From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Proliferation and Cytolytic Function of Anti-CD3 + Interleukin-2 Stimulated
Peripheral Blood Mononuclear Cells Following Bone Marrow Transplantation
By Emmanuel Katsanis, Peter M. Anderson, Alexandra H. Filipovich, Diane E. Hasz, Mary L. Rich,
Cynthia M. Loeffler, August0 C. Ochoa. and Daniel J. Weisdorf
We evaluated the proliferation, cytolytic function, and phenotypic characteristics of antLCD3 plus interleukin-2 (IL-2)
stimulated peripheral blood mononuclear cells (PBMCs) from
44 patients with leukemia or non-Hodgkin's lymphoma (NHL)
treated with multiagent chemotherapy or following bone
marrow transplantation (BMT). BMT patients had decreased
cell growth with only a 1.35 f 0.25 (autologous BMTfor acute
lymphoblastic leukemia [ALL]), 1.24 f 0.25 (autologous BMT
for NHL), and 0.8 f 0.1 (allogeneic BMT for leukemia) mean
fold increase by day 5 of culture compared with controls
(4.0 f 0.4). P < .001. Anti-CD3 + IL-2 activated cells from
patients with ALL and NHL who had received autologous
BMT and cells from patients with leukemia who underwent
allogeneic BMT were more effective in lysing the natural
killer (NK) sensitive target, K562, and the NK-resistant target,
Daudi, compared with controls. In contrast, cytolysis of K562
and Daudi by cultured PBMCs from patients with ALL and
NHL receiving multi-agent chemotherapy was similar t o that
of controls. Cultures from BMT recipients had a significant
increase in CD16' (autologous ALL 5.7 f 1.5%. P < .01;
3.5%, P < .001; allogeneic
autologous NHL 12.4
14.3 f 2.9%. P < .001) and CD56+ cells (autologous ALL
27.6 f 12.0%. P < .01; autologous NHL 39.3 -c 9.5%,
P < .001; allogeneic 42.7 -c 7.4%. P < .001) compared with
controls (CD16' 2.5 f 0.4%; CD56' 6.9 0.9%). Stimulation
of PBMCs with anti-CD3 + IL-2 is effective in generating cells
with high cytolytic function post-BMT.
o 1991by The American Society of Hematology.
A
Adoptive transfer of IL-2 stimulated cells in combination
with systemically administered IL-2 has been associated
with the regression of melanoma and renal cell carcinoma
in humans'0''' and in therapeutic responses of a large variety
of murine tumor^.'*^'^ Recent trials have shown complete or
partial responses in patients with advanced lymphoma after
systemic administration of IL-2 with or without adoptively
transferred cells.'o~ll
We and others have shown that the anti-CD3 (OKT3)
monoclonal antibody (MoAb) can be used in conjunction
with IL-2 in vitro to stimulate peripheral blood mononuclear cells (PBMCs) to acquire LAK activity.'"" This
method also results in large increases in cell numbers in
both human and murine systems compared with conventional IL-Zstimulated c u l t ~ r e s . ' Furthermore,
~-~
on a percell basis, cytolytic activity against various targets is comparable with that of IL-2-stimulated cells. In the current study
we have shown that anti-CD3 + IL-2 activated cells from
autologous or allogeneic BMT recipients have enhanced
cytolytic function compared with those from healthy controls and from patients receiving multiagent chemotherapy
for leukemia and lymphoma. These results indicate that
LAK function can be increased in the early post-BMT
period and that anti-CD3 + IL-2 stimulated cells may be
useful for adoptive transfer in patients with lymphoid
malignancies undergoing BMT.
LTHOUGH high-dose chemotherapy and total body
irradiation followed by autologous or allogeneic bone
marrow transplantation (BMT) has been shown to be the
best salvage regimen for patients with relapsed leukemia
and lymphoma, the risk of relapse remains high.'-3Because
allogeneic BMT for acute lymphoblastic leukemia is also
associated with high rates of relapse, this suggests failure to
completely eradicate malignant lymphoblasts in the recipient despite lethal doses of chemoradi~therapy.'~~~~
Clinical
studies have documented that patients developing graftversus-host disease (GVHD) posttransplant have lower
relapse rates than those without GVHD.'6 This is termed
graft-versus-leukemia (GVL) effect and is believed to be
secondary to effects of alloreactive T cells of donor origin
against residual host tumor cells. Recipients of T-depleted
marrows' and those receiving significant immunosuppression for prevention of GVHD have increased relapse rates,
presumably due to a lack of GVL effect?
