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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2024-2030
IMMUNOBIOLOGY
Cyclophosphamide induces type I interferon and augments the
number of CD44hi T lymphocytes in mice:
implications for strategies of chemoimmunotherapy of cancer
Giovanna Schiavoni,
Fabrizio Mattei,
Tiziana Di Pucchio,
Stefano M. Santini,
Laura Bracci,
Filippo Belardelli, and
Enrico Proietti
From the Laboratory of Virology, Istituto Superiore di Sanità,
Rome, Italy.
 |
Abstract |
In a previous study, we reported that a single injection of
cyclophosphamide (CTX) in tumor-bearing mice resulted in tumor eradication when the animals were subsequently injected with
tumor-sensitized lymphocytes. Notably, CTX acted by inducing bystander
effects on T cells, and the response to the combined CTX/adoptive
immunotherapy regimen was inhibited in mice treated with antibodies to
mouse interferon (IFN)- / . In the present study, we have
investigated whether CTX induced the expression of type I IFN, and we
have characterized the CTX effects on the phenotype of T cells in
normal mice. CTX injection resulted in an accumulation of type I IFN messenger RNA in the spleen of inoculated mice, at 24 to 48 hours, that
was associated with IFN detection in the majority of the animals. CTX
also enhanced the expression of the Ly-6C on spleen lymphocytes. This
enhancement was inhibited in mice treated with anti-type I IFN
antibodies. Moreover, CTX induced a long-lasting increase in in vivo
lymphocyte proliferation and in the percentage of
CD44hiCD4+ and CD44hiCD8+
T lymphocytes. These results demonstrate that
CTX is an inducer of type I IFN in vivo and enhances the number of T
cells exhibiting the CD44hi memory phenotype. Since type I
IFN has been recently recognized as the important cytokine for the in
vivo expansion and long-term survival of memory T cells, we suggest
that induction of this cytokine may explain at least part of the
immunomodulatory effects observed after CTX treatment. Finally, these
findings provide a new rationale for combined treatments with CTX and
adoptive immunotherapy in cancer patients.
(Blood. 2000;95:2024-2030)
© 2000 by The American Society of Hematology.
| |
Introduction |
Cyclophosphamide (CTX) is a widely used
chemotherapeutic agent in cancer therapy1 and in some
autoimmune diseases.2 Combined regimens with CTX and
immunotherapy are used in clinical trials with cancer
patients.3-6 However, the mechanisms of CTX action are not
fully understood. On the one hand, CTX can act as a conventional
anticancer drug by affecting the in vivo proliferation of tumor
cells.1 On the other hand, CTX can exhibit immunomodulatory effects, which may play an important role in the antitumor
response.7-20 In this regard, many studies had reported
that CTX can increase the efficacy of immunotherapeutic agents by
removing tumor-induced suppressor T cells.21-25 However,
the nature of these suppressor cells is still a matter of
debate.26
By using transplantable mouse tumor models, we have recently
reported that CTX can induce marked effects on T cells, which are
important for a successful tumor eradication in response to adoptive
immunotherapy.27 In particular, the results of an ensemble of experiments aimed at understanding the mechanisms underlying the
synergistic antitumor response observed in tumor-bearing mice injected
with CTX and tumor-sensitized lymphocytes have suggested that CTX acts
by means of bystander effects (possibly through production of T-cell
growth factors occurring during the rebound events after drug
administration) that may sustain the proliferation, survival, and
activity of the transferred
lymphocytes.27 In considering which CTX-induced
factor(s) can be important for explaining the effects on T cells and
the antitumor response observed in tumor models, we focused our
attention on type I interferon (IFN). In fact, the working hypothesis
that this cytokine could be induced by CTX was suggested by 2 major
considerations: (1) In our previous study,27 the
synergistic antitumor response induced by the combined treatment with
CTX and immune lymphocytes was abolished when mice were treated
with a potent preparation of antibodies to mouse IFN- /,27 suggesting that this cytokine was somehow
induced in our experimental system with tumor-bearing mice. (2) Recent reports have clearly indicated that type I IFN is the major factor responsible for the in vivo proliferation and long-term survival of
certain subsets of T cells (especially CD44hiCD8 T
lymphocytes) in response to viruses or other stimuli.28,29
All of this prompted us to investigate whether the injection of CTX in
normal mice could result in any induction of type I IFN and to
characterize the effects of CTX on T cells.
