Figure 2 shows a
representative karyotype of this clone.
1
2
6
7
38
3
8
9
19
14
20
4
5
l1
12
P8 II 56
46
13
10
In addition to the t(4;6), t(5;13). another chromosome
aberration, t(1;12) (q42;q15), was observed in 1 CD4' Tand 2 B-lymphocyte colonies, and a t(1;2) (q23;q37), t(2; 15)
(p23;q15) aberration was present in 2 B-lymphocyte colonies (Table 2). These latter 2 aberrations were also detected
in a whole-blood PHA culture (2 of 100 metaphases in each
case) from the same blood sample.
Because identical karyotypic changes were observed in T
and B lymphocytes, the aberration most likely occurred in
a precursor cell before differentiation into T and B cells. In
fact, the results accord with the observation that such colonies bearing the identical chromosomal changes all showed
different patterns of TCRD or IgH gene rearrangement for
T- or B-cell colonies, respectively (Figs 3 and 4). That is,
these cells had undergone independent differentiation processes, and none of them were daughter cells in the periphery.
To confirm that the aberration was derived from a bone
marrow stem cell, two sets of experiments were conducted.
In these experiments, FISH method was usedto detect trans-
18
16
15
&l,
#B
21
22
17
-" -
xx
Fig 2. Representative karyotype of a clonal aberration t(4;6)(qll;qll),
t(5;13)(pl3;ql2)
with
G-band.
Arrows indicate abnormal chromosomes.
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CLONAL EXPANSION OF STEM CELL INTO T AND B CELLS
D
kb
1
2
3
4
5
6
F-
23.l+i
2109
ony produced no detectable L-2. Furthermore, one colony
(T-89) showed natural killer (NK) cell-like activity (cytotoxicity against K562 cells without ligands), and three colonies
(T-16, T-24 and T-89) clearly showed strong lectin-dependent cell-mediated cytotoxicity. Because these six colonies
showed L 2 production and/or lectin-dependent cell-mediated cytotoxicity activities, it was concluded that these T
lymphocytes were functionally differentiated and heterogeneous.
DISCUSSION
Fig 3. Southern blot analysis of TCR P-chain genes from sixTlymphocyte colonies with t(4;6)(qll;qll), t(5;13)lp13;q12). DNAs
from a Daudi B-cell line (lane D) and from the colonies (lanes 1-6)
were digestedwith B a d 1 restrictionenzyme and probed with cDNA
from a TCR P-chain constant region.
locations by using DNA probes for chromosomes 4 and 13
(Fig 5). The first set of experiments used PBMCs to form
BFU-E andCFU-GM colonies in methycellulose culture.
Subsequently, colonies were pooled, and metaphases were
examined to find that 20 cells of 300 carried double translocations involving chromosomes 4 and 13. In the second set
of experiments, CD34' cells (a surface marker of stem cells
in bone marrow) were sorted from PBMCs by means of
flowcytometry to avoid contamination of PBLs and were
subsequently cultured in vitro. Resulting colonies mostly
consisting of BFU-E but also of granulocytic/myelocytic
cells as well were mixed, and metaphases were examined.
Again, 12 cells of 100 examined carried double translocations involving chromosomes 4 and 13 (Fig 4).
A chromosome of group B (ie, chromosome 4 or 5 ) was
consistently involved in the translocation involving chromosome 13. Because chromosome 4 is already painted, we
concluded that this unpainted group-B chromosome was
chromosome 5. Additionally, the breakpoint in chromosome
13 was characteristically near the centromere of the long
arm; therefore, the centromeric portion of chromosome 13
was too small to produce a positive signal on the translocated
chromosome. Thus, all the translocations involving chromosome 13 were totally compatible with t(5; 13)(p13;q12). As
for the other translocation involving chromosome 4, a chromosome of group C (ie, chromosomes 6 to 12 and X) was
consistently involved. The exchange occurred near the centromere of both chromosomes, and these are fully compatible
with t(4;6)(qll;qlI). With regard to the rarity of double
translocations within a cell, characteristically similar patterns
of breakpoints of the chromosomes involved, and compatibility with the G-band karyotype, we concluded that they
are most likely the same clonal derivatives as observed in T
and B cells.
