1. No category

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1994 84: 3550-3557
Soluble CD4, soluble CD8, soluble CD25, lymphopoieitic recovery,
and endogenous cytokines after high-dose chemotherapy and blood
stem cell transplantation
AD Ho, M Maruyama, A Maghazachi, JR Mason, S Gluck and RE Corringham
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Soluble CD4, Soluble CDS, Soluble CD25, Lymphopoieitic Recovery, and
Endogenous Cytokines After High-Dose Chemotherapy
and Blood Stem Cell Transplantation
By Anthony D. Ho, Midori Maruyama, Azzam Maghazachi, James R. Mason,
Stefan Gluck, and Robert E.T. Corringham
Mononuclear cell preparations from peripheral blood after
mobilization with hematopoietic growth factors have been
shown t o induce accelerated neutrophil and plateletrecovery as compared with that induced by autologous bone marrow transplantation after myeloablative chemotherapy. Because these mononuclear cell productscontainmany
immunocompetent cells other than hematopoietic progenitors, these accessory cells might contributeto therapid immunohematopoietic reconstitution. We have monitored the
concentrations of solubleCD4 (sCD4). sCD8, and sCD25; the
recovery of the lymphocyte
subsets and of natural killer (NK)
cells; and theendogenous levels of granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3). IL-6, and granulocyte-macrophage-CSF (GM-CSF) in 12 patients who underwent high-dose chemotherapy supported by blood stem
cells that wereobtained by mobilization with
chemotherapy
and GM-CSF. The concentrations of both G-CSF and IL-6
peaked at 7 days after reinfusionof stemcells, and this transient elevationpreceded the increase in the white blood cell
count byapproximately 5 t o 7 days. The levels of sCD4 and
sCD8 increased t o a maximum on day 21, and the time to
peak levels coincided with the maximum increase in white
blood cell count, absolute neutrophil count, or lymphocytes.
The levels of sCD25 were found t o be elevated from day 7
t o day 21. Statistically, the increases in sCD4, sCD8, sCD25,
G-CSF, and IL-6 were highlysignificant, whereas there were
no significant changes in IL-3 and GM-CSF. A rapid recovery
of the NK activity was found in all 8 of the patients who
could be monitored for this
assay. Therefore, our study suggests that recovery of CD4' cells, CD8' cells, and NK activity
coincided with that of neutrophils, which is preceded by a
marked, but transient, elevation of IL-6 and G-CSF.
0 1994 by The American Society of Hematology.
H
mobilized by GM-CSF as the source of stem cells for hematopoietic support might induce
a rapid immune as well as hematopoietic reconstitution. However, reliable measurements of
absolute lymphocytes and their function during severe leukopenia and early recovery is frequently not feasible.
Activated T lymphocytes release various molecules including soluble CD4 antigen ( S C D ~ ) , ~sCD8,I""
-"
and soluble interleukin-2 receptor (sIL-2R) or s C D ~ ~ . 'Elevated
~.'~
levels of sCD4 have been found in patients with diseases
associated with activation of T helper cells (eg, Sjogren's
syndrome,I6 systemic lupus erythemato~us,'~
or rheumatoid
arthritis'*) or in malignant hematologic diseases (eg, acute
myeloidleukemia" or progressive B-chronic lymphocytic
leukemia"). Similarly, elevated sCD8 and sCD25 levels
havebeenfoundin
patients withviral infections, autoimmune diseases, and various hematologic malignancies.'2~'4.2"
In patients with acute Epstein-Barr virus infection, the serum
sCD8 levels correlated withthe percentage of activated
CD8' (CDV/HLA-DR') cells." In hairy cell leukemia, tumor burden has been reported to correlate with the plasma
or serum levels of sIL-2R and CD8.'5.20Measurements of
concentrations of these T-cell products might serve as an
index for activation of the corresponding T lymphocytes.
In this report, we have monitored the levels of soluble
antigens CD4, CD8, and CD25 as indicators for lymphopoietic reconstitution after high-dose chemotherapy and blood
stem cell support in 12 patients with metastatic breast cancer.
Moreover, their relationships to naturalkiller (NK) cell activity, torecovery of neutrophil and lymphocyte subpopulations, and to endogenous concentrations of granulocyte-CSF
(G-CSF), IL-3, IL-6, and GM-CSF are examined. These kinetic data might provide a better insight into the sequence of
immunohematopoietic reconstitution after autologous blood
stem cell transplantation (ABSCT).