Lymphocytes cultured in vitro with interleukin-2 (IL-2)
acquire the ability (lymphokine-activated killer [LAK] activity) to lyse natural killer (NK)-resistant tumor targets?
From the Departments of Pediatrics, Internal Medicine, Laboratory
Medicine, Surgery and the Bone Marrow Transplantation Program,
University of Minnesota, Minneapolis; and the fiogram Resources Inc,
National Cancer Institute, Frederick, MD.
Submitted March 30, 1990; accepted May 3, 1991.
Supported in part by National Institutes of Health Grant No. CA
21 737, the Bone Marrow Transplant Research Fund, and the Children's Cancer Research Fund. E.K. is supported by a fellowship from
the Medical Research Council of Canada. P.M.A. is a recipient of the
American Cancer Society Clinical Oncology Career Development
Award.
Address reprint requests to Emmanuel Katsanis, MD, University of
Minnesota, Box 484 UMHC, 420 Delaware St SE, Minneapolis, MN
55455.
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 1991 by 7?zeAmerican SocieQ of Hemaiologv.
0006-4971I9117805-0025$3.OOIO
1286
*
*
MATERIALS AND METHODS
Patients. Forty-four patients (22 male, 22 female) with ages
ranging from 5 to 68 years (median 37 years) were studied.
Seventeen healthy volunteers were used as controls. Six patients
with acute lymphoblastic leukemia (ALL) and 10 with nonHodgkin's lymphoma (NHL) had received multiagent chemotherapy within the year before study, while seven ALL and nine NHL
patients underwent autologous BMT. An additional 12 patients
with various hematologic malignancies (six acute nonlymphoblastic
leukemia [ANLL], three chronic myelogenous leukemia, two ALL,
1chronic lymphocytic leukemia) received allogeneic BMT. Pretrans;
plant conditioning included total body irradiation and high-dose
cyclophosphamide plus additional etoposide (VP-16) for five
Five of the allogeneic BMT patients received T-depleted marrow with TlOl MoAb (anti-CD5) linked to ricin immuBlood, Vol78, No 5 (September I ) , 1991: pp 1286-1291
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ANTI-CD3
+ IL-2 PEMCs AFTER EMT
1287
notoximu Autologous BMT recipients received cryopreserved
marrow purged with B43 (anti-CD19 MoAb) linked to pokeweed
antiviral protein (PAP) immunotoxin.’,24 Peripheral blood samples
were drawn at 28 to 159 days (95 f 20) after BMT in the
autologous group and 22 to 201 days (92 f 18) in the allogeneic
group (P = not significant).
Cellpreparation and culture. Heparinized blood was diluted 1:l
with Hank’s Balanced Salt Solution 1X (HBSS) (GIBCO, Grand
Island, NY) and layered over Ficoll-Hypaque (Pharmacia, Piscataway, NJ). Interface PBMCs were washed three times in tissue
culture media (TCM), consisting of RPMI 1640 (GIBCO) supplemented with 25 mmol/L HEPES, 2 mmol/L L-glutamine, 100 U/mL
of penicillin, 100 &mL of streptomycin (Sigma, St Louis, MO),
and 5% pooled heat-inactivated human serum. Cultures were
incubated at 37°C in a humidified atmosphere of 5% CO, in 75-cmZ
flasks (Corning, Corning, NY) at a concentration of 0.5 X 106
cells/mL in TCM with 10 ngimL antLCD3 MoAb (OKT3; Ortho,
Raritan, NJ) and recombinant IL-2 100 U/mL (HoRmann LaRoche, Nutley NJ; specific activity 1.5 X lo7U/mg) or IL-2 alone.
Anti-CD3 MoAb was added on the first day of culture only. Cells
were subcultured to 0.2 to 0.4 x 106 cells/mL in TCM containing
IL-2 when they exceeded 1 x lo6cells/mL.
Estimation of cell number increase and viability. Cultures were
sampled on day 5 and 7 of culture; 10 pL of culture aliquots were
mixed with 10 pL of trypan blue (GIBCO) and counted using a
standard hemocytometer. Viability of cells was consistently greater
than 90%.
Tumor lines. Human tumor cell lines, K562 (NK-sensitive;
erythroleukemia cell line) and Daudi (NK-resistant; Burkitt lymphoma cell line) were maintained in culture with TCM and
subcultured at 0.5 to 1 x 106/mLin fresh media two to three times
per week.
