[CANCER RESEARCH 48, 534-538, February 1. 1987]
Effect of Lymphokine-activated Killer Cell Fraction on the Development of Human
Hematopoietic Progenitor Cells1
Yoshihiro Fujimori,2 Hiroshi Hará,and Kiyoyasu Nagai
Second Department of Internal Medicine fY. F., K. N.1 and Division of Blood Transfusion [H. H.], Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan
trifugation (450 x g for 20 min). BM-cells of consenting healthy adults
were aspirated and incubated with silica particles (KAC; Japan Immuno-Research Laboratories Inc., Takasaki, Japan) for l h at 37°Cto
ABSTRACT
Lymphokine-activated killer (LAK) cells from cultures of human
peripheral blood mononuclear cells with recombinant interleukin-2 (rlL2) have been clinically used in adoptive immunotherapy for cancer
patients. To study their influence on human hematopoiesis, the LAK cell
fraction was cocultured with marrow nonphagocytic cells from normal
subjects in an assay system of hematopoietic progenitors. The fraction
suppressed colony growth from relatively mature erythroid progenitors
in a dose-dependent manner. Although unactivated cells, which were
produced without IL-2, augmented the growth of early erythroid progen
itors, the LAK cell fraction did not. This fraction suppressed colony
growth from mature granulocyte-macrophage progenitors (day 7 CFUGM) especially with an 18-h preincubation prior to coculture. It also
suppressed both immature granulocyte-macrophage progenitors (day 14
CFU-GM) and multipotential hematopoietic progenitors. The suppressive effects were observed on colony growth from autologous marrow
cells as well as allogeneic marrow cells. The suppression of day 7 CFUGM colony growth by supernatants due to preincubation with marrow
cells and the LAK cell fraction suggested that the humoral factor con
tributes to the suppression by the LAK cell fraction. These data suggest
that the LAK cell fraction suppresses the development of human hema
topoietic progenitor cells.
INTRODUCTION
Incubation of PBMC3 with a lymphokine, IL-2, leads to the
remove phagocytic cells as previously described (6). BM nonphagocytic
cells were then separated by Ficoll-Conray gradient centrifugation.
Preparation of LAK Cells. LAK cells were obtained according to the
methods described by Rosenberg et al. (5) with slight modification.
PBMC were incubated at 2 x IO6 cells/ml in RPMI 1640 medium
(Flow Laboratories, Rockville, MD) containing 10% autologous human
serum, 300 ¿tg/mlglutamine (GIBCO Laboratories, Grand Island, NY)
100 U/ml penicillin G (Banyu Pharmaceutical Co., Ltd., Tokyo, Japan),
100 Mg/ml streptomycin (Meiji Seika Co., Ltd., Tokyo, Japan) and 100
U/ml of human rIL-2 (Shionogi & Co., Ltd., Osaka, Japan). The rlL2 was isolated from Escherichia coli cells expressing the human IL-2
gene (7). Our preliminary experiments revealed that 100 U/ml of rlL2 was enough to induce a sufficient level of LAK activity. After 5 days
of incubation in air with 5% CO2 at 37°C,the cells were harvested and
washed three times with PRMI 1640 medium. The unactivated cells
were those subjected to the same procedure without the addition of
rIL-2.
Cytotoxicity Assay. A 4-h chromium 51-release assay in roundbottom microtiter plates (Delta, Denmark) was used to measure cytotoxicity. Daudi cells of 1 x IO7, which are of the natural killer cellresistant Barkitt lymphoma cell line, were labeled with 100 ¿iCi5lCr
(NEN Research Products, PA) for l h at 37°Cas target cells. Effector
induction of LAK cells which are cytotoxic to a wide variety of
fresh tumor cells (1-4). LAK cells can lyse targets without
deliberate immunization or MHC restriction. They can lyse not
only NK-sensitive but also NK-resistant tumor cells (1-4).