IGH-DOSE CHEMOTHERAPY supported by peripheral blood (PB) stem cells has been increasingly used
for patients with cancer.',2 There isnow little doubt that
leukocyte and platelet recovery is more rapid withblood
versus bone marrow(BM) autografts, provided that the blood
mononuclear cells (MNC) are collected after mobilization
with hematopoietic growth factors, chemotherapy, or both.
Because the MNC products obtained by leukapheresis procedures contain a large number of immunocompetent cells, it
has been suggested that these accessory cells might have
therapeutic as well as restorative potential.'
So far, most of the studies have focused mainly on the
neutrophil and platelet recovery after blood stem cell transplant, and only a few reports have provided evidence for an
accelerated recovery of lymphocyte^.'.^ In previous studies,
we and others have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) has the potential to activate T lymphocytes as well as to mobilize hematopoietic
progenitor cells.'-7 Therefore, the use of MNC preparations
From the Division of Hematology/Oncology, University of California, San Diego, CA;and the Northeastern Ontario Regional Cancer Centre, Sudbury, Ontario, Canada.
Submitted February 16, 1994; accepted July 21, 1994.
Supported by grants of the Ontario Cancer Foundation, Canada;
the Northern Heritage Fund of the Ministry of Northern Ontario
Development and Mines; and the Canadian Red CrossICutter Biologics Inc, Canada.
Address reprint requests to Anthony D. Ho, MD, PhD, USCD
CancerCenter,Division
of Hematology/Oncology,University of
California, San Diego, 200 West Arbor Dr, #8421, San Diego, CA
92103-8421.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore behereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solelv to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8410-0017$3.00/0
3550
MATERIALS AND METHODS
Patients. Female patients with metastatic breast cancer who underwent high-dose chemotherapy supported by reinfusion of blood
Blood, Vol84, No 10 (November 15). 1994:pp 3550-3557
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3551
CYTOKINES sCD4, sCD8, sCD25 AFTERABSCT
stem cells were monitored in this study. The age ofthe patients
ranged from 18 to 60 years, and performance status had to be less
than 2 according to the World Health Organization scale.” The
protocol was approved by the Institutional Review Board, and all
patients gave written informed consent.
Blood stem cells were harvested after induction chemotherapy
that consisted of cyclophosphamide at 750 m g h ’ administered intravenously (IV), epirubicin at 100 mg/m2 IV, and 5-fluorouracil at
750 mg/m’ IV (CEF) all on day 1. The chemotherapy cycle was to
be repeated every 21to28
days, and 2 to 3 cycles were to be
administered. Starting on day 2 of each treatment cycle, GM-CSF
(Sandoz Canada Inc, Montreal, Canada) at a daily dose of 5 pgkg
was administered subcutaneously until the leukapheresis procedures
were completed. The high-dose chemotherapy regimen consisted of
carboplatin (200 mg/mz/d), mitoxantrone (12.5 to 17.5 mg/m’/d),
and cyclophosphamide (1.5 g/mz/d), all administered IV, and all
administered for 4 days from day -5 to day -2. After 1 day of rest,
ABSCs were reinfused on day 0. Within 24 hours of reinfusion,
GM-CSF was administered daily at a dosage of 5 pg/kg/d by a
continuous IV infusion over 6 hours until the absolute neutrophil
count (ANC) was greater than 3.0 X IO9 for 3 consecutive days.
Leukapheresis, cryopreservation, and reinfusion ofstem cells. Peripheral blood progenitor cells were collected as soon as the white
blood cell (WBC) counts approached a level of greater than 2.0 X
109L and when the platelet counts were greater than 50 X 109L
after induction chemotherapy. Four leukaphereses were performed
on 4 consecutive days, unless the patient was febrile or a weekend
or holiday intervened. Leukaphereses were performed by means of
a blood cell separator (CS3000 Plus; Fenwal Laboratories, Deerfield,
IL). A total blood volume of 10 L per apheresis was processed at a
flow rate of 60 to 70 mL per minute. The apheresis product was
centrifuged, part of the plasma was removed, and the product was
resuspended with minimal essential medium and was supplemented
with 20% dimethylsulfoxide. The cells were then frozen to - 100°C
using a computer-controlled device, were transferred into the liquid
phase of nitrogen, and were stored at - 196°C. Thawing of the cryopreserved cells was performed by immersing the freezing bag into
a 42°C water bath. The cell suspension was immediately injected
into a central line. The target was to obtain a minimum of 1 X lo6/
kg of CD34’ cells or 2 X 105kgof colony-forming unit granulocytemacrophage (CFU-GM) for reinfusion.