Cytotoxicity assay. Tumor cell line targets (K562, Daudi) (1 to
2 x 106 in 0.5 mL TCM) were incubated with 500 pCi Na”Cr0,
(5,000 kCi/mL) (New England Nuclear Research Products, Boston, MA) for 1 to 1% hours at 37°C. Targets were washed three
times in cold TCM and resuspended at a concentration of 1 x lo4
cells/mL. Fifty microliters (500 targetdwell) were then added to
96-well V-bottomed microtiter plates (Costar, Cambridge, MA)
into which effectors (day 5 anti-CD3 + IL-2 or IL-2 stimulated
PBMCs) had been previously added in triplicate and serially
diluted in TCM to yield effector to target ratios (E:T ratio) of
1W1, 33.3:1, 11.1:1, and 3.7:l. Spontaneous release wells contained only TCM and ”Cr-labeled targets while maximal release
wells contained detergent and targets. The microtiter plates were
gently centrifuged at 5% for 5 minutes and incubated at 37°C and
5% CO, for 4 hours. The plates were then centrifuged at 15% for
10 minutes after which 100-pL aliquots of supernatant were
harvested into glass scintillation vials (Dynalon, Rochester, NY)
and 2.5 mL of scintillation fluid added (Cytoscint; ICN Biomedicals, INine, CA). Radioactivity was counted using an LKB 1216
liquid scintillation counter. Cytotoxicity was determined by the
formula:
RESULTS
Comparative proliferation and cytolytic function ofIL-2 and
anti-CD3 IL-2 stimulated cells after BMT. PBMCs from
+
11 patients who underwent BMT (seven autologous BMT
for NHL, two autologous BMT for ALL, and two allogeneic
BMT for ANLL) were obtained to evaluate if the addition
of anti-CD3 to IL-2 increases cell proliferation after BMT.
Samples were taken 20 to 180 days (93 f 31) after transplant. The mean fold increase in cell number of anti-CD3
IL-2 stimulated cultures on day 7 was significantly greater
(3.5 2 1.0) than cultures stimulated with IL-2 alone
(0.5 rt 0.1) (P < .Ol) (Fig 1). Percent cytolysis at 33 to 1
effector to target ratio was comparable with a mean f
S.E.M. of 65.1% 2 3.4% (IL-2 stimulated cultures) versus
63.7% f 2.1% (anti-CD3 IL-2 stimulated cultures)
against K562 target (P = not significant) and 63.0% rt 9.3%
versus 68.2% 2 7.2% against Daudi target (P = not significant). Due to the superior growth of anti-CD3
IL-2
stimulated cultures, all further experiments in this report
were performed using this method of cell activation.
Proliferation of anti-CD3
IL-2 stimulated cells. AntiCD3 IL-2 stimulated PBMCs from patients with ALL
treated with chemotherapy had significantly less growth
after 5 days of culture with a 2.0 f 0.5 mean fold increase
compared with 4.0 2 0.4 in controls (P < .02) (Fig 2).
Proliferation of PBMCs from NHL patients who received
multiagent chemotherapy (3.5 2 0.6) did not differ from
that of controls. Bone marrow transplant patients had a
strikingly blunted cell growth when compared to controls
with only a 1.35 2 0.25 (autologous BMT for ALL), 1.24 rt
0.25 (autologous BMT for NHL), and 0.8 f 0.1 (allogeneic
BMT for leukemia) mean fold increase by day 5 of culture,
P < .001 (Fig 2).
Generation of cells with cytolytic effector function. To
determine the cytolytic function of day 5 anti-CD3 IL-2
stimulated cells, cytotoxicity against NK-sensitive K562 and
NK-resistant Daudi cell lines was assessed using a 4-hour
V r release assay. The cytolytic function of effectors from
BMT recipients was generally higher than controls. In
contrast, cytolytic function of cells derived from patients
treated with chemotherapy was similar to controls (Fig 3).
Due to the similar cytotoxicity demonstrated by cultures
from ALL and NHL patients receiving chemotherapy and
the comparable cytotoxicity between cells from ALL and
+
+
+
+
+
+
% Cytotoxicity = 100
X
were placed in 96-well V-bottomed microtiter plates. MoAbs used
for double-color analysis included FITC-conjugated or PEconjugated MoAbs anti-Leu-4 (CD3), anti-Leu-3 (CD4), antiLeu-2 (CDS), anti-Leu-11 (CD16), anti-leu-19 (CD56), and
anti-IL-2 R (CD25) (Becton Dickinson, Mountain View, CA).