The results reported in this article demonstrate that CTX can induce
type I IFN expression, which may represent an important mediator of the
immunomodulatory effects of CTX, especially with regard to the
expansion and persistence of memory T cells.
| |
Materials and methods |
Mice and in vivo treatments
Six- to 8-week-old male DBA/2 mice were obtained from Charles River
Breeding Laboratories (Calco, Italy). All mice were treated in
accordance with the European Community Guidelines. For
bromodeoxyuridine (BrdU)-incorporation studies, mice were given
drinking water containing BrdU (Sigma Chemical, St Louis, MO) at
0.8 mg/mL, which was made fresh and
changed daily. CTX (Sigma) was dissolved in 0.15 mol/L NaCl (saline)
and filter sterilized, and 0.5 mL of freshly prepared solution was
injected intraperitoneally (CTX-treated mice).
Polyinosinic-polycytidylic acid (poly [I:C]) (Sigma) was dissolved in
saline at a concentration of 10 mg/mL. Frozen aliquots were thawed just
before each experiment, and 0.15 mg of poly (I:C) was injected
intraperitoneally in a volume of 0.15 mL of saline
solution. Control preparations consisted of saline unless otherwise stated.
Antibody to mouse interferon /
Sheep antibodies to mouse interferon / and normal sheep
immunoglobulin were a generous gift from Dr Ion Gresser (Villejuif, France). The origin of sheep antibodies to mouse interferon / (sheep no. 1) and normal sheep immunoglobulin, their purification, and
their assay have been previously described in detail.30,31 Antibody titer was 1.6 × 106 neutralizing units
against 8 interferon units; mice were injected with 0.2 mL of a 1:10
dilution on day  1, +2, and +4 with respect to
CTX administration.
Preparation of spleen and lymph node cells suspensions
Mice were killed and spleen and lymph nodes were removed
aseptically. Lymph node cells were pooled from cervical, axillary, inguinal, and mesenteric nodes. Tissues were gently disaggregated with
a tissue homogenizer for 3 minutes at room temperature in lysis buffer
(0.16 mol/L tromethamine [Tris]-buffered
NH4Cl). After erythrocyte lysis, cells
were washed in Rosewell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum (FCS), passed through
a cell strainer (Falcon 2350, Becton Dickinson, Orlando,
FL), and resuspended to approximately
1 × 107 viable cells/mL (determined by trypan blue
exclusion) in RPMI 1640 medium with 2% FCS.
Flow cytometry
Monoclonal antibodies (mAbs) used to stain cell surface antigens
were the following: biotinylated
anti-Ly-6C (104-2.1, mouse immunoglobulin [Ig]-G) (PharMingen, San Diego, CA), biotinylated anti-CD44 (IM7, rat IgG)
(PharMingen), phycoerythrin (PE)-conjugated anti-CD4
(GIBCO-BRL, Gaithersburg, MD), PE-conjugated anti-CD8 (GIBCO-BRL).
Bound biotinilated L antibodies were detected with red
670-streptavidin (GIBCO-BRL). After surface staining by conventional techniques,32 cells were washed, resuspended in cold
saline, and fixed by dropwise addition of cold 95% ethanol for 30 minutes on ice. For BrdU-incorporation studies, an intracellular
staining method33 was used. Briefly, the cells were washed
with PBS, then incubated with PBS containing 1% paraformaldehyde and
0.01% Tween 20 for 30 minutes. Cells were pelleted,
then incubated with 50 Kunitz units DNAse I (Sigma) in 0.15 mol/L NaCl,
4.2 mmol/L MgCl2, pH 5.0, for 10 minutes.