Clonogenic assay for hematopoietic stem cells. A total of 2 X
105/mL mononuclear cells was mixed with Iscove’s modified Dulbecco’s medium (GIBCO, Grand Island, NY) containing 0.9% methylcellulose, 5 ng/mL each of GM-CSF and IL-3 (Sandoz AG, Basel,
Switzerland), 2.5 U/mL erythropoietin (Behringweike AG, Marberg,
m o m 2-mercaptoethanol. The cultures
Germany), and 5 X
were plated in duplicate and were incubated for 14 days at 37°C in
5% COz in a humidified atmosphere. After 14 days, the cultures were
scored using an inverted microscope. The granulocyte-macrophage
colonies and erythroid bursts were identified by their typical properties.
Plasma levels of sCD4, sCD8, and sCD2.5. The levels of CD4
or CD8 antigen in plasma were determined by a sandwich enzyme
immunoassay (Cellfree CD4/CD8 Test Kits; T Cell Sciences, Cambridge, MA).” An anti-CD4 or anti-CD8 monoclonal antibody
(MoAb) was first absorbed onto the wells of a polystyrene 96-well
microtiter plate; aliquots of sample diluent and of plasma sample
were added into each well and were incubated at 37°C. After washing, a horseradish peroxidase-conjugated MoAb directed against a
second epitope on the corresponding molecule was added. After
further incubation, the wells were washed, and 100 pL of a substrate
solution containing 0-phenylenediamine was pipetted into each well.
After an incubation of 30 minutes at room temperature, the reaction
was quenched with a 2 N solution of sulfuric acid, and the ab-
sorbance was measured at 490 nm versus substrate blank. A standard
curve was prepared using serial dilutions of a reference standard
supplied in the corresponding assay kits. The absorbance of the test
wells was then compared with the standard curve and was converted
to a numerical value. CD4 or CD8 concentrations were expressed
in units per milliliter (U/mL); 1,000 U of CD8 was defined as the
amount of released CD8 present in 1 .O mL of a reference preparation
provided by the supplier. A unit of CD4 is defined as the amount
of CD4 found in lo3 Jurkat T cells lysed with 1% NP-40.
Similarly, the levels of C D 2 5 were determined in the plasma
samples by a sandwich enzyme immunoassay (Cellfree IL-2 Test
Kit, T Cell Sciences). The details have been described elsewhere.’”
Plasma levels of hematopoietic growth factors. All samples were
assayed using sandwich enzyme immunoassays (IL-3, IL-6, GMCSF, and G-CSF; Quantikine; R & D Systems, Minneapolis, MN).
The assay protocols as delineated by the supplier were followed.
Polystyrene 96-well microtiter plates were coated with MoAbs
against the corresponding growth factor. Recombinant human CSFs
(rhCSFs) were used as standards in geometric dilution within a range
of 15 to 2,000 pg/mL for IL-3, of 3 to 300 pg/mL for IL-6, of 7 to
1,000 pg/mL for GM-CSF, and of 39 to 5,000 pg/mL for G-CSF,
respectively (all supplied together with the assay kits). In addition,
growth factors from other suppliers have been used to countercheck
the quality ofthe assays: IL-3 and GM-CSF from Immunex, Inc
(Seattle, WA); IL-6 from Genetics Institute (Cambridge, MA); and
G-CSF from Amgen (Thousand Oaks, CA).
The serial dilutions of the rhCSFs and the patient samples were
added to the wells and incubated for 2 hours at room temperature.
After washing, a polyclonal murine antibody against a second epitope was added and further incubated for 2 hours at room temperature. The antibodies were directly conjugated to horseradish peroxidase. The color-reaction with a solution of tetramethylbenzidine was
stopped with 2 N sulfuric acid after incubation for 20 minutes at
room temperature in the dark. The optical density of each well was
then determined within 30 minutes using a spectrophometer set to
450 nm.