L 2 production and cytotoxic activities were further analyzed for the T-lymphocyte colonies bearing t(4;6), t(5; 13)
to confirm that these T-lymphocytes were functionally mature. The results are summarized in Table 3. Among these
colonies, two colonies produced large amounts (>50 U/mL)
of IL-2 after stimulation with anti-CD3 MoAb. Three other
colonies produced IL-2 weakly, and the remaining one col-
In the present study, three kinds of chromosome aberrations were observed in both B and T cells and, thus, are of
precursor-cell origin in one A-bomb survivor. Furthermore,
the aberration t(4;C), t(5;13), perfectly compatible with the
largest clone [t(4;6), t(5; 13)] observed in B and T cells, was
found in cultures of hematopoietic stem cells (ie, CD34+
cells). Surprisingly, the frequencies were very similar among
the different lineages of cells, ie, 3% to 8% in PBL, 8% in
T-cell colonies, 12% in B-cell colonies, 7% and 12% among
cells forming BFU-E, CFU-G, or CFU-GM colonies derived
from PBMCs and CD34+ cells, respectively. Because the
BFU-E or CFU colonies derived from PBMC or CD34+ cell
cultures were mixed for FISH analysis, differences in in vitro
growth rate among the colonies could have introduced a bias
in the proportion of cells with t(4;6), t(5; 13) as compared
with that in the in vivo growth rate. Nonetheless, the results
are in accord with the hypothesis that the aberration is derived from a single stem cell in bone marrow.
We have previously reported that one A-bomb survivor
(estimated dose of 1.99 Gy, exposed at age 17) possessed a
markedly increased frequency of mutants at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus (2 X
in an IL-Zdependent colony assay of lymphocytes [T
andNK cells] and 7 X
in B cells). Because most of
the mutants accompanied a partial deletion of chromosome
20q and new bands in Southern hybridization using cDNA
of the HPRT gene as a probe, the mutants were concluded
T 1 2 3 4 5 6 7 8 9 1 0
kb
?
23.1I
9.4+
Fig 4. Southern blot analysis of IgH chain genes from six B-lymphocyte colonies witht(4;6)(qll;qll),
t(5;13)(p13;q12) (lanes l
through 6). t w o B-lymphocyte colonies with t(1;12)lq42;q15) (lanes
7and
S), and two B-lymphocytecolonies with t(1;2)(q23;q37),
t(2;15)(p23;q15) (lanes 9 and 10). DNAs from a T-lymphocyte colony
(lane T) and from the B-lymphocytecolonies were digested with
EcoRl restriction enzyme and probed with a germline IgCp gene
clone.
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KUSUNOKI ET AL
Fig 5. A
metaphase
from
stem
cell
cultures
the FISH method
(mainly BFU-E colonies) stained by
using composite probes for chromosomes
4 and 13.
The painted chromosomes4 and 13 are seenas yellow, whereasthe remaining chromosomes are counterstained as red. Two arrows indicatethe two abnormalchromosomesconsisting
of a reciprocal
4 and a chromotranslocation between chromosome
some of group C, most likely number 6. An arrowhead showsa derivative chromosome froma translocation between chromosomes
5 and 13. The other
abnormal chromosome bearing
the centromeric portion ofchromosome13 is not detectable because
the breakpoint in chromosome 13 is so close to the
centromere.Normalchromosomes
4 and13are
shown by numbers, respectively.
to be monoclonal derivatives of a mutant that occurred in a
stem cell." Compared with that report, the present case is
remarkable becauseof its large clonal size. The largest clone
comprised 3%to 8% of total PBLs, and the remaining two,
2%. Evidences shown in this report suggest that the clone
size seems to reflect the potential of one stem cell to generate
lymphocyte progenies in adults. This does not necessarily
imply that such a large clonal expansion occurs normallyin
adults. Probably,extensive stem-cell depletion and preferential proliferation at some early stage of recovery from radiationinjury may underlie such an extensive monoclonal
expansion.
one may argue that the currentobservation of clonal
expansion of certain translocation is a result of positive selection caused by a selective growth advantage. However,
the frequency appears to have remained constant duringthe
past 10 years. Thus, the association of the clonally expanded
Table 3. Functional Analyses of T-cell Colonies With Aberrations:
t(4;6)(ql1;qlI),t(5;13)~pl3;qlZ)
11-2
Production (UlrnL)'
Clone
Phenotype
No Stimulus
Anti-CD3
T-l6
T-40
T-64
T-92
T-24
T-89
CD4
CD4
CD4
CD4
CD8
CD8
15
<5
22
64
196
<5
17
<5
53
10
49
<5
<5
<5
<5
Cytotoxic Activity
(% "Cr Release)t
No Stimulus
7
15
<5
<5
<5
17
PHA
35
5
6
*T-cell clones (5 x IO5)were cultured for 24 hours in the presence
of a B-cell line, OKlB (2 x lo5), with or without stimulation by 1 pg/
mL of anti-CD3 (Leu 4) antibody, IL-2 activity of the culturesupernatant was quantified by measuring proliferative response in an 1L-2dependent cell line, CTLL-2.
t A leukemia cell line, K562, was used as target cells. The effectorto-target ratio ( E n ratio) was 10:l.
translocation with preleukemic conditions, for example, is
highly unlikely.