Their effective antineoplastic activities offer a new approach to
the treatment of cancer. Rosenberg et al. (5) showed that the
adoptive transfer of LAK cells with IL-2 to cancer patients can
mediate the regression of metastatic tumors. However, severe
side effects have also been reported (5). Thus, the effects of
LAK cells on various human tissues must be elucidated. Very
little information is available at present as to influence of LAK
cells on human organs or tissues other than their cytotoxic
activity on tumor cells.
The present experiments examined the influence of the LAK
cell fraction on human hematopoiesis in vitro. The fraction
prepared by incubating PBMC with human RIL-2 suppressed
the colony formation from CFU-E, CFU-GM, and CPU-Mix.
MATERIALS
AND
METHODS
Preparation of Cells. PBMC were obtained from heparinized blood
of consenting healthy volunteers by Ficoll-Conray density gradient cenReceived 4/13/87; revised 10/8/87; accepted 10/12/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science and Culture of Japan.
2To whom requests for reprints should be addressed, at the Second Depart
ment of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho,
Nishinomiya, Hyogo 663, Japan.
3 The abbreviations used are: PBMC, peripheral blood mononuclear cells; IL2, interleukin-2; rIL-2, recombinant interleukin-2; LAK, lymphokine-activated
killer; MHC, major histocompatibility complex; NK, natural killer; CFU-E,
erythroid colony-forming units; BFU-E, erythroid burst-forming units; CFU-GM,
granulocyte/macrophage colony-forming units; CPU-Mix, mixed hemopoietic
colony-forming units; BM, bone marrow, PCS, fetal calf serum.
cells were coincubated with the targets at effectortarget (E:T) cell ratios
of 10:1 for 4 h at 37°Cin humidified air with 5% CO2. The radioactive
supernatants were harvested and counted with a gamma counter (United
Technologies Packard, IL). Maximum isotope release was produced by
incubating the targets with Triton X-100 (Katayama Chemical Co.,
Ltd., Japan). Spontaneous release was determined by incubating the
targets with medium alone. The percentage of specific lysis was calcu
lated by the formula:
(Experimental cpm —spontaneous cpm)
(Maximal cpm —spontaneous cpm) X 100%
Cell Surface Marker Analysis. The unactivated cell and the LAK cell
fractions were characterized by their reaction with monoclonal antibod
ies. A series of reagents including anti-Leu2a (suppressor/cytotoxic Tlymphocytes), anti-Leu3a (helper/inducer T-lymphocytes), anti-LeuS
(T-cells, B-cells, granulocytes, and monocytes), anti-Leu 15 (C3bi recep
tor), anti-Leu7 (LGL and NK cells), and anti-Leu 1le (neutrophil and
NK-cell Fc IgG receptor) (Becton Dickinson Monoclonal Center,
Mountain View, CA) were used. The monoclonal antibodies had been
conjugated with either fluorescein isothiocyanate or phycoerythrin. The
two-color direct immunofluorescence experiments were performed with
a fluorescence-activated cell sorter (Becton Dickinson). Anti-Tac (IL-2
receptor) (8) antibody, which was provided kindly by Dr. T. Uchiyama
(Kyoto University, Kyoto, Japan), was used in an indirect immunoflu
orescence assay with fluorescein isothiocyanate-labeled anti-mouse IgG
(Cappel, PA) as a secondary antibody. The indirect immunofluores
cence experiments were performed by flow cytofluorometry (Spectrum
III; Ortho Diagnostic Systems, Westwood, MA).
Assay for Hematopoietic Progenitors. BFU-E assay was performed
according to a method previously described (9) with slight modification.