After washing, cells were incubated with fluorescein
isothiocyanate-conjugated anti-BrdU mAb (Becton Dickinson, Mountain
View, CA) and analyzed on FACsort flow cytometer (Becton Dickinson). A
total of 10 000 events per sample were collected. Erythrocytes, dead
cells, and tissue debris were excluded according to forward- and
side-scatter properties in order to gate only live lymphocyte populations.
Interferon titration
Biological activity of serum IFN was determined as described
elsewhere.34 One of the units, as expressed in the text, is the equivalent of 4 IFN reference units.
Detection of murine and interferon messenger RNAs in the
spleen by reverse transcriptase (RT)-polymerase chain reaction
(PCR)
At different times after treatments, mice were killed and their
spleens were immediately removed and directly homogenized with a tissue
homogenizer in 2mL RNAzol B (Bioteck, Houston, TX) in an ice bath.
Total RNA was then subjected to chloroform extraction and isopropanol
precipitation. RNA was resuspended and treated with ribonuclease-free
DNAse (Boehringer Mannheim, Germany), further purified, and then
quantitated by UV absorbance at 260 nm. One microgram of total RNA was
incubated for 5 minutes with oligo-(dt) 12-18 (Pharmacia, Uppsala,
Sweden) at 75°C, cooled at room temperature, and
reverse-transcribed by 200 U of Moloney murine leukemia virus reverse
transcriptase (Bethesda Research Laboratories, Bethesda, MD) for 1 hour
at 37°C in a final volume of 20 µL. We
amplified 2 µL of complementary DNA
in a final volume of 20 µL (10 mmol/L Tris-HCL, pH 8.3, 50 mmol/L
KCl, 1.5 mmol/L MgCl2, 0.01% gelatin, 200 µmol/L deoxyribonucleoside triphosphate
(dNTP), and 10 pmol of each primer) using a Perkin
Elmer Thermal Cycler (Perkin Elmer, Norwalk, CT).
Samples were heated at 94°C for 5 minutes, and each cycle was
performed as follows: 40 seconds denaturation at 94°C, 40 seconds
annealing at 62°C, and 1 minute extension at 72°C. At the end,
samples were further incubated at 72°C for an additional 10 minutes. Table 1 reports the cytokine
primer sequences, the number of amplification cycles, and the size of
the fragment amplified in this study. For reaction product
visualization, 10 µL of each PCR product was run on
1% agarose gel in 0.5 × tris borate dectrophoresis (TBE)
buffer. As
molecular weight markers, 1 µg of 132 bp DNA ladder (GIBCO-BRL) were
run in parallel. As positive controls for IFN- or IFN- RT-PCR, we
used messenger RNA (mRNA) extracted from
IFN-1-transduced Cl-11 cells35 and IFN
-transduced TSA/Cl-A4 cells,36 respectively. PCR
products were visualized by means of ethidium bromide staining and UV
transillumination. After electrophoresis, the relative density of mRNA
bands stained with ethidium bromide was determined by LKB 2202 Ultrascan densitometer (Pharmacia, LKB, Uppsala,
Sweden). Messenger RNA transcripts were expressed in absorbance units.
Statistical analyses
Data were analyzed by Student t test.
| |
Results |
Detection of interferon activity in sera of mice treated with CTX
In a first set of experiments, mice were injected
intraperitoneally with 2 doses of CTX (83 or 150 mg/kg body weight), with saline or poly (I:C)
(positive control for IFN production). Sera were collected at different
times after injection and tested for IFN activity by a standard
biological assay on L929 cells. Table 2
shows the IFN activity detected in sera from individual mice at 12, 24, and 48 hours after injection. No IFN activity was detected in sera from
saline-treated control mice. In contrast, sera from poly (I:C)-treated
mice exhibited high levels of IFN activity at 12 hours. Considerable
levels of IFN (64 U/mL) were also found at 24 hours after poly (I:C)
injection, while no IFN activity was detected at the subsequent time
point. None of the 6 CTX-treated mice showed any presence of serum IFN
at 12 hours after injection. However, at 24 hours, detectable IFN
activity was found in 3 out of the 6 CTX-treated mice. At 48 hours, IFN
activity (24 to 48 U/mL) was detected in the serum of 4 out of the 6 CTX-treated mice, with no significant difference with respect to the
dose of CTX injected. At subsequent times, no IFN activity could be found in the sera of CTX-treated mice (data not shown).