N K cell activity. Peripheral blood cells of patients were layered
over Ficoll-Hypaque (Pharmacia Chemicals, Ontario, Canada) and
were then centrifuged for 25 minutes at 700g to remove red blood
cells. The erythrocyte-depleted cell preparation was tested for its
ability to kill tumor target cells. Target cells include the NK-sensitive
K562 (a human myelogenous leukemia cell line), the NK-resistant
but lymphokine-activated killer (LAK)-sensitive targets; R A J 1 (a
human Burkitt lymphoma cell line), and EM-3 (a human leukemia
cell line). Target cells (1 to 5 X lo6) were labeled with 100 pCi
”Cr (sodium chromate; New England NuclearlDupont, Ontario,
Canada) for 1 hour at 37“C, washed, and incubated with various
numbers of effector cells in triplicate in round-bottomed wells of
microtiter plates (Coming Glass Works, Coming, NY) at 1 X lo4
target cells per well in a total volume of 200 pL. The plates were
centrifuged and then incubated for 4 hours at 37°C in a humidified
atmosphere of5% COz. After the incubation period, supernatants
were removed from wells and then counted in a Beckman LS6OOOIC
(Beckman Instruments, Fullerton, CA). Percentage of cytotoxicity
was calculated according to the following formula:
(Mean cpm released from test wells Mean cpm of spontaneous release)
x 100.
(Mean total cpm Mean cpm of spontaneous release)
Flow cytometry. Flow cytometry was performed to determine
the lymphocyte subpopulations. Leukocytes in whole blood were
stained at room temperature with appropriate antibodies for 20 minutes (fluorescein isothiocyanate- or phycoerythrin-labeled MoAbs
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H 0 ET AL
3552
Table 1. Clinical Characteristicsof Patients Studied
~
Previous Treatment
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
Age
(yrl
43
33
44
40
45
36
33
54
38
44
40
Metastatic Sites
Chemotherapy
Radiation
WBC per nL
LN
LN
+
-
Lung, bone
Bone
Bone
+
2.3
3.7
4.6
3.9
4.4
7.8
7.2
4.3
2.6
4.1
5.4
3.9
LN
LN
Lung, bone
LN, bone
Lung
Inflammatory
LN, lung
0.46
0.78
+
-
+
+
-
+
+
+
+
+
+
+
0.68
0.72
0.52
~
~
+
0.49
0.38
0.32
~~~
Pretransplant
~
+
Lymphocytes per nL
0.61
0.66
0.86
0.47
Abbreviation: LN, lymph node.
against CD4, CD8, CD20 and CD25, or negative controls). Erythrocytes were lysed, and leukocytes were fixed with lysis reagent supplied by Coulter Electronics (Hialeah, l%) and according to the
instructions of the manufacturer. All MoAbs were obtained from
Coulter Electronics, unless otherwise noted. Two-color, four-parameter flow cytometric analysis was performed with a Coulter Elite I1
as previously described.**Mononuclear cells were gated by forwardand side-scatter signals. At least 30,000 events were gated, acquired,
and stored as list-mode data. The stained cell samples were analyzed
within 72 hours after preparation. The absolute numbers were calculated by multiplying the corresponding percentages with theabsolute
number of lymphocytes.
Statistical analysis. For statistical analysis, a personal computer
program, Testimate ( D V Datenanalyse, Gauting-Munich, Germany), was used. The Friedmann analysisz3was applied to test the
differences in levels of sCD4, sCD8, and sCD2.5 as well as inplasma
levels of endogenous CSFs (IL-3, IL-6, GM-CSF, G-CSF) or lymphocyte subpopulations before and after high-dose chemotherapy
and ABSCT.
Recovery of lymphocyte subpopulations. Parallel to the
recovery of the WBCs and neutrophils, the absolute number
of lymphocytes also increased within the same time frame
(see Fig I). The numbers of total lymphocytes, of ceIIspositive for CD4, CD8, CD20, and CD25 before transplantation
(baseline) and at the time of maximum increase in WBCs (21
days after ABSCT) are shown in Fig 2. With the exception of
a significant reduction in CD20+ cells (P = .0020, Wilcoxon
two-sided test), there were no significant differences in the
absolute numbers of cells positive for CD4, CD8, and CD25.
NK activity of the MNC before and after ABSCT. Complete serial monitoring of the NK activity could be performed
in 8 of 12 patients. The percentage of cytotoxicity activity
against K562 cells before induction chemotherapy was
32.8% 2 8.0% (range, 4.1% to 52.0%), and the percentage
before high-dose chemotherapy and ABSCT was 26.9% ?
5.3% (range, <0.1% to 52.1%). Whenever possible, the NK
RESULTS
Patients. A total of 12 patients were included in the
present study. Clinical characteristics of the patients are
shown in Table 1.All patients were women, and allhad
metastatic breast cancer (median age, 41.5 years). The pretransplant WBC count was 4.5 ? 0.41nL (mean ? SEM),
and the lymphocyte count was 0.58 ? 0.05InL.