Briefly, bone marrow nonphagocytic cells were plated in a 35-mm
culture dish (Miles Laboratories, Inc., Naperville, IL) in 1 ml of a
mixture containing Iscove's modified Dulbecco's medium (GIBCO
Laboratories, Grand Island, NY), 0.92% methylcellulose (Shinetsu
Chemicals Co., Tokyo, Japan), 1% bovine serum albumin (Armour
Pharmaceutical Co., Kankakee, IL, USA), 30% PCS (Microbiological
534
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SUPPRESSION
OF HEMATOPOIESIS
Associates, Bethesda, MD), 10 4 M 2-mercaptoethanol (Wako Pure
Chemicals, Osaka, Japan), l l "/nil of human urinary erythropoietin
(Toyobo Co., Ltd., Osaka, Japan) and 5% (vol/vol) phytohemagglutinin-stimulated leukocyte-conditioned medium (10) as a source of burstpromoting activity. Cultures were set up in triplicate and maintained
at 37°Cin humidified air with 5% CO2. Cell aggregates of red color
containing more than 500 cells were scored as BFU-E colonies with an
inverted microscope on day 14 of culture. CFU-E (erythroid colonyforming units) were scored from these same plates on day 6 of culture
as colonies containing 8-150 cells of red color. CPU-Mix (mixed
hemopoietic colony-forming units) assays were carried out under almost
the same conditions as for BFU-E except that 2 U/ml of erythropoietin
was added. Cultures were set up in quadruplicate and scored on day 16
as colonies containing both erythrocytic subcolonies with red color and
nonhemoglobinized cells as previously described (9). In the CFU-GM
assay, bone marrow nonphagocytic cells were cultured in a mixture
containing Iscove's modified Dulbecco's medium, 0.92% methylcellulose, 20% PCS, and 200 U/ml of CSF-Chugai (Chugai Pharmaceutical
Co., Ltd., Tokyo, Japan). Cultures were performed in triplicate and
cell aggregates containing >50 cells were scored on days 7 and 14 of
culture; day 7 CFU-GM are more mature than day 14 CFU-GM (11).
Effect of Cell Fractions and Supernatants on Colony Growth. To test
the effects of LAK cell fraction on colony formation of marrow cells, 1
x 10"-2 x 10s effector cells were cocultured with 1 x 10s cells of
BY LAK CELL FRACTION
in comparison with unactivated cell fraction, but the difference
was not statistically significant. Tac-positive (8), i.e., IL-2 re
ceptor-positive, cells increased in the LAK cell fraction (P <
0.05). On the other hand, both Leull+Leu7~ cells, which have
a high active NK function in PBMC (13), and Leull*Leu7+
cells, which have weak NK activity in PBMC (13), did not
increase in the LAK cell fraction (1.6 and 6.2%, respectively).
These findings showed that this LAK cell fraction, which was
obtained by almost the same procedure as clinically used LAK
cells (5), had high cytotoxic activity and was composed of a
heterogenous population.
Effect of LAK Cell Fraction on CFU-E Proliferation. Fig. 1
shows the effects of the LAK cell or the unactivated cell fraction
on CFU-E colony growth, when 1 x 10"-2 x 10s effector cells
were cocultured with 1 x IO5allogeneic marrow nonphagocytic
cells. Whereas the unactivated cell fraction produced no signif
icant effects, the LAK cell fraction suppressed the growth in a
dose-dependent manner. At E:T ratios of 2:1, the LAK cell
fraction suppressed it to 44% of the control (P< 0.001). These
suppressive effects did not change with 18 h incubation prior
to coculture (data not shown). Autologous marrow cells were
marrow nonphagocytic cells under the conditions described above. In
preincubation experiments, 1 x 10*-2 x IO6 effector cells/ml were
incubated with 1 x 10" marrow cells for 18 h at 37°Cin RPMI 1640
medium containing 10% FCS, and then the cells were washed twice
and resuspended in RPMI 1640 containing 10% PCS at the same
volume as before and plated (1 x 10s bone marrow cells per dish) for
different types of colonies as described above. The effect of soluble
factor(s) on CFU-GM colony formation was tested by adding supernatants at 30% of the culture volume.
Preparation of Supernatant Fluids. After the LAK cell or the unactivated cell fraction had been obtained by incubating PBMC with or
without IL-2, respectively, the cells were washed three times with RPMI
1640 medium. The last supernatant fluids from the cell washings were
stored. After preincubation of BM:LAK cells or BM:unactivated cells
for 18 h, the cells were centrifuged and the cell-free superna tan is were
harvested. All supernatants were stored at —80°C
until use.
Statistics. Data were analyzed with Student's / test.