Double-color analysis was performed by incubating 10 pL of
FITC-conjugated and PE-conjugated MoAbs with the cells for 30
minutes at 4°C. The cells were then washed three times with PBS
containing 2% fetal calf serum and 0.1% sodium azide and fixed
with PBS containing 2% paraf~rmaldehyde?~
Analysis was performed using a Becton Dickinson FACScan.
Statistical analysis. The Student’s t-test was used to compare
growth, percent cytolysis, and immunophenotype of cells. Statistical comparisons of the distribution of lytic units between groups
were made using the nonparametric Mann-Whitney U test.
Experimental Mean cpm - Spontaneous Release Mean cpm
Maximal Release Mean cpm - Spontaneous Release Mean cpm
One lytic unit (LU) was defined as the number of effectors
required to lyse 30% of targets; cytotoxicity is presented as LU per
lo6effector cells.
Phenotypic analysis by immunofluorescence. Fresh PBMCs or
cells in culture for 5 days with anti-CD3 IL-2 were washed in
phosphate-buffered saline (PBS) containing 2% heat-inactivated
fetal calf serum and 0.1% sodium azide (Sigma) and 1 x lo5 cells
+
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
KATSANIS ET AL
with anti-CD3 + IL-2, cell cultures from BMT recipients
continued to have a lower percentage of CD3+ cells. The
proportion of CD16' and CD56' in cultured PBMCs was
significantly higher in patients receiving BMT compared
with controls. Expression of the p55 IL-2 receptor (CD25)
was infrequent in all groups' fresh cells. After stimulation
with anti-CD3 + IL-2 for 5 days, CD25 expression was
similar in controls and chemotherapy-treated patients but
significantlylower in patients after BMT (Table 2 ) .
DISCUSSION
There has recently been increased interest in investigation of immunotherapy regimens after BMT in an attempt
to decrease disease recurrence. Enhancement of the immune response against cancer at a time of minimal residual
disease may be a promising adjuvant to high-dose chemotherapy and total body irradiation as currently used in
BMT. Recent studies have demonstrated the presence of
circulating cells with LAK activity in the early posttransplant period?6 IL-2 stimulation of these cells augmented
their capacity to lyse tumor target^.^' Others have shown
0
T
1
-2
n/2 + a n t i a s
Fig 1. Comparative mean fold increase in cell number between IL-2
and anti-CD3 IL-2 stimulated cultures on day 7 of culture. PBMCs
were stimulated with IL-2 100 U/mL or activated with anti-CD3 MoAb
(OKT3 10 ng/mL) on day 0 and placed in TCM containing 100 U/mL
IL-2. The mean fold increase in cell number of anti-CD3
IL-2
stimulated cultures on day 7 was significantly greater (3.5 ? 1.0) than
cultures stimulated with IL-2 alone (0.5 2 0.1). (P < .01). Data expressed as mean ? SEM.
+
+
NHL recipients of autologous BMT, patients were combined into chemotherapy (chemo) and autologous BMT
(auto BMT) groups for statistical analysis (Mann-Whitney
U test; Fig 3). Cytotoxicity of effectors from patients
receiving chemotherapy was not significantlydifferent from
that of controls; however, cultured cells from autologous
and allogeneic BMT recipients showed a significant increase in cytolyticfunction (K562; auto BMT P < .001, allo
BMT P < .01) (Daudi; auto BMT P = .05, allo BMT
P = .06). Similarly, the percent cytotoxicity of effectors
from patients receiving chemotherapy was not significantly
different from that of controls (t-test); however, cultured
cells from autologousand allogeneicBMT recipients showed
a significant increase in cytolytic function (Table 1).There
was no association between the time interval from chemotherapy or BMT to initiation of anti-CD3 + IL-2 cultures
and degree of cytolytic function (data not shown).
Phenotype of fresh PBMC and anti-CD3 + IL-2 stimulated
cells. As shown in Table 2, the proportion of CD3'
PBMCs did not differ significantly between controls and
patients treated with chemotherapy but BMT patients had
a significantlylower percentage of CD3' cells. This was due
to a decreased number of CD4+ cells, resulting in an
inverted CD4/CD8 ratio post BMT. After 5 days in culture
51
a
8
B
I
A
3
la
Qa
d"
MtoBMT
Fig 2. Mean fold increase in cell number on day 5 of culture.