Hematopoietic reconstitution. The recovery of the
WBCs after high-dose chemotherapy and ABSCT in the 12
patients is summarized in Fig 1. The median time to a WBC
of greater than 1.0 X lo9& was 12 days (range, 8to 16
days), to absolute neutrophil count of greater than 0.5 X lo9/
L was 13 days (range, 9 to 18 days), and to an unmaintained
platelet count of greater than 20 X lo9& was 10 days (range,
7 to 27 days). The median number of CFU-GM reinfused
was 3.9 X 105/kgof body weight (range, 0.8 to 12.4 X IO5/
kg), and the median number of CD34+ cells reinfused was
8.9 X lO%g (range, 1.4 to 13.8 X 106/kg). The median
number of days for GM-CSF administration was 19 days
(range, 15 to 23 days). The correlation between CFU-GM
or CD34+ cell dose and hematopoietic recovery has been
reported in detail elsewhere.*’
WBC InL
’*m
10 -
1.
0.1 .
0.01 I
-10
I
0
10
I
I
20
30
40
3
DAYS AFTER TRANSPLANT
Fig l . Recovery of WBC and lymphocytesafter high-dow chemotherapy andABSCT. Day 0 is the day of ABSCT. Values below 0.05 are
beneath detection limits. The means k SEM (error bars) are shown.
(U),WBC; (x), lymphocyte count.
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CYTOKINES sCD4,sCD8,
sCD25 AFTER ABSCT
3553
ABSOLUTE NUMBERS PER uL
1,000
1
SCW LEVELS (UlmL)
12
l
l0
\
8
6
-
l
Pre MerPreAfterPreAfterPreAfterPreAfter
CD25CD20
LymCD0 CD4
Fig 2. Recoveryof lymphocyte subpopulations after high-dose
chemotherapy and ABSCT. "Pre" stands for baseline values before
transplant; "After" stands for values at the peak of WEC recovery
(day 211 after transplant. The columns represent the means and the
error bars ofthe SEM.
activity was monitored weekly for 6 to 8 weeks after reinfusion of stem cells, but the numbers of MNC available were
usually not adequate for the assay until the third week. The
recovery of N K activity was also rapid and reached 41.6%
2 8.9% (range, 7.8% to 73.9%) by the third to fourth week
after ABSCT. At the same time, cytotoxicity activity against
RAJ1 or EM-3 cells was not detected in all 8 patients before
ABSCT. In 3 of 8 patients, a slight increase to 7.3%, 8.1 %,
and 15.0% of cytotoxicity to RAJ1 cells was found, suggesting a generation of LAK-cell activity in a small proportion of the patients after ABSCT and administration of GMCSF.
Kinetics of sCD4,
sCD8,
and sCD25. The plasma level
of sCD4 before induction chemotherapy was 8.7 t 1.9 U/
mL, and before high-dose chemotherapy and ABSCT was
5.5 t 0.4 U/mL. In our laboratory, both levels were significantly lower than the normal control value of 2 1.5 t 1.9 U/
mL (range, 10.0 to 36.4 U/mL; n = 1 1 ; P < .0001 for both
comparisons). There was no significant change in the sCD4
levels within the first 3 weeks after high-dose chemotherapy.
The sCD4 level increased significantly in the thirdweek
(day 21 ) after ABSCT to a median of 1 1 .O 5 0.9 U/mL
(range, 7.0 to 15 U/mL; P = .0004, according to Friedmann
analysis for matched set of data). The levels of sCD4 returnedreadily to the pretransplant baseline range in the
fourth week. Although the sCD4 levels from weeks 4 to 15
were slightly higher than those before high-dose treatment,
the changes were statistically not significant. The results are
summarized in Fig 3A.
Before induction chemotherapy with CEF, the plasma
level of sCD8 was 308 5 49 U/mL, and before high-dose
chemotherapy and stem cell transplant, it was 258 5 23 U/
mL. The controls in our laboratory showed sCD8 level of
355 2 23 U/mL (n = 12). The differences between controls
and patients in both circumstances were not significant. The
4
5
AFTER
WEEKS
0
A
10
TRANSPLANT
15
SCD8 SERUM LEVELS (UlrnL)
1,000.
800 -
T
400
0'
I
I
I
0
5
c
10
15
B
WEEKS AFTER TRANSPLANT
SCD25 LEVELS fUlrnLI
100'
C
I
0
5
10
15
WEEKS AFTER TRANSPLANT
Fig 3. Kinetics of soluble
CD4 (A), of solubleCD8 (B), and of soluble
CD25 (C) before AESCT and after transplant. The corresponding
means k SEM (error ban) are shown. The shaded areas represent
the normal range.