1x10* 2x104 1x1052x105
Number of added cells
Fig. 1. Dose-response effects of LAK cell (D) or unactivated cell fraction (•)
on CFU-E growth from 1 x 105 allogeneic marrow nonphagocytic cells. Data,
percentage of control CFU-E growth; values, mean ±SD for three experiments.
Control CFU-E growth was obtained from marrow cells without addition of
effector cells and ranged from 96 ±10 to 168 ±14 CFU-E colonies/10s marrow
cells in three experiments.
RESULTS
Characterization of Cell Fractions. Antigenic and functional
characteristics of unactivated and LAK fractions are shown in
Table 1. The unactivated cell fraction had very low cytotoxic
activity to NK-resistant Daudi cells (3.9%), whereas the LAK
cell fraction had high activity (54.6%). In the LAK cell fraction,
Leu2a+Leul5~ cells (cytotoxic T-lymphocytes) (12) increased
100-
s"
è
u
Ill
Table 1 Antigenic and functional characteristics of unactivated cell and LAK cell
fraction
TestLeu2a*
(5)35.2
±8.5*
cellsLeu2a*Leu
11.3(5)42.3
±
cellsLeu2a*Leu
IS"
(5)3.6
±7.4
1.8(5)3.8
±
cellsLeu3a* 15*
1.8(5)44.3
±
(5)38.0
±3.3
cellsLeu3a*Leu8*
(5)30.6
±2.9
(5)14.5
±4.8
cellsLeu3a*Leu8~
(5)23.5
±5.1
(5)13.7
±7.4
cellsLeu
(5)6.2
±8.2
(5)5.7
±6.3
cellsLeu
ll*Leu7*
(5)0.8
±2.4
1.6(5)1.6
±
cellsTac*
!TLeu7(5)4.2
±0.5
(5)29.2
±0.7
12.7C(3)54.6
±
cellsLAK
(3)3.9
±4.1
±18.0' (3)
activity*Unactivated38.9
±3.3 (3)LAK"45.7
60¡fi
1
40-
20-
" Unactivated cell or LAK cell fraction was obtained by incubating PBMC
without or with 100 U/ml of rIL-2, respectively, for 5 days.
* Values, percentage of positive or cytotoxicity; mean ±SD given for (N)
observations in parentheses.
' /' •0.05 in comparison with unactivated cell fraction.
* LAK activity was measured by a 4-h "Cr-release assay against Daudi cells at
an E:T ratio of 10:1.
' f< 0.001 in comparison with unactivated cell fraction.
Allogeneic
Autologous
Fig. 2. Effect of LAK cell fraction on CFU-E colonies from allogeneic or
autologous marrow cells. Marrow nonphagocytic cells at 1 x 10* cells were
cocultured with 2x10" effector cells. Points, percentage of inhibition calculated
according to the following formula: (No. of CFU-E colonies in control culture No. of CFU-E colonies in cocultured with effector cells)/No. of CFU-E colonies
in control culture x 100.
535
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SUPPRESSION OF HEMATOPOIESIS BY LAK CELL FRACTION
subjected to the same coculture experiments. A similar degree
of inhibition was observed with the LAK cell fraction on CFUE grown from autologous marrow cells as well as allogeneic
marrow cells (Fig. 2). These results indicated that the LAK cell
fraction suppressed CFU-E growth and the suppression was
not restricted by the MHC.
Effect of LAK Cell Fraction on BFU-E Proliferation. To
determine the effects of the LAK cell fraction on BFU-E
proliferation, similar dose-response experiments were per
formed. As shown in Fig. 3, the unactivated cell fraction aug
mented BFU-E colony number to 155% (at E:T ratios of 2:1)
in a dose-dependent manner (P < 0.001), but the LAK cell
fraction did not.