PBMCswere activated with anti-CD3 MoAb (OW3 10 ng/mL) on day 0
and cultured in TCM containing 100 U/mL IL-2. PBMCs from patients
with ALL treated with chemotherapy had significantly less growth
after 5 days of culture compared t o controls (P< .02). Proliferation of
PBMCs from NHL patients who received multiagent chemotherapy
(chemo) did not differ from that of controls. Bone marrow transplant
patients had significantly less cell growth than controls (auto BMT for
ALL, NHLand allo BMTfor leukemia); allP < .001). Data expressed as
mean 2 SEM.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ANTI-CD3
+ IL-2 PBMCS AFTER BMT
1289
K562
Daudi
25Ooo-
0
#x)o-
0
lBoo0
-
.
0
-1
loo00
Fig 3. Cytotoxicity against
K562 and Daudi targets. PBMCs
were activated with anti-CD3
MoAb (Om3 10 ng/mL) on day 0
and cultured in TCM containing
100 U/mL IL-2. Cytotoxicity was
reassessed using a 4-hour
lease assay on day 5 of culture.
One lytic unit (LU) = the number
of effectors required to lyse 30%
of targets; cytotoxicitj is presented as LU per 1Q effector
cells.
0 .
-d
e
.
..
0
0
.
I
0
8
:
4- +
+
d 4
si01
8
..
. .
. .
. .
i
c
8
-L -F
7
i
4 d 4 8 Em
showed very poor growth; therefore, we decided to assess
that IL-2 stimulated cells can be effective in purging
the effectiveness of anti-CD3 + IL-2 stimulation in generatmarrow of neoplastic C ~ I I S . ~ ~ . ~ ~
ing cells with LAK activity post BMT. Using this approach
We have previously reported on the use of anti-CD3
MoAb (OKT3) in combination with IL-2 to generate
we have evaluated the proliferation, cytolytic function, and
rapidly proliferating cultures of cells with LAK a~tivity.'~-'~ phenotype of PBMCs from BMT recipients and compared
These culture conditions resulted in an average 1,000-fold
these findings with those of healthy controls and with
increase in cell number over 21 days compared with less
patients receiving multi-agent chemotherapy but not BMT.
Proliferation of anti-CD3 + IL-2 stimulated PBMCs from
than 100-fold increase when IL-2 was used alone. In this
report we first compared proliferation of PBMCs from
patients receiving autologous or allogeneic BMT was reliable but lower than that of controls (Fig 2). However, the
BMT patients cultured with IL-2 to those activated with
anti-CD3 + IL-2 (Fig 1). Cultures containing IL-2 alone
cytolytic function on a per-cell basis of day 5 cultured cells
Table 1. Cytolytic Function of Anti-CD3
+ IL-2 Stimulated PBMCsAgainst K562 and Daudi
Daudi
K562
Controls
Chemotherapy ALL
Chemotherapy NHL
Autologous BMT ALL
Autologous BMT NHL
Allogeneic BMT
*
44.9 f 3.1
50.5 ? 9.9
57.0 6.8
64.7 ? 6.0
63.6 f 1.7
60.3 f 5.6
P=NS
P=NS
P < .004
P < a01
P < .015
Shown are the mean SEM cytolysis of the respectivetarget at 33:l effector to target ratio.
Abbreviation: NS, not significant.