1.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
3554
H 0 ET AL
G-CSF (pGlmL)
1,000 -
100 -
I
I
0
lo
10
5
WEEKS AFTER TRANSPLANT
A
I
15
SERUM
LEVELS
(pG/mL)
IL6
10 -
A
.
1
B
,
15
0
10
I
I
5
WEEKS AFTER TRANSPLANT
Fig 4. Endogenouslevelsof G-CSF (A) and 11-6 (B)before and after
ABSCT.Thecorrespondingmeans f SEM (errorbars)areshown.
day
limits of detection of the respective assays. The endogenous
levels of G-CSF in the 12 patients before induction chemotherapy with CEF and before ABSCT were also in the minimum detectable range (13.9 ? 8.1 pg/mL and 14.5 t 2.2
pg/mL, respectively) but increased to 690 -t 490 pg/mL
(range, 128 to 5,758 pg/mL) in the first week. This increase
was statistically significant with a P value of less than ,0001
according to Friedmann analysis. Thereafter, endogenous GCSF levels decreased rapidly and were within baseline range
after day 21. The changes in G-CSF levels are summarized
in Fig 4A.
Parallel to the increase in G-CSF, the plasma IL-6 levels
also increased significantly (P = .0014) from less than 10
pg/mL to 21.1 f 4.0 pg/mL (range, 3.0 to42.5 pg/mL)
within the first 7 days. After the maximum increase in the
first week, endogenous IL-6 decreased to
rapidly
less than
10 pg/mL and remained at this level. The kinetics of IL-6
levels are shown in Fig 4B. Both GM-CSF and IL-3 were
below detection limits before induction chemotherapy, before ABSCT, or after high-dose chemotherapy and ABSCT
in all patients.
Relationship between sCD4, sCD8, sCD2.5, cytokines, and
lymphohematopoietic recovery. The time course of the soluble antigens CD4, CD8, and CD25 and of the cytokines GCSF and IL-6 in relationship to the WBC counts of the 12
patients is shown inFig 5. There was a synchronous and
transient elevation in both G-CSF and IL-6 levels within the
first week after ABSCT that preceded the reconstitution of
WBCs, whereas the increase in sCD4 and sCD8 levels on
day21 coincided with the maximum increase in WBCs,
neutrophils, and lymphocytes. The concentrations of C D 2 5
remained elevated from days 7 to21, as compared with
baseline, and are probably associated with the administration
of GM-CSF, as described previously.6
sCD8 levels seemed to decrease slightly in the first 3 weeks
after chemotherapy but then increased significantly ( P =
.Owl, Friedmann analysis) to a mean of 638 ? 164 U/mL
on
The sCD8 levels
week
remained
until high
10,
where it returned to the pretransplant baseline range of the
patients. The results are shown in Fig 3B.
The plasma level of sCD25 before induction chemotherapywas 1,945 2 560 U/mL. After induction chemotherapy
and pretransplant, it was 780 ? 117 U/mL . Both were
significantly higher than the normal mean of 325 2 87 U/
mL (n = 16; P < .W1 for both comparisons). Within the
first week after ABSCT, sCD25 levels increased significantly
to 2,459 ? 294 U/mL and remained elevated until the fourth
week after reinfusion. The sCD25 levels returned to a pretransplant baseline of 750 2 76 U/mL after week 7, and the
induction chemotherapy and before ABSCT are beneath the
DISCUSSION
Defects of immune function, as indicated by an imbalance
of CD4+ and CD8+ cells, diminished T-cell proliferative
SCD~.SCD25(U/ml).lLE,G-CSF(W/ml)
SCM(Ulml). WEClnl
12
10.000
10
1.000
8
100
4
10
2
1
-5
0
5
10
15
20
25
30
DAYS A F E R TRANSPIANT
beforetransplant,during recovery after ABSCT.