Effect of LAK Cell Fraction on the Growth of Day 7 and Day
14 CFU-GM. The first series of experiments were performed
by coculturing 2 x 10s effector cells with 1 x IO5 allogeneic
marrow cells without preincubation. The LAK cell fraction
suppressed day 7 CFU-GM colony growth to 84% without
preincubation (P < 0.05) (Fig. 4), but the degree of the inhibi
tory effect on CFU-GM growth was lower than that on CFUE growth. As the suppressive effect of NK cells on CFU-GM
has been reported to be dependent on preincubation time (14),
we incubated the LAK cell fraction with BM cells for 18 h,
washed them twice, then cultured them in a CFU-GM assay
system. The unactivated cell fraction had no effect, but the
LAK cell fraction suppressed day 7 CFU-GM proliferation to
45% of the control values (P < 0.001) (Fig. 4). These results
suggest that the inhibitory effects depended on the time of cellto-cell contact. To confirm the suppressor effects on CFU-GM,
the LAK cell or the unactivated cell fraction was cocultured
with marrow cells after 18 h preincubation. The unactivated
cell fraction had no significant effects on day 7 CFU-GM, while
the LAK cell fraction suppressed it in a dose-dependent manner
(Fig. 5). As for day 14 CFU-GM, which are considered to be
less mature than day 7 CFU-GM (11), similar inhibitory effects
by LAK cells were observed (Fig. 6). Similar suppressive effects
were also detected with CFU-GM colony growth from autolo
gous marrow cells (data not shown).
Effect of LAK Cell Fraction on CPU-Mix Growth. The effect
of the LAK cell fraction on immature multipotential hematopoietic progenitors (CPU-Mix) was examined (Table 2). Al
though the unactivated fraction produced no significant effect,
the LAK cell fraction suppressed the growth of CPU-Mix (P <
0.05).
Effects of Supernatant Fluids. To determine the contribution
of humoral factor to the inhibitory effects of the LAK cell
fraction, various supernatant fluids were added to the CFU-
160
1xl04
2x104 1xl05 2X105
Number of added cells
1x10* 2x10* 1X105 2x105
Fig. 3. Dose-response effects of LAK cell (D) or unactivated cell fraction (•)
on BFU-E growth from 1 x 10* allogeneic marrow nonphagocytic cells. Values,
Number of added cells
percentage of mean ±SD of control values for three experiments. The number
of BFU-E growth in control culture ranged from 75 ±7 to 126 ±6 bursts/10*
Fig. 5. Dose-response effects of LAK cell (D) or unactivated cell fraction (•)
marrow cells.
on day 7 CFU-GM colony growth after 18 h preincubation. Marrow nonphago
cytic cells at 1 x 10' cells were cocultured with each batch of effector cells. Values,
percentage of mean ±SD of control values for three experiments.
"o1
100
80'S
60SC3£
£
«
Ür*»£
20
O•JLl
\JLi
without
18hr
preincubation
preincubation
Fig. 4. Effect of LAK cell <ß)
or unactivated cell fraction (d) on day 7 CFUGM growth with or without preincubation. Marrow nonphagocytic cells at 1 x
10' cells were cocultured with 2 x IO5 cells of LAK cell or unactivated cell
fraction. Bars, percentage of mean ±SD of control. Control day 7 CFU-GM
growth ranged from 168 ±15 to 234 ±14 colonies/10* marrow cells without
preincubation (in three experiments) and from 149 ±6 to 194 ±9 colonies/10*
marrow cells with 18 h preincubation (in three experiments).
1X104 2x104 1x105 2x106
Number of added cells
Fig. 6. Dose-response effects of LAK cell (D) or unactivated cell fraction (•)
on day 14 CFU-GM colony growth after 18 h preincubation. Marrow nonphag
ocytic cells at 1 x IO5 cells were cocultured with each batch of effector cells.
Values, percentage of mean ±SD of control values. Control culture ranged from
105 ±8 to 154 ±13 day 14 CFU-GM colonies/10s marrow cells.
536
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SUPPRESSION
OF HEMATOPO1ESIS
Table 2 Effect of LAK cell fraction on CPU-Mix colony formation
Number of mixed colonies
per4x 10s cells"
+Unactivated1591814.0
LAK4374.6
+
Experiment123Meanonly21121716.7
±2.1*
+ 4.5BM
±SDBM
±4.6BM
a Marrow nonphagocytic cells at 1 x 10s cells were cultured with or without 2
x IO5effector cells. Values, sum of colonies from four dishes.