58.6 f 2.7
55.8 f 11.0
55.5 f 9.5
71.1 f 5.1
76.3 f 5.9
73.8 * 7.1
P=
P=
P <
P <
P<
NS
NS
.03
.03
.01
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1290
KATSANIS ET AL
Table 2. Phenotype of Fresh PBMCs and Day 5 Cells in Culture With Anti-CDS + IL-2
Day 0
Day 5
Chemotherapy
ALL
Chemotherapy
NHL
Auto BMT
Auto BMT
Controls
ALL
NHL
Allo BMT
Leukemia
70.2 -t 2.9
94.8 f 0.8
60.4 f 7.9
91.7 f 2.4
63.0 f 4.9
79.1 f 10.4
44.4 f 11.9t
57.8 f 14.4*
56.7 f 6.0*
67.0 -t 8.4$
52.2 f 7.2'
52.7 f 6.5$
47.0 f 3.3
59.3 f 4.6
33.2 f 6.2'
57.3 f 7.2
34.7 f 4.8'
39.4 f 7.1*
15.2 f 3.7$
18.3 f 4.6$
25.6 f 3.0$
38.8 f 7.2'
11.9 f 2.6$
16.8 f 4.3$
22.0 f 2.5
38.1 f 2.5
26.7 f 4.2
39.0 7.3
30.9 f 6.0
52.7 f 5.8t
33.9 f 9.7
49.8 f 10.6
44.8 2 5.1$
39.8 f 6.2
40.9 f 6.9t
46.1 7.1
11.7 f 1.6
2.5 f 0.4
21.0 f 9.6
1.8 2 0.3
17.4 f 4.4
5.1 f 1.9
12.2 f 3.2
5.7 f 1.5t
25.8 f 5.8t
12.4 f 3.5$
17.1 f 3.8
14.3 f 2.9$
18.7 f 2.3
6.9 f 0.9
27.0 f 16.1
7.1 f 1.5
21.9 f 3.6
22.1 f 7.3t
24.2 f 7.3
27.6 f 12.0t
31.6 f 4.5t
39.3 2 9.5*
23.5 f 5.4
42.7 f 7.4$
9.1 -c 1.4
88.0 f 4.6
6.6 f 2.2
90.0 f 3.0
17.5 f 3.5t
87.8 f 7.1
6.9 f 2.4
58.5 f 5.0t
13.2 3.6
69.5 f 8.6*
3.8 f 1.2
39.9 f 7.6$
CD4
Day 0
Day 5
CD8
Day 0
Day 5
*
CD16
Day 0
Day 5
CD56
Day 0
Day 5
CD25
Day 0
Day 5
Shown are the mean percent f SEM of PBMCs expressing the antigens indicated. Day 0 is fresh uncultured cells and Day 5 is cells from 5-day
cultures of anti-CD3 + IL-2.
*Significantly different from controls: P < .05.
tsignificantly differentfrom controls: P < .01.
Significantly different from controls: P < .001.
from transplant patients was enhanced compared with
controls (Table 1). This is in contrast to the report by
Higuchi et alMin which LAK cells generated postautologous BMT did not have higher cytolytic function when
compared with those from healthy controls. This data may
suggest that anti-CD3 + IL-2 activation may be a promising
technique to generate larger numbers of potent cytolytic
effectors for adoptive immunotherapy post BMT.
Fresh PBMCs from healthy volunteers and patient groups
studied had a similar number of CD16' and CD56' cells.
However, after 5 days in culture BMT patients had a
significantly higher number of CD16' and CD56' cells
(Table 2). It has been reported that CD16' and CD56' cells
mediate most of the LAK activity in IL-2 stimulated
c ~ l t u r e s . "We
~ ~ have
~ previously reported that at least three
different cell subpopulations develop LAK activity in longterm cultures with anti-CD3 + IL-2; (1) CD3-, CD16+;(2)
CD3-, CD16-, CD56'; (3) CD3', CD4-, CD8-.I5 In this
study we did not evaluate the contribution of each cell
subpopulation to total LAK activity generated. However,
given the previous data it appears that the higher cytolytic
function of cultured PBMCs from BMT patients was related to their higher percentage of CD16+and CD56' cells.
Thus, anti-CD3 + IL-2 activation appears to be a
promising technique to generate cells that may be used in
an adoptive immunotherapy program as adjuvant therapy
in post BMT patients. Further studies are needed to define
signals beyond anti-CD3 and IL-2 that may improve proliferation of these cells posttransplant while retaining their
enhanced lytic function.
ACKNOWLEDGMENT
The authors thank Michelle R. Jenkins for her excellent secretarial assistance, HoiTmann LaRoche for the generous gift of IL-2,
Genia Gordon and the Immunobiology Research Center staff for
their invaluable logistical assistance. We would also like to thank
Drs Les Robison and Ann Mertens for their assistance in the
statistical analysis of the data.