35
40
0
45
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CYTOKINES sCD4, sCD8, sCD25 AFTER ABSCT
responses, depressed IL-2 production, and failure to respond
to endogenous L - 2 have all been reported after allogeneic
or autologous BM transplantation (ABMT)."'" We have
monitored the levels of sCD4, sCD8, sCD25, the lymphocyte
subpopulations, and the NK activities before and after
ABSCT and have found a rapid reconstitution of the T cells
as well as NK cell function that occurred parallel toan
accelerated WBC and neutrophil recovery. As previously
shown by studies from our group and from others, measurements of the soluble antigens serve as a more sensitive index
of activation of the cells expressing the corresponding surface antigen.'3"8.zo
The sCD4 levels in the patients before induction chemotherapy (CEF), before high-dose chemotherapy and ABSCT,
and after hematopoietic recovery were significantly lower
than that in the normal controls, whereas levels of sCD25
were significantly elevated in the patients under all circumstances. Elevations of sCD25 in various malignancies is
well-established and might represent the activation of CD25'
cells as an immune resp~nse'~-~'
or, for lymphoid malignancies such as hairy cell leukemia, as a tumor marker.I5 The
finding of significantly reduced sCD4 levels in patients with
metastatic breast cancer is novel and might reflect the suppression of CD4+ lymphocytes, which could be either associated with advanced di~ease'~.~'
or could represent a consequence of previous chemotherapy and/or irradiation
therapy.32Irrespective of the underlying cause, changes in
sCD4 or sCD8 have been shown to reflect the activation or
suppression of the cells expressing the corresponding antiAnalysis of the kinetic data of sCD4 and sCD8 in relationship to endogenous levels of IL-3, IL-6, G-CSF, and GMCSF showed that the increase in sCD4, sCD8, and in Tlymphocyte subpopulations were preceded by a transient increase in IL-6 and G-CSF levels by 2 weeks, whereas no
significant changes in IL-3 or GM-CSF levels were found.
Similar elevation of endogenous G-CSF levels has been reported by other investigators after ABSCT or ABMT. Cairo
et aP3 reported a significant increase in G-CSF production
around day 5 after ABMT, whereas Kawano et al" found
an increase of G-CSF immediately after graft infusion but
no significant changes in IL-3 or GM-CSF levels." We found
that GM-CSF levels were beneath detectable limits at all
times despite administration of GM-CSF after ABSCT. In
our study, blood samples for soluble antigens and endogenous cytokines wre collected at6 AM, and GM-CSF was
administered as a 6-hour continuous infusion between 2 PM
to8 PM. Other investigators have also reported failure to
detect significant increases in endogenous GM-CSF levels
after ABMT.34-36
Reports on endogenous IL-3 levels after high-dose chemotherapy and ABMT or ABSCT are contradictory. Whereas
Kawano et a134and our present report showed no significant
change in IL-3 levels, Mangan et a137 detected a burst of IL3 (peak levels, 1,500 to 6,000 pg/mL) in the immediate
posttransplant period between day 0 and day 14.37 Furthermore, a lower percentage of T-cell-depleted transplant recipients had detectable IL-3 levels, and lower peak levels of
IL-3 were observed in 60% of the recipients of ABSCT.
3555
They suggested that a larger infusion of T cells in ABSCT
recipients might downregulate IL-3 release, and the more
rapid engraftment observed in ABSCT recipients might also
downregulate IL-3 release through a feedback mechanism.
At present, we have no satisfactory explanation for the lack
of detection of IL-3 in our patients, but the extensive amount
of T cells in our MNC preparations stimulated by GM-CSF
might suppress IL-3 release, as suggested by Mangan et al.37
Because the levels of these growth factors were determined weekly, we could have missed a very early surge in
IL-3 or GM-CSF. However, in 2 of the patients, we monitored the G-CSF, GM-CSF, IL-3, and IL-6 levels every other
day for the first 14 days after ABSCT, and we noted asurge
in G-CSF and L - 6 levels from days 2 to 12. The peak values
were within the range measured at day 7, and there were no
increases in IL-3 or GM-CSF levels during this period. For
this reason, we have subsequently monitored these parameters on a weekly basis.
Contrary to our present observation, an increase in IL-6
was not reported by Kawano et al.34 The blood stem cells
in their series were collected after chemotherapy without the
addition of cytokines, whereas our patients all received GMCSF after induction chemotherapy. Rabinowitz et a13' also
reported an increase in IL-6 levels after ABSCT. Based on
a multivariate regression analysis comparing the number of
platelet transfusions required with levels of tumor necrosis
factor, IL-6, and macrophage-CSF (M-CSF), they concluded
that high M-CSF and IL-6 levels on day + l 2 after ABMT
correlated with anincreased demand for platelet transfusions
and increased toxi~ity.~'
All our patients have recovered from
ABSCT without major nonhematologic toxicity, and the
platelet recovery has been extremely rapid. In previous studies, we have shown both in vitro and in vivo that GM-CSF
activates T lymphocyte^.^.^ IL-6 is produced by lymphocytes,
monocytes, and mesenchymal stroma cells, and it is likely
that MNC preparations mobilized by chemotherapy and GMCSF contain more activated lymphocytes that produce IL-6
after PBSCT and, thus, induce a more rapid reconstitution
of platelets.