* P < 0.05 in comparison with BM + unactivated cell fraction.
Supernatants fron
freshly produced
and washed cells
Supernatants after
18hr premcubation
Unactivated cells
LAK cells
BM : Unactivated cells
_H
BM LAK cells
0
50
Day 7 CFU-GM (% of Control)
Fig. 7. Effect of supernatant fluids on day 7 CFU-GM growth. The Superna
tants were added to 30% of the culture volume when 1 x 10* bone marrow
nonphagocytic cells were cultured. Bars, percentage of mean ±SD of control
values for three experiments. Control culture ranged from 93 ±3 to 153 ±10
day 7 CFU-GM colonies/10* marrow cells.
GM culture system. No effects were observed when the super
natant fluids from freshly produced and washed LAK cell or
unactivated cell fraction were added to 30% of the culture
volume (P > 0.05) (Fig. 7). However, the Supernatants obtained
after 18 h BM:LAK preincubation inhibited day 7 CFU-GM
colony growth (P < 0.001), while those from BM:unactivated
cells produced no effects. These results suggest that suppression
of day 7 CFU-GM colony growth was mediated at least partially
by the factor(s) produced by the LAK cell fraction.
DISCUSSION
Precise estimation of the effects of LAK cells on human
tissues is needed from the viewpoint of adoptive immunotherapy in which LAK cells are administered in vivo. The present
results elucidated the effect of the LAK cell fraction on human
hematopoietic progenitor cells.
Although the LAK cell population had high cytotoxic activity
against NK-resistant tumor cells, it was heterogenous. When it
was cocultured with marrow nonphagocytic cells at various cell
ratios, inhibition of late erythroid (CFU-E), myeloid progeni
tors (day 7 and day 14 CFU-GM), and mult ¡potentialprogeni
tors (CPU-Mix) was observed. The inhibitory effect was ob
served with colonies from autologous marrow cells as well as
from allogeneic marrow cells. The LAK cell fraction suppressed
CFU-E almost to the same degree with or without preincuba
tion prior to coculture, but it suppressed CFU-GM slightly
without preincubation and significantly with 18 h preincuba
tion. The sensitivity to the LAK cell fraction may differ between
CFU-E and CFU-GM. Thus, the LAK cell fraction seemed to
suppress both mature and immature hematopoietic progenitors.
The LAK cell fraction may also affect early erythroid progen
itors (BFU-E); however, experimental observation with BFU-E
cultures is more complicated than with CFU-E and CFU-GM
cultures because peripheral blood T-cells contain two opposing
subpopulations, one which enhances BFU-E growth and one
which limits it (15). Unfractionated blood T-cells have enhanc
ing effects on BFU-E (15, 16), which agree with our finding
BY LAK CELL FRACTION
that the unactivated cell fraction augmented BFU-E growth. In
the LAK cell fraction, such an enhancing effect was not ob
served, suggesting that the LAK cell fraction suppresses BFUE growth. The enhancing effect of the unactivated cell fraction
may be eliminated by the suppressive effect of the LAK cell
fraction.
Some T-cells and NK cells are known to regulate hematopoiesis through soluble factor(s) (17, 18). A similar phenome
non occurred for the LAK cell fraction. The supernatant from
18-h LAK:BM preincubation contained factor(s) that inhibited
CFU-GM growth, suggesting that the effect of the LAK cell
fraction on hematopoietic progenitors is at least partially me
diated through the release of the humoral factor(s). However,
it remains unclear to what degree the humoral factor(s) is
responsible for suppression of CFU-GM by the LAK cell frac
tion. As the effect depends on contact time during preincuba
tion, it may be partially due to cell-mediated lysis. Both humoral
factor(s) and cell-mediated lysis may contribute to the suppres
sion of hemopoietic progenitors by the LAK cell fraction.