Kinetics of accumulation of IFN- and IFN- messenger RNAs in
the spleen of mice treated with cyclophosphamide
We then evaluated the levels of type I IFN mRNA expression in
splenocytes harvested at different times after CTX injection. Thus,
total RNA was extracted from spleen cells of mice treated with saline,
CTX (83 or 150 mg/kg), or poly (I:C) and processed for
RT-PCR by using 2 sets of primers specific for mouse
IFN-1-2 and IFN-, and for actin as a control
(Figure 1). Injection of Poly (I:C) caused
a transient but marked increase in the expression of both mRNAs for
IFN- and IFN-, compared with untreated control mice (Figure 1A).
In CTX-treated mice, the peak of mRNA expression for both IFN- and
IFN- was almost comparable to that obtained with poly (I:C), but
the kinetics was quite different. As shown by densitometric analyses
(Figure 1B and 1C), IFN- and IFN- mRNA expression in spleen cells
from mice injected with poly (I:C) reached its maximum level 6 to 12 hours after treatment. In CTX-treated mice, the maximal expression for
both IFN mRNAs peaked between 12 and 24 hours. The apparent delay in
the induction of IFN mRNA expression in mice injected with the higher
dose of CTX (especially observed for IFN-) may be somehow dependent
on a CTX-induced toxicity. In this regard, there was a consistent
decrease in the spleen weight in CTX-treated mice; this appeared more
marked in animals treated with the higher dose of CTX. (Figure 1D).
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| Fig 1.
RT-PCR analysis of IFN- and IFN- mRNA levels in the
spleen of mice treated with CTX or poly (I:C).
Seven-week-old DBA/2 mice were treated
intraperitoneally with CTX (83 mg/kg,  , and 150 mg/kg,  ); poly (I:C) (0.15 mg/mouse),  ; or
saline,  . After 6, 12, 24, and 48 hours, mice were killed, and total
spleen RNA was isolated and assayed for the presence of IFN- and
IFN- mRNAs by RT-PCR as described in "Materials and methods."
Reaction products were run on 1% agarose gel in the presence of
molecular markers (not shown). Values represent the mean ± SE of 3 mice per group. (A) Each band corresponds to a single
mouse spleen. Densitometric values of ethidium bromide-stained bands,
expressed as absorbance units (OD) and normalized to the control
values, are reported, (B) for IFN- mRNAs and (C) for IFN- mRNAs.
(D) Spleen weight of mice treated as above was measured 6, 12, 24, and
48 hours after treatment.
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CTX injection results in the up-regulation of Ly-6C expression and
bromodeoxyuridine incorporation in spleen lymphocytes
Ly-6C is a lymphocyte activation and differentiation antigen
normally present on minor subsets of mature T cells, monocytes, macrophages, and endothelial cells. Previous studies have demonstrated that IFN-/ is a cytokine capable of specifically enhancing Ly-6C expression.37 By cytofluorimetric analyses, we then
evaluated the percentage of Ly-6C positive T cells with respect to the
number of total T lymphocytes in the spleens of mice treated with a
single injection of CTX (83 mg/kg) 3, 6, 9, and 15 days before sacrifice. In the same experiment, spleen lymphocytes were
also analyzed for BrdU uptake. As shown in Figure
2, there was a significant increase of
Ly-6C positive cells in CTX-treated mice in comparison with controls at
6 days after CTX treatment. Cell proliferation data, obtained by the
BrdU-labeling technique, showed a substantial decrease in the
percentage of proliferating cells at day 3 after CTX administration,
followed by a significant and long-lasting increase at the subsequent
time points. In a subsequent experiment, illustrated in Figure
3, we compared the Ly-6C expression in
spleen lymphocytes at various times after treatment of mice with either poly (I:C) or CTX. Ly-6C expression was maximal in splenic lymphocytes 6 days after CTX treatment, whereas poly (I:C) induced a more intense
but more transient Ly-6C antigen enhancement that peaked at day 3.
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| Fig 2.