REFERE1UCES
1. Woods WG, Nesbit ME, Ramsay NKC, Krivit W, Goldman
A, McGlave PB, Kersey JH: Intensive therapy followed by bone
marrow transplantation for patients with acute lymphocytic leukemia in secondary or subsequent remission. Determination of
prognostic factors. Blood 61:1182,1983
2. Johnson FL,Thomas ED, Clark BS, Chard RL, Hartmann
JR, Storb R A comparison of marrow transplantation with
chemotherapy for children with acute lymphoblastic leukemia in
second or subsequent remission. N Engl J Med 305:846,1981
3. Applebaum FR, Sullivan KM, Buckner CD, Clift RA, Deep
HJ, Fefer A, Hill R, Mortimer J, Neiman PE, Sanders JE, Singer J,
Stewart P, Storb R, Thomas ED: Treatment of malignant lym-
phoma in 100 patients with chemotherapy, total body irradiation
and marrow transplantation. J Clin Oncol5:1340,1987
4. Kersey JH, Weisdorf D, Nesbit ME, LeBien T, Woods WG,
McGlave PB, Kim T, Vallera DA, Goldman AI, Bostrom B, Hurd
D, Ramsay NKC: Comparison of autologous and allogeneic bone
marrow transplantation for treatment of high risk refractory acute
lymphoblastic leukemia. N Engl J Med 317:461,1987
5. Weiden PL, Flourney N, Thomas ED, Prentice R, Fefer A,
Buckner CD, Storb R: Anti-leukemic effect of graft versus host
disease in human recipients of allogeneic marrow grafts. N Engl J
Med 300:1068,1979
6. Weisdorf DJ, Nesbit ME, Ramsay NKC, Woods WG, Gold-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
ANTI-CD3
+ IL-2 PBMCS AFTER BMT
man AI, Kim TH, Hurd DD, McGlave PB, Kersey JH: Allogeneic
bone marrow transplantation for acute lymphoblastic leukemia in
remission: Prolonged survival associated with acute graft-versushost disease. J Clin Oncol5:1348,1987
7. Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J,
Kolb HJ, Rimm AA, Ringden 0, Rozman C, Speck B, Truitt RL,
Zwaan FE, Bortin MM: Graft versus leukemia reactions after
BMT. Blood 75:555,1990
8. Goldman JM, Gale RP, Horowitz MM, Bigs JC, Champlin
RE, Gluckman E, Hoffmann RG, Jacobsen SJ, Marmont AM,
McGlave PB, Messner HA, Rimm AA, Rosman C, Speck B, Tura
S, Weiner RS, Bortin MM: BMT for CML in chronic phase:
Increased risk of relapse associated with T-cell depletion. Ann
Intern Med 108:806,1988
9. Grimm EA, Mazumder A, Zhang H, Rosenberg S A Lymphokine activated killer phenomenon: Lysis of natural killer-resistant
fresh solid tumor cells by interleukin-2activated autologous human
peripheral blood lymphocytes. J Exp Med 155:1823,1982
10. Rosenberg SA, Lotze MT, Muul LM, Chang AE, Avis FP,
Leitman S, Linehan WM, Robertson CN, Lee RE, Rubin JT, Seipp
CA, Simpson CG, White D E A progress report on the treatment
of 157 patients with advanced cancer using lymphokine-activated
killer cells and interleukin-2 or high dose interleukin-2 alone. N
Engl J Med 316:889,1987
11. West WH, Tauer KW, Yannelli JR, Marshall GD, Orr DW,
Thurman GB, Oldham R K Constant-infusion recombinant interleukin-2 in adoptive immunotherapy of advanced cancer. N Engl J
Med 316:898,1987
12. Mule JJ, Shu S, Schwarz SL, Rosenberg S A Adoptive
immunotherapy of established pulmonary metastases with LAK
cells and recombinant interleukin-2. Science 225:1487,1984
13. Lafreniere R, Rosenberg S A Successfulimmunotherapy of
murine experimental hepatic metastases with lymphokine activated killer cells and recombinant interleukin-2. Cancer Res
45:3735,1985
14. Ochoa AC, Gromo G, Alter BJ, Sondel PM, Bach FH:
Long-term growth of lymphokine activated killer (LAK) cells: Role
of anti-CD3, f3-IL-1,interferon y and f3. J Immunol138:2728,1987
15. Ochoa AC, Hasz DE, Rezonzew R, Anderson PM, Bach
FH: Lymphokine-activatedkiller activity in long-termcultures with
anti-CD3 plus interleukin-2:Identificationand isolation of effector
subsets. Cancer Res 49:963,1989
16. Anderson PM, Bach FH, Ochoa A C Augmentation of cell
number and LAK activity in peripheral blood mononuclear cells
activatedwith anti-CD3 and interleukin-2.Cancer Immunol Immunother 27:82,1988