Rosenberg et al. (5) showed the therapeutic significance of
adoptive immunotherapy with LAK cells and IL-2 on cancer
patients. Although the results were promising, significant side
effects were noted, such as fever, chills, malaise, diarrhea,
mental confusion, fluid retention, and anemia (5). The anemia
was serious enough to necessitate blood transfusion, and thrombocytopenia (<50,000/mnr1) was also observed (5). The anemia
is thought to have been due to the administration of IL-2 which
caused release of 7 interferon (5). Our results indicate that both
allogeneic and autologous LAK cell fractions can cause hema
topoietic suppression even if IL-2 has already disappeared.
Therefore, the LAK cell fraction itself may also contribute to
this anemia. This inhibitory effect of the LAK cell fraction on
hematopoiesis should be taken into account when it is used
together with IL-2 for adoptive immunotherapy.
Recent reports have shown that LAK cells are composed of
a heterogenous population and that Leu 11 activated NK cells,
in spite of their small number, have a major role in the LAK
phenomenon (19-21). The present study indicated that the bulk
LAK cell fraction contains a compartment that suppresses
hematopoiesis. In our preliminary experiments, treatment of
the LAK cell fraction by Leu lie and complement partially
abolished the suppression on day 7 CFU-GM," which suggests
that Leull* NK-cells were responsible for this suppression.
There is the possibility that LAK cells themselves suppress
hematopoiesis. More detailed studies on the effects of LAK
cells on hematopoiesis are now underway in our laboratory.
Our results suggest that the LAK cell fraction suppresses
erythroid, myeloid and multipotential hematopoietic progeni
tors and that caution should be exercised in adoptive immuno
therapy with LAK cells because they may cause bone marrow
suppression.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Yoshihiro Okada for his measure
ments of cytotoxicity. We also thank Dr. Masatoshi Kohsaki, Dr.
Teruaki Damano, and Dr. Akihisa Kanamaru for their valuable discus
sions and suggestions and Judy Noguchi for her assistance with the
preparation of this manuscript.
REFERENCES
1. Lotze, M. T., Grimm, E. A., Mazumder, A., Strausser, J. L., and Rosenberg,
S. A. Lysis of fresh and cultured autologous tumor by human lymphocytes
4 Unpublished observation.
537
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SUPPRESSION
2.
3.
4.
5.
6.
7.
8.
9.
OF HEMATOPOIESIS
BY LAK CELL FRACTION
cultured in T-cell growth factor. Cancer Res., 41: 4420-4425, 1981.
Grimm, E. A., Mazumder, A., Zhang, H. Z., and Rosenberg, S. A. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh
solid tumor cells by interleukin 2-activated autologous human peripheral
blood lymphocytes. J. Exp. Med., 155: 1823-1841, 1982.
Grimm, E. A., Ramsey, K. M., Mazumder, A., Wilson, D. J., Djeu, J. Y.,
and Rosenberg, S. A. Lymphokine-activated killer cell phenomenon. II.
Precursor phenotype is serologically distinct from peripheral T lymphocytes,
memory cytotoxic thymus-derived lymphocytes, and natural killer cells. J.
Exp. Med., ¡57:884-897, 1983.
Grimm, E. A., and Wilson, D. J. The human lymphokine-activated killer
cell system. V. Purified recombinant interleukin 2 activates cytotoxic lym
phocytes which lyse both natural killer-resistant autologous and allogeneic
tumors and triniirophcnyl modified autologous peripheral blood lympho
cytes. Cell. Immunol., 94: 568-578, 1985.
Rosenberg, S. A., Lotze, M. T., Muul, L. M., Leitman, S., Chang, A. E.,
Ettinghausen, S. E., Matory, Y. L., Skibber, J. M., Shiloni, E., Vetto, J. T.,
Seipp, C. A., Simpson, C., and Reichert, C. M. Observations on the systemic
administration of autologous lymphokine-activated killer cells and recombi
nant interleukin-2 to patients with metastatic cancer. N. Engl. J. Med., 313:
1485-1492, 1985.
Ohe, Y., Hará, H., and Nagai, K. Ill,A antigens expressed on human
pluripotent hemopoietic precursors in vitro (CFUMn). Exp. Hematol., 10:
467-471, 1982.