Amenomori et alz5 reported two survivors who had possibly identical chromosome changes both in PHA culture of
PBLs and in myeloid colonies. In view of the total number
of examinees (n= 21) andthe average frequency of chromosome aberrations (23%)
in PHA culture, the results suggest
that the number of stem cells actively involvedin hematopoiesis at a given time may not be quite large, perhaps on the
order of lo3 to lo4 among the high-dose-exposed people.
The frequency of survivors showingthree or more identical
karyotypic changes in each PHA culture of whole blood is
aboutandtends
to increase withradiation dose (A.A.
Awa, Y. Kodama, unpublished data).The results agree with
the above estimate, although it remains to be determined
whether such clonal aberrationsin PBLs are indeed derived
from a precursor stem cell or, alternatively, from a mature
T lymphocyte in the periphery. Examples of monoclonal
expansion in the periphery have been found be
to in a subset
of T lymphocyte^"*^ and in HPRT mutants of T lymphocyte~.~'
Are the present monoclonal derivatives long-lived lymphocytes and not produced continuously from the precursor
stem cell? Circumstantial evidence suggests thatthis is probably not the case. For example, the patterns of TCR-gene
rearrangement are all different among the clonal T cells.
Thus, it is highly unlikely that they are memoryT cells
sharing the specific reactivity to certain common antigen(s)
and are continuously stimulatedto proliferate in the periphery. Rather, we suspect that
this subject's memory T-cell
pool had been mostly established at the time of radiation
exposure (20 years old), and the present clonal T cells are
more likely naive T cells.
This comes from our recent finding
on the afore-mentioned A-bomb survivor who carried the
clonal HPRT mutants in T,
NK,
and B cells.zzThe abnormally elevated mutant frequency was confined to CD45RAf
naive T cells, and the frequency was markedly lower (less
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CLONAL EXPANSION OF STEM CELL INTO T AND B CELLS
than 1 of 10) in CD45RO’ memory T cells.28All mutants
examined so far derived from CD45RA’ T cells were descendants of the single mutant stem cell, whereas mutants
derived from CD45RO’ cells were mostly unrelated.Michie
et alZ9reported that decay of dicentric-chromosome frequencyamongradiotherapy patients occurred not onlyin
CD45RO+ but also in CD45RA+ populations, suggesting
that CD45RA’ T cells are continuously replenished from the
precursor-cell pool. In the study subject’s case, cytogenetic
examination of sorted CD45RA’ and CD45RO’ T cells is
expected to provide a definitive answer to the question.
Kay” originally proposed a clonal succession theory for
hematopoiesis, in which a small number of short-lived stem
cells are sequentially triggered to produce a large number
of differentiated functional cells. Some experiments using a
reconstituted hematopoietic system in irradiated mice have
shown clonal changes of stem cells with time, which is in
agreement with the the01y.I.~Recently, however, Keller et
reported that a population of murine stem cells, tagged
by a neomycin-resistant gene randomly integrated in the
genome, could persistently produce progeny cells for a total
of at least 15 months in two successive generations of the
transplanted animals and suggested thatthe transplanted
stem cells can proliferate (self-renew) during reconstruction
processes of the radiation-sterilized bone marrow of the recipients. Furthermore, Nakahata et a13’ reported that bone
marrow-derived cells constituting blast cell colonies in vitro
were able to form colonies of two types, differentiated and
secondary blast cell colonies. The latter are considered to be
self-renewable stem cells. On the basis of these observations,
Ogawa3* proposed that
stem cells both differentiate into various lineages and self-renew stochastically and claimed that
this model may reconcile the experimental evidence in mice
for both the short- and long-term persistence of hematopoiesis by a single stem cell. Usually, it wouldbe extremely
difficult to test the hypothesis in humans unless some of the
stem cells were properly marked. In this regard, the present
case is rare and important. If, inthe future, the identical
chromosome aberrations are detectable in both a dormant
stem cell population and in differentiated cells of several
lineages, the conditions for the stochastic model would be
fulfilled in adult human hematopoiesis. Progress in the identification of human hematopoietic stem cells will be indispensable in further proving this thesis.
a’l
ACKNOWLEDGMENT
The authors are grateful to Dr A.A. Awa (Radiation Effects Research Foundation) for valuable comments. We thank Drs T.W. Mak
and T. Honjo for providing DNA probes; H. Onishi, K. Takahashi,
M. Yamaoka, and M. Saito for their excellent technical assistance;
and M. Takagi and N. Saito for preparing of the manuscript.
REFERENCES
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Cytogenetic and immunologic identification of clonal expansion of
stem cells into T and B lymphocytes in one Atomic-bomb survivor
Y Kusunoki, Y Kodama, Y Hirai, S Kyoizumi, N Nakamura and M Akiyama
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