The data from our present study support the notion that
there is a fast recovery of lymphocyte counts, in addition to
accelerated reconstitution of neutrophil and platelet counts
after ABSCT. Although the absolute numbers of lymphocytes, CD4+ cells, and CD8+ cells were relatively low after
ABSCT, the baseline ranges before high-dose chemotherapy
were already low and were reached within3 weeks. In
agreement with other studies on lymphocyte recovery after
ABSCT,3.4.27.39 we
also found a tendency for the CD4+ cells
to decrease and for the CD8+ cells to increase after ABSCT,
resulting in a decrease in CD4/CD8 ratio. However, the differences were statistically not significant. Henon et a14found
a severe imbalance in CD4/CD8 ratio of 0.3 ? 0.2 but found
a normal number of B cells after ABSCT, whereas Kiesel
et a139reported a CD4/CD8 ratio of slightly less than 1.0 in
most patients. We found a moderate (but not significant)
decrease of numbers of CD4+ cells and of CD4/CD8 ratio
among our patients. In contrast to the studies reported by
the patients examined in the present investigation were homogeneous in their primary tumor diagnosis
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
3556
H 0 ET AL
(breast cancer) as well as in the induction chemotherapy.
However, the fact that a similar trend was observed in patients with acute leukemias, Hodgkin’s disease, andnonHodgkin’s lymphomas undergoing ABSCT indicates that the
phenomenon of rapid lymphocyte recovery is universally
observed in patients undergoing ABSCT.
MNC preparations, obtained by leukapheresis after mobilization regimens, contain numerous immunocompetent
cells, especially T helper cells (Ho et al, unpublished results).
It has been suggested that such accessory cells might have
therapeutic as well as reconstitutive potential and might be
responsible for the accelerated neutrophil and platelet recovery after high-dose chemotherapy. This notion is supported
by our present study and by a few other comparative studies
which show that immunologic as well as hematologic reconstitution is accelerated with the use of PB-versus BM-derived
stem cell^.'.^.^^ However, because we have no comparative
control data from patients who received mononuclear cell
preparations alone without GM-CSF, the possibility cannot
be excluded that the present data on immune reconstitution
are associated with GM-CSF rather than with PB-derived
stem cells. The results might eventually be different in patients receiving ABSCT without GM-CSF.
NK cells are lymphoid cells with spontaneous cytotoxic
activity against a variety of tumor cell lines or primary and
metastatic tumor cells. Animal models and clinical observations have demonstrated their vital role in the immune surveillance mechanism. NK cells were found to control established and induced metastases in animal models. A strong
correlation was observed between low levels of NK cytotoxicity and diminished time of manifestation of metastatic disease or decreased survival in patients with solid tumors.40A
rapid reconstitution ofNK activity after ABSCT suggests
that the immunocompetent cells in the MNC preparations
might possess therapeutic potential. The fast recovery of
NK cells in patients receiving ABSCT may facilitate the
destruction of the remaining tumor cells that may be chemoresistant. NK or IL-2-activated NK (LAK) cells are potent
antitumor effector cells4’ and have been used for the treatment of cancer patients4* NK cells could be specifically
activated ex vivo by cytokines targeted at stimulation of T
lymphocyte subsets, eg, by IL-2, and utilized simultaneously
for immunotherapy. In conjunction with ABSCT, this strategy might ultimately improve the long-term outcome of
high-dose chemotherapy with stem cell support.
ACKNOWLEDGMENT
The authors thank Drs L. Beauregard-Zollinger and F. Romeyer
(Sandoz Canada Inc. Montreal, Canada) and Drs F. Philbrook and
A. Wagner (Lederle Oncology Division, Cyanamid Inc, Toronto,
Ontario, Canada) for their assistance in this study; Dr A. Mazzuchin
(Laurentian Hospital, Sudbury, Ontario, Canada) for his expertise
with preparation of the graphic materials; Drs A. Arnold (Hamilton
Regional Cancer Center, Hamilton, Ontario, Canada), C. Vergidis
(Thunder Bay Regional Cancer Center, Thunder Bay, Ontario, Canada), and J. Yau (Ottawa Regional Cancer Center, Ottawa, Canada)
for referral of their patients; and Barbara Vickers (UCSD Cancer
Center, San Diego, CA) for her assistance in the preparation of the
manuscript.
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