Devos,
R., Plaetinck,
G., Cheroutre,
H., Simons,
G., ofDegrave,
Tavernier,2
J.,
Remaut,
!•'..,and
Fiers,
W. Molecular
cloning
human W.,
interleukin
10. Aye, M. T., Niho, Y., Till, J. E., and McCulloch, E. A. Studies of leukemic
cell populations in culture. Blood, 44: 205-219, 1974.
11. Ferrerò, D., Broxmeyer, H. E., Pagliardi, G. L., Venuta, S., Lange, B.,
Pessano. S., and Rovera, G. Antigenically distinct subpopulations of myeloid
progenitor cells (CFU-GM) in human peripheral blood and marrow. Proc.
Nati. Acad. Sci. USA, 80:4114-4118, 1983.
12. Landay, A., <.an land. G. L., and Clement, L. T. Characterization of a
phenotypically distinct subpopulation of Leu-2* cells that suppresses T cell
proliferative responses. J. Immunol., 131: 2757-2761, 1983.
13. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F.
Subpopulations of human natural killer cells defined by expression of the
Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J. Immunol., 131:1789-1796,
1983.
14. Hansson, M., Heran, M., Andersson, B., and Kiessling, R. Inhibition of in
vitro granulopoiesis by autologous allogeneic human NK cells. J. Immunol.,
129: 126-132, 1982.
15. Torok-Storb, B., Martin, P. J., and Hansen, J. A. Regulation of in vitro
erythropoiesis by normal T cells: Evidence for two T-cell subsets with
opposing function. Blood, 58: 171-174, 1981.
16. Nathan, D. G., Chess, L., Hillman, D. G., Clarke, B., Breard, J., Merler, E.,
and Housman, D. E. Human erythroid burst-forming unit: T-cell requirement
for proliferation in vitro. J. Exp. Med., 147: 324-339, 1978.
17. Mangan, K. F. T-Cell-mediated suppression of hematopoiesis. N. Engl. J.
Med., 312: 306-307, 1985.
18. Degliantoni, G., Perussia, B., Mangoni, L., and Trinchieri, G. Inhibition of
bone marrow colony formation by human natural killer cells and by natural
killer cell-derived colony-inhibiting activity. J. Exp. Med., 161: 1152-1168,
cDNA and its expression in E. coli. Nucleic Acids Res., //: 4307-4323,
1985.
1983.
19. Phillips, J. H., and Lanier, L. L. Dissection of the lymphokine-activated
Uchiyama, T., Broder, S., and Waldmann, T. A. A monoclonal antibody
killer phenomenon. Relative contribution of peripheral blood natural killer
(anti-Tac) reactive with activated and functionally mature human T cells. I.
cells and T lymphocytes to cytolysis. J. Exp. Med., 164: 814-825, 1986.
Production of anti-Tac monoclonal antibody and distribution of Tac(+) cells.
20. Itoh, K., Tilden, A. B., Kumagai, K., and Batch, C. M. Leu-11* lymphocytes
J. Immunol., 126: 1393-1397, 1981.
with natural killer (NK) activity are precursors of recombinant interleukin 2
(rIL 2(-induced activated killer (AK) cells. J. Immunol., 134: 802-807, 1985.
Hará,H., Kai, S., Fushimi, M., Taniwaki. S., Okamoto, T., Ohe, Y., Fujita,
21. Damle, N. K., Doyle, L. V., and Bradley, E. C. Interleukin 2-activated human
S., Noguchi, K., Senba, M., Marnano, T., Kanamaru, A., and Nagai, K.
Pluripotent hemopoietic precursors in vitro (CFUMix) in aplastic anémia.
killer cells are derived from phenotypically heterogeneous precursors. J.
Exp. Hematol., 8: 1165-1171, 1980.
Immunol., 137: 2814-2822, 1986.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1988 American Association for Cancer Research.
Effect of Lymphokine-activated Killer Cell Fraction on the
Development of Human Hematopoietic Progenitor Cells
Yoshihiro Fujimori, Hiroshi Hara and Kiyoyasu Nagai
Cancer Res 1988;48:534-538.
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