Effects of CTX injection on the expression of Ly-6C
antigen and on BrdU incorporation in spleen lymphocytes at different
times after treatment.
Seven- to 8-week-old DBA/2 mice were placed on BrdU water from day 0 to
sacrifice. Mice were injected intraperitoneally with
CTX (83 mg/kg) (dotted bars) or saline (white bars) at
day 0. At different time intervals, spleens were taken and
disaggregated. Cells were stained for Ly-6C; this was followed, after
fixation, by nuclear staining for BrdU incorporation. Cell fluorescence
was evaluated by fluorescence-activated cell sorter (FACS). There were
3 mice per group. The data show the mean (± SE) of the percentage
of Ly-6Chi cells (upper panel) or BrdUhi cells
(lower panel) with respect to the total number of spleen lymphocytes.
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| Fig 3.
Effect of CTX versus poly (I:C) injection on Ly-6C
expression in spleen lymphocytes at different times after treatment.
Seven- to 8-week-old DBA/2 mice were injected
intraperitoneally with CTX (83 mg/kg)
(dotted bars), poly (I:C) (0.15 mg/mouse) (striped bars), or saline
(white bars). At different time intervals, spleens
were taken and cells were stained for Ly-6C expression and processed
for FACS analysis. There were 3 mice per group. The data show the mean
(± SE) of the percentage of Ly-6Chi cells with respect
to the number of total spleen lymphocytes. *P  .05 versus
controls; **P .001 versus controls.
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As shown in Figure 4, injection of mice
with a potent preparation of anti-IFN-/ antibodies resulted in a
significant inhibition of the CTX-induced up-regulation of Ly-6C
expression on spleen lymphocytes, suggesting that CTX-induced type I
IFN was involved in enhancing the expression of this antigen.
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| Fig 4.
Effects of injection of anti-IFN-/ antibodies on
the CTX-induced up-regulation of Ly-6C expression in spleen lymphocytes
from mice.
Seven- to 8-week-old DBA/2 mice were injected
intraperitoneally with CTX (83 mg/kg)
(dotted bars) or saline (white bars) at day 0. One day
before and 2 and 4 days after CTX treatment, some mice were also
injected intraperitoneally with 0.2 mL of an
anti-murine IFN-/ antibody preparation (160 000
IFN-neutralizing units/mouse/injection) (striped bars). At different
time intervals, spleens were collected and cells were stained for Ly-6C
expression and processed for FACS analysis. There were 3 mice per
group. The data show the mean (± SE) of the percentage of
Ly-6Chi cells with respect to the total number of spleen
lymphocytes.
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Increase of the number of CD44hi T lymphocytes
in the spleen and lymph nodes of CTX-treated mice
We then evaluated whether injection of CTX could affect the number
of T cells in the spleen and lymph nodes at different times after
injection. In general, no statistically significant differences were
observed in the total number of CD4 or CD8 T lymphocytes recovered from
the spleen or pooled lymph nodes of CTX-treated mice with respect to
control injected mice on days 6 and 10, with the exception of a slight
increase in the number of CD4 T cells detected in the spleen at 6 days
after CTX injection (data not shown). Spleen or lymph node cells from
mice treated with a single injection of CTX (83 mg/kg), poly (I:C), or saline were also labeled with
biotinylated anti-CD44 and PE-conjugated anti-CD4 or
anti-CD8 antibodies and assessed by cytofluorimetric analysis. As shown in Figure 5, there was a marked increase in
the percentage of CD44hi T lymphocytes in both the spleen
and the lymph nodes of CTX-injected mice, compared with saline-injected
control animals. This increase reached its maximum level 10 days after
treatment for both CD4+ and CD8+ T-cell
subsets. In poly (I:C)-injected mice, there was also a marked increase
in CD44hi cell percentage, but the kinetics of this
increase appeared to be slightly different in spleen cells as compared
with CTX-treated animals.
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| Fig 5.
Increase in the percentage of CD44hi T
lymphocytes in the CD4+ and CD8+ T cells from spleens and lymph nodes
of mice treated with CTX or poly (I:C).