17. Ting CC, Hargrove ME, Yun YS: Augmentation by anti-T3
antibody of the lymphokine-activatedkiller cell mediated cytotoxicity. J Immunol141:741,1988
18. Anderson PM, Blazar BR, Bach FH, Ochoa A C Anti-CD3
+ IL-2 stimulated murine killer cells: In vitro generation and in
vivo antitumor activity. J Immunol142:1383,1989
19. Yun YS, Hargrove ME, Ting C C In vivo anti-tumor activity
of anti-CD3 induced activated killer cells. Cancer Res 494770,
1989
1291
20. Loeffler CM, Platt JL, Anderson PM, Katsanis E, Ochoa JB,
Urba WJ, Longo DL, Leonard AS, Ochoa A C Antitumor effects
of IL-2 liposomes and anti-CD3 stimulated T cells against murine
MCA-38 hepatic metastases. Cancer Res 51:2127,1991
21. Weisdorf DJ, McGlave PB, Ramsay NKC, Miller WJ, Nesbit
M, Woods W, Goldman A, Kim TH, Kersey JH: Allogeneic bone
marrow transplantation for acute leukemia: Comparative outcomes for adults and children. Br J Haematol69:351,1988
22. Filipovich AH, Vallera DA, Youle RJ, Haake R, Blazar BR,
Arthur D, Neville DM, Ramsay NKC, McGlave PB, Kersey J H
Graft-versus-host disease prevention in allogeneic bone marrow
transplantation from histocompatible siblings. Transplantation
44:62,1987
23. Uckun FM, Gajl-Paczalska KJ, Kersey JH, Houston LL,
Vallera D A Use of a novel colony assay to evaluate the cytotoxicity
of an immunotoxin containing pokeweed antiviral protein against
blast progenitor cells freshly obtained from patients with common
B-lineage acute lymphoblastic leukemia. J Exp Med 163:347, 1986
24. Uckun FM, Jaszcz W, Ambrus JL, Fauci A, Gajl-Peczalska
K, Song CW,Wick MR, Myers DE, Waddick K, Ledbetter J A
Detailed studies on expression and function of CD19 surface
determined by using B43 monoclonal antibody and the clinical
potential of anti-CD19 immunotoxins. Blood 71:13,1988
25. Lanier U, Womer NL: Paraformaldehyde fixation of hematopoietic cells for qualitative flow cytometry (FACS) analysis. J
Immunol Methods 47:25,1981
26. Reittie JE, Gottlieb D, Heslop HE, Leger 0, Drexler HG,
Hazlehurst G, Hofirand AV, Prentice HG, Brenner MK Endogenously generated activated killer cells circulate after autologous
and allogeneic marrow transplantation but not after chemotherapy. Blood 73:1352,1989
27. Gottlieb DJ, Prentice HG, Heslop HE, Bello-Fernandez C,
Bianchi AC, Galazka AR, Brenner MK Effects of recombinant
interleukin-2 administration on cytotoxic function following highdose chemo-radiotherapy for hematological malignancy. Blood
742335,1989
28. Long GS, Hiserodt JC, Hamaha JB, Cramer DV: Lymphokine-activated killer cell purging of leukemia cells from bone
marrow prior to syngeneictransplantation. Transplantation 46:433,
1988
29. Agah R, Malloy B, Kerner M, Mazumder A Generation and
characterization of IL-2 activated bone marrow cells as a potent
graft vs. tumor effector in transplantation. J Immunol 143:3093,
1989
30. Higuchi CM, Thompson JA, Cox T, Lindgren CG, Buckner
CD, Fefer A Lymphokine-activated killer function following
autologous bone marrow transplantation for refractory hematological malignancies. Cancer Res 49:5509, 1989
31. Ortaldo JR, Mason A, Overton R: Lymphokine-activated
killer cells: Analysis of progenitors and effectors. J Exp Med
164:1193,1986
32. Ferrini S, Miescher S, Zocchi MR, Fliedner V, Moretta A
Phenotypic and functional characterization of recombinant interleukin-2 induced activated killer cells: Analysis at the population
and clonal levels. J Immunol138:1297,1987
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1991 78: 1286-1291
Proliferation and cytolytic function of anti-CD3 + interleukin-2
stimulated peripheral blood mononuclear cells following bone marrow
transplantation
E Katsanis, PM Anderson, AH Filipovich, DE Hasz, ML Rich, CM Loeffler, AC Ochoa and DJ
Weisdorf
Updated information and services can be found at:
http://www.bloodjournal.org/content/78/5/1286.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.