Seven- to 8-week-old DBA/2 mice were injected
intraperitoneally with CTX (83 mg/kg)
(dotted bars), poly (I:C) (0.15 mg/mouse) (striped bars), or saline
(white bars). At different time intervals, spleens and a pool of lymph
nodes were taken and disaggregated. Cells were stained for CD44 and for
CD4 or CD8 surface antigen expression before being processed for
cytofluorimetric analysis. There were 3 mice per group. The data show
the mean (± SE) value of the percentage of CD44hi cells
on CD4 (left panels) or CD8 (right panels) lymphocytes, in the spleen
(upper panels) or in the pooled lymph nodes (lower panels).
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| |
Discussion |
In this study, we have provided the first evidence indicating that
CTX is an inducer of type I IFN expression in mice. The induction of
type I IFN by CTX was demonstrated not only by the presence of
biologically active IFN in the serum of the injected mice at 24 to
48 hours, but also by a progressive accumulation of mRNAs for IFN-
and IFN- in the spleens, starting at 6 hours and lasting for at
least 48 hours after injection. The comparison of the kinetics of
induction by CTX with that observed after injection of poly (I:C) (a
typical strong inducer of type I IFN in mice) revealed that the peak of
IFN- mRNA induction by poly (I:C) occurred earlier (at 6 hours),
but appeared to decrease more rapidly than that observed after CTX
injection. As for IFN- mRNA induction, the kinetics of accumulation
of IFN- mRNA in mice injected with poly (I:C) was similar to that
observed in mice treated with the lower dose of CTX (83 mg/kg), whereas
a certain delay in the induction kinetics was detected in mice injected
with the higher dose of CTX (150 mg/kg). On the whole, these results
indicate that CTX is an inducer of type I IFN at a time early after
injection, before drug treatment might result in a strong
myelosuppression27(Figure7A) or in a detectable decrease in
the spleen weight (Figure 1D), substantially before the rebound
phenomenon, which starts 5 to 6 days after CTX injection.27
Thus, although the mechanisms of type I IFN induction by CTX remain
unclear, our results suggest that the type I IFN induction is a direct
effect of the chemotherapeutic agent on some mouse cells and does not
represent a secondary response to drug-induced myelosuppression or to
cell-stimulation events occurring during the rebound phase. This
CTX-induced IFN production appears to be capable of directly affecting
some lymphocyte functions. In fact, CTX induced an up-regulation in the
expression of Ly-6C antigen (a marker known to be specifically induced
by type I IFN), and this induction was inhibited by injecting the mice
with a potent preparation of anti-IFN-/ antibodies. Of interest,
CTX was also capable of inducing an enhancement of the percentage of
proliferating (ie, BrdU+) spleen lymphocytes as well as of
modulating the phenotype of T cells, especially by enhancing the number
of CD4+ and CD8+ T cells exhibiting a memory
(CD44hi) phenotype in both the spleens and the lymph nodes
of the injected mice.
The notion that CTX can induce release of cytokines and growth factors
during the rebound events after drug-induced immunosuppression is well
documented.38,39 Some studies have recently suggested that
CTX can induce a pattern shift of cytokines from TH2 to
TH1 in tumor models several days after
injection.40 However, our finding that CTX can induce an
early and long-lasting expression of type I IFN suggests that this
event can be directly important in mediating some of the
immunomodulatory effects of CTX observed in a number of experimental systems.
CTX is a currently used drug in cancer therapy. Although CTX is
generally considered to be a typical chemotherapeutic agent capable of
directly inhibiting tumor cell proliferation, many studies have shown
that CTX can also induce multiple immunomodulatory effects.7-20 In mouse tumor models, some groups had
suggested that CTX can act by inhibiting T suppressor
cells.21-25 However, the nature of these suppressor cells
assumed to be inhibited by CTX has remained elusive.26 In a
recent study, in which we have investigated the mechanisms underlying
the impressive antitumor response in tumor-bearing mice subjected to a
single injection of CTX followed by adoptive
immunotherapy,27 we provided evidence suggesting
that CTX did not act by inhibiting T suppressor cells, but by rendering
the mouse host capable of allowing survival and proliferation of the
transferred immune lymphocytes, probably as a result of CTX-induced
cytokines affecting T-cell functions.27,41 Notably, type I
IFN was important in the response to the combined CTX/adoptive
immunotherapy regimen, since the antitumor activity was abolished by
injection of antibodies to IFN-/.27 Other groups have
recently provided additional examples of CTX effects on T cells. For
instance, Li and coworkers have recently reported that CTX given after
active specific immunization augments antitumor immunity by modulation
of TH1 commitment of CD4 T cells.42 Likewise, Apostolopoulos et al43 have reported a CTX-induced
enhancement of cytotoxic lymphocyte (CTL) precursors'
frequency in mice immunized with mucin 1-mannan fusion protein. In
this regard, it is of interest to note that recent studies have
revealed that type I IFN is an important cytokine in the regulation of
T-cell response.44 Early studies had shown that type I IFN
can inhibit T suppressor cells in mice.45 Type I IFN is
involved in the polarization toward a TH1 type of immune
response and has been shown to augment the generation and activity of
CTL.46,47 Of interest, recent studies have shown that type
I IFN can specifically induce in mice the proliferation and persistence
of CD8+ T lymphocytes exhibiting the memory
(CD44hi) phenotype.28,48 In light of all this,
we may assume that the induction of type I IFN by CTX is important in
inducing proliferation and persistence of T cells (especially CD8 T
cells) and a TH1 type of immune response. There are some
intriguing similarities in the effects induced by CTX and type I IFN,
which may suggest, in light of the results reported in this article,
that an induction of this cytokine may be involved in some of the
responses observed after CTX treatment. CTX accelerates diabetes
progression in non obese diabetic mice inducing a
polarization toward a TH1 type of immune
response49; notably, it has been postulated that type I IFN
plays a role in diabetes as well as in the pathogenesis of other
autoimmune diseases.50,51 In a different context adoptive immunotherapy protocols in tumor modelsit is worth mentioning that
the antitumor efficacy of transferred lymphocytes has been shown to be
markedly enhanced by CTX18,19 as well as by type I
IFN.52 In the light of the collection of recent data
showing the importance of type I IFN in the regulation of the T-cell
turnover in mice,48,53,54 we suggest that, although CTX can
induce several cytokines capable of affecting the antitumor activity of
the transferred lymphocytes, type I IFN is the major factor that may
allow the proliferation and long-term survival of at least some subsets
of the injected immune cells (especially CD8+ T cells
exhibiting the memory phenotype) when adoptive immunotherapy is
performed after CTX injection. Finally, we believe that the finding
that type I IFN, a cytokine endowed with well-recognized antitumor
activity, is induced early after CTX injection may provide new insight
for the definition of more selective strategies for the combined
treatment with CTX and adoptive immunotherapy in cancer patients. In
our previous work, we suggested that in order to attain the optimal
effects of adoptive immunotherapy, it should be performed following
chemotherapy at a time point when immune function is
rebounding.27 Notably, we found that the
antitumor response was optimal when immune cells were transferred in
tumor-bearing mice approximately 6 hours after a single CTX injection,
when an induction of type I IFN mRNA expression is
observed in the spleen (Figure 1). We suggest, therefore, that adoptive
immunotherapy should be performed shortly after chemotherapy (at a time
when type I IFN is initially expressed in some lymphoid tissues),
before the rebound overshoot is observed.
| |
Acknowledgments |
We are grateful to Dr Ion Gresser (Paris, France) for providing us with
the sheep antibody to mouse IFN-/ and for the helpful discussion
and suggestions. We thank Ms C. Gasparrini for secretarial aid.
| |
Footnotes |
Submitted October 4, 1999; accepted October 26, 1999.
Supported in part by grants from the Associazione Italiana
Ricerca sul Cancro (AIRC, Milan) and from the Italian Ministry of
Health (Progetto di Ricerca sull'AIDS 1998, 30B/I).
Reprints: Enrico Proietti, Laboratory of Virology, Istituto
Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy;
e-mail: Proietti{at}virus1.net.iss.it.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction