ARTICLE
Auteur(s) : S Fiorentino1,4, M
Chopin1, H Dastot1, N Boissel2, M
Reboul1, L Legrès1, A Janin1, P
Aplan3, F Sigaux1, Armelle
Regnault1
1Unité U728 INSERM-Laboratoire de Pathologie
Université Paris VII, Hôpital Saint-Louis – Institut Universitaire
d’Hématologie, 1 avenue Claude-Vellefaux, 75475 Paris Cedex 10,
France
2Unité INSERM U396, Institut Universitaire
d’Hématologie, Hôpital Saint-Louis, Paris, France
3NIH/NCI/Genetics Branch, Navy 8, Room 5101, 8901
Wisconsin Ave. Bethesda, MD 20889-5105, USA
4Present address: Universidad Javeriana. Departamento de
Microbiologia, Bogota, Colombia
In the last decades, therapeutic progress has dramatically improved
the outcome of children with ALL. Fewer benefits were observed in
adults [1]. The oncogene TAL1 is over-expressed by leukemia cells
in about 50% of children and adults with T-ALL. In this disease,
TAL1 over-expression is associated with an unfavourable outcome;
the ectopic expression of the LMO1 oncogene is also found in a high
percentage of TAL1-expressing cases [2]. The response of patients
to chemotherapy has been identified as the major prognostic factor.
Patients with high, persistent residual disease after the first
course of chemotherapy have a high risk of relapse [3]. For these
at-risk patients, there is hope that immunotherapy could elicit CTL
responses capable of eliminating the residual leukemia cells after
chemotherapy. The importance of T cell-mediated immune
responses for eradication of residual disease is documented by the
GVL effect that leads to lower relapse rates in AML patients who
received allogeneic stem cell transplantation (ASCT) [4]. The fact
that relapses are more frequent after T cell graft depletion
further underscores the essential role of the immune system in
controlling residual CML cells. This allogeneic effect is obviously
weaker in ALL than in myeloid malignancies. Limited benefits of
donor lymphocyte infusions were observed in patients with recurrent
ALL after ASCT [5]. However, the relationship between these
disparities and the poor immunogenicity of ALL blasts remains
speculative.One major obstacle to tumor-specific immunotherapy is
the low efficiency of many cell types in antigen presentation, but
the systematic comparison of efficiency obtained by different
antigen presentation systems within a unique tumor model has not
been performed. It is believed that dendritic cells are the most
efficient antigen-presenting cells, but their ability to generate
anti-tumor immune responses in vivo has been documented in only a
few therapeutic models. When tumor-specific antigens are not known,
DC can be efficiently loaded with exosomes, tumor lysates,
apoptotic or necrotic bodies, or with synthetic MHC class
I-restricted peptides i.e. tumor-associated antigen-derived
peptides. Many of these cancer antigen sources have been shown to
induce tumor-specific immune responses [6-10].A second obstacle to
the generation of efficient tumor-specific immune response is the
possibility of T cell tolerance towards tumor antigens. Of
particular interest in this regard are the intensively studied
sub-populations of CD4+ T cells, which have an inhibitory role
in the control of autoreactive CD8+ T cells [11]. The tumor
regression induced by injection of anti-CD25 antibodies has been
documented [12, 13]. However, in these studies, the effect of CD25
depletion was only analyzed over a short period of 30 days. On the
other hand, the role of CD4+CD25+ suppressive T cells in
limiting autoreactive immune responses during anti-tumor therapy
has been shown in mice, but the effect of CD25 depletion was
studied in experiments with GM-CSF-secreting tumor cells as
antigen-presenting cells (APC), not with DC [14]. Recent results
have shown that it is important to separate the role of CD4+ CD25+
suppressive T cells and of CD4+ CD25- helper T cells
because CD4 CD25+ T cells have been shown to suppress
anti-tumor CTL responses and to permit the progressive growth of
tumor cells [15, 16].Efficient T-cell activation also depends on
various costimulatory molecules on the surface of DC that enhance
interactions with the corresponding molecules on T cells. The
most efficient costimulatory molecule expressed by T cells is
CD28, which shares its ligand with the “down-regulator” of
T cell responsiveness, the CTL-associated antigen (CTLA-4) [17
1198]. CTLA-4 is expressed by antigen-presenting cells such as DC
[18]. Anti-CTLA-4 antibodies have been successfully used for murine
melanoma therapy [19]. However, the combined effect of CD25
depletion and CTLA-4 blockade was investigated in this paper with
GM-CSF-secreting tumor cells, as opposed to potent APC such as DC
[14].Since no efficient immunotherapy protocol has been published
for T-ALL up to now, we first defined an immunotherapy protocol
based upon injection of DC loaded with leukemia necrotic bodies,
and demonstrated that it elicited a partial elimination of T-ALL
cells. We then compared this DC-based immunotherapy protocol to a
combined treatment associating tumor antigen-pulsed DC with
anti-CD25 depletion and CTLA-4 blockade. We deliberately selected
this mAb combination since it had been shown to be the most
efficient in association, in the B16 melanoma mouse model [14]. We
show here that the disruption of those regulatory mechanisms,
synergized with DC-based immunotherapy for eliminating poorly
immunogenic T-ALL.
Materials and methods
Mice
6-week-old (C57BL/6xC3H) F1 mice were purchased from Harlan,
France. TAL1 and LMO1 transgenic mice were generated as described
[20]. All animal manipulations and housing were in accordance with
our Research Institute Animal Care Committee guidelines.
Murine model of T-ALL: in vivo adoptive transfer of leukemia
cells; leukemia challenge
For short term experiments, 2 x 106 T-ALL
72C18 cells, a subcloned line from a previously described
leukemia tumor clone [21], were adoptively transferred by i.v.
injection into the retro-orbital sinus of normal (C57BL/6xC3H) F1
mice. We verified that leukemia cells were detected in vivo at the
time of DC-based immunotherapy by RQ-PCR with a TCRβ
clonotype-specific probe. T-ALL 72C18 cells that expressed Vβ9 TCR
were detected by quantitative PCR in the lungs, bone marrow and
kidney, 3 and 6 days after injection.
For combined treatment experiments, we transferred T-ALL lines
expressing undetectable levels of CD25 to avoid interference with
the in vivo depletion protocol. Two T-ALL lines H39 and H535, fresh
ex vivo leukemia cells from leukemic transgenic mice, were
selected. They were CD4+8+-,
CD25-- CD44Low-, CD3+-,
CD95+- and MHC class I- low. They were used respectively
for primary adoptive leukemia transfer (2 x 106 H39
cells injected i.v. at day 0), and challenge injections to
demonstrate anti-leukemia long term memory (8 x 106
H535 cells injected i.v. at 32 weeks). When necessary, they were
maintained for less than 24h in RPMI supplemented with 10% FCS, 2mM
glutamine, penicillin (0.1 U/mL), and streptomycin
(0.1 mg/mL), before the in vivo injections.
RQ-PCR analysis
BM and kidney samples were obtained after euthanasia of the mice.
DNA was extracted as previously described. Primer and probe were
designed using the Primer Express 1.0 software (Applied Biosystems,
Foster City CA, USA).
Forward Vβ9 primer sequence: 5’-TACATTGGCTCTGCAGGCCTA-3’
Reverse Jβ2.5 primer sequence: 5’-GAGTGCCTGGCCCAAAGTAC-3’
Clonotypic probe sequence:
5’-Fam-TGTGCTACGAGTAGAGGGACAGGGGGCCAA-Tamra-3’
Forward GAPDH primer sequence: 5’-GGGAAGCCCATCACCATCTT-3’
Reverse GAPDH primer sequence: 5’-GCCTTCTCCATGGTGGTGAA-3’
GAPDH probe sequence: 5’-CAGGAGCGAGACCCCACTAACATCAAATG-3’
RQ-PCR was performed using standard procedures. Efficiency and
specificity of the Vβ9-Jβ2.5 RQ-PCR system, was assessed on
diagnostic DNA (50 ng/μL) serially diluted from
10-1 to 10-5 into spleen cell DNA.
Calibration curves were performed using 10-fold serial dilutions of
diagnostic DNA into organ-related DNA. No-template controls
(H2O) as well as non-amplification controls (irrelevant
leukemia cell line DNA) were included in each assay. Cell numbers
were calculated using the ratio 670 ng of DNA for
105 cells.
Biological reagents
Antibodies for CD25 T cell depletion: PC61 (anti-CD25)
and 4F10 (anti-CTLA4) hybridomas were kindly provided by Dr A.
Bandeira (Pasteur Institute, Paris, France) and PC61 and 4F10 mAbs
were purified from culture supernatants with Econopack 10 DG
(BIORAD, Hercules, CA, USA). 400μg of PC61 mAb were injected i.p. 5
days before tumor cell injection. 2x105 H39 lymph node
leukemia cells were injected i.v. into F1 mice at day 0; 100 μg
4F10 anti-CTLA4 mAb were injected i.p. at days 3 and 6 post-tumor
injection.
GM-CSF-rich supernatants were obtained from cultures of the
GM-CSF-transfected J558 cell line provided by D. Gray (London, UK)
[22]. Cells were maintained in IMDM (Sigma, St Louis, MI, USA)
supplemented with 10 % heat-inactivated foetal calf serum
(Biowest, Nuaillé, France), 2 mM glutamine, penicillin
(0.1 U/mL), and streptomycin (0.1 mg/mL) (Invitrogen,
Carlsbad, CA, USA).
Immunophenotype of fresh murine T-ALL. CD4, CD8, CD3,
CD25, CD44, CD95 antibodies were purchased from BD Biosciences (San
Diego, CA, USA). Phenotype analyses were performed on
105 cells using APC-, PE-, FITC-conjugated monoclonal
antibodies.
Generation of dendritic cells (DC) in vitro, leukemia
antigens preparation, and loading DC.
Murine femur, bone marrow cells were plated in untreated plastic
Petri dishes at 4 x 105/mL in IMDM, supplemented
with 10 % FCS and 30 % of GM-CSF-transfected J558 cell
culture supernatant for at least 18 days before use. The activation
capacity of each DC preparation was tested as previously described
[23].
Loading DC with leukemia necrotic bodies was as follow: necrotic
bodies were prepared by freezing cell suspensions in liquid
nitrogen followed by thawing at 37°C. After 4 freeze-thaw cycles,
necrotic bodies were centrifuged at 1500 rpm for 10 minutes and
supernatants were sterilized using a 0.22 μm filter(Millipore,
Billerica, MA, USA).
DC loading was achieved by incubating in vitro-generated DC with
necrotic body supernatants in a 1:1 cell number equivalent ratio
for 20 hours [24].
Maturation of loaded DC was induced by incubation with
lipopolysaccharide (LPS) (100 ng/mL) (Sigma) 12 hours before
injection. Maturation by LPS induced a strong activation of the Th1
response by DC with secretion of IL2, IL12 and INFγ [25]. Before
injection, matured, loaded DC were washed, and 2
x 105 cells, resuspended in 100 μL of
serum-free RPMI, were injected subcutanously in the flank of each
mouce.
For anti-CD25 depletion experiments, pulsed DC were injected at
day 3 and 6 post-tumor injection.
Th1/Th2 cytokine secretion levels in immunized animals
Each serum was tested with the BD Mouse th1/Th2 Cytokine CBA Kit
(BD Biosciences) as recommended.
Results
Description of our murine model of T-ALL, obtained by adoptive
transfer of leukemia cells, in normal mice in vivo
One of us had previously developed a transgenic mouse model that
demonstrated the synergic oncogenic role of the TAL1 and LMO1 genes
[20]. After 4 months, 100% of the transgenic mice developed clonal
T cell tumors, with diverse phenotypes that represent all
stages of thymocyte development. However, since very little is
known about a possible role of these two oncogenes in mature
T cell functions in transgenic animals, we decided to evaluate
our immuno-intervention protocols using a murine model of T-ALL
obtained by adoptive transfer of leukemia cells in normal mice.
T-ALL cells from TAL1 x LMO1 transgenic animals were
transferred into immunocompetent, syngeneic, immuno-competent
(B6xC3H) F1 mice. To determine the invasion pattern in these mice,
hyperleukocytosis (> 10 x 106 cell/mL)
was measured in the blood of mice after 3 weeks (( figure 1 )A). Leukemia
cells were observed in many locations, including lymph nodes, brain
and spleen (( figure
1 )B-D). The results showed that our leukemia transfer
model reproduced the invasion pattern seen in double transgenic
mice, which in turn is very similar to the invasion pattern
observed in patients.
Injections of DC loaded with leukemia-derived antigens alone
induced a partial regression of T-ALL as evaluated at 30 days
Having tested various protocols with different sources of antigens
for DC loading and co-stimulatory signals, we selected the most
efficient protocol against T-ALL (not shown), which was based on DC
loaded with leukemia necrotic bodies (LNB) and then incubated with
LPS. Indeed, maturation by LPS induced a DC activation that ensured
a better activation of Th1 response by DC, with secretion of IL2,
IL12 and INF-γ [25].
We generated immature DC that retained their phenotype, their
antigen internalisation capacity and their sensitivity to
activation, for about two months in culture [23]; 48h after loading
and LPS treatment, DC-LNB maturation was revealed by their high
levels of CD40 molecule expression, the ligand of the costimulatory
molecule CD40L that is required for cytotoxic T cell
generation [26] (( figure 2 )A). We also
observed increased expressions of MHC class II, CD80 and CD86
molecules (not shown). Antigen-loaded, LPS-matured DC were injected
at days +3 and +6 post-tumor injection. LPS-activated unloaded DC
were injected as a negative control.
Our first experiments monitored short-term leukemia cell
invasion in recipient animals using RQ-PCR with a T-ALL
cell-specific Vβ-Dβ-Jβ probe. We first injected cells of the T-ALL
clone 72C18, and then treated the animals with two injections of
the immunotherapeutic DC at days +3 and +6. Then, we measured
residual disease one month later. 72C18 T-ALL cells expressed CD3,
CD4 and CD8 molecules, and Vβ9-Jβ2.5 gene segments. Its clonality
was verified by RT-PCR for the 23Vβ in Vβ-Cβ PCR, followed by
direct sequencing of the PCR products (not shown). Since there were
no residual leukemia cells circulating in the blood, their
detection was performed using RQ-PCR and the DNA from the bone
marrow and from a cryptic site, the kidneys.
We observed that the immunotherapeutic protocol with two
injections of DC-LNB induced partial regression of T-ALL, with
1 % of leukemia cells, or near complete regression with less
than 0.01 % of leukemia cells,. The overall anti-leukemia
efficiency was one or two logs better in the bone marrow than in a
cryptic site such as the kidneys (( figure 2 )B). Consistent
results were obtained in duplicate evaluation of kidney and bone
marrow samples (not shown). In view of these encouraging, but only
partially successful results, we set up a more efficient
immunotherapy protocol to induce long term survival.
Disruption of the CTL-A4 pathway and depletion of CD25+
suppressive T cells synergized with DC-based therapy in the
long term cure of T-ALL
We attempted to improve the efficiency of the protocol by combining
anti-CD25 depletion and blockade of CTLA-4 with DC-LNB injections
(combined treatment) and compared its efficiency to DC-LNB
treatment alone. To perform this experiment, we used as leukemia,
cells the T-ALL H39 cell line, which does not expressed significant
levels of CD25 molecule at the cell surface. Also, CD25 depletion
was carried out before leukemia cell injection to avoid tumor cell
depletion. We also randomly examined the efficacy of the CD25 cell
depletion protocol. The number of CD4+CD25+ cells was measured by
flow-cytometry in the blood of 6 out of 38 mice that had received
CD25-depleting antibody, 5 days before tumor injection. The
depletion was not total, but there was a five-fold reduction in the
CD4+CD25+ T cell population in the blood (( figure 3 )A).
The combined treatment was as follows: five days after the
CD25-depleting antibody injection, T-ALL H39 leukemia cells were
adoptively transferred in CD25-depleted recipient mice, and then
DC-LNB were injected at days +3 and +10 post-tumor injection.
CTLA-4 antibodies were injected at day 3 with DC-LNB, and alone at
day +6. Control groups of animal received either PBS injection
alone, or unloaded LPS-matured DC (DC-LPS), or antigen-loaded and
LPS-matured DC alone (DC-LNB-LPS) or treatment with depleting
antibodies only (anti-CD25+ anti-CTLA-4),
We observed that depletion of CD25 T cells, combined with
blockade of CTLA-4 without DC injection, resulted in a small
increase in the number of surviving mice (( figure 3 )B), indicating
either a marginal effect of antibody treatment on the growth of
T-ALL cells, or that treatment with these two antibodies improved
the natural immune response of these mice. DC-LNB injections alone
resulted in survival of 50% of the mice at 13 weeks, then of 30% of
the mice at 20 weeks post-injection. This result indicated that
although DC-LNB could induce a therapeutic effect at one month
post-injection (( figure
2 )B), this effect did not last long enough to ensure
sufficient leukemia cell elimination (p = 0.09 at 20 weeks). Most
interestingly, combined treatment, associating DC-LNB and
mAb-induced disruption of suppressive pathways, resulted in a
striking increase in treatment efficacy, with 65% of mice capable
of rejecting the T-ALL cells and surviving over 32 weeks (p = 0.003
and p = 0.0006) (( figure 3 )B). This suggests
that combined treatment, significantly improved the overall,
long-term survival of mice when compared to DC-LNB-LPS treatment (p
= 0.03) and when compared to unloaded DC or to antibodies (p =
0.0006).
Our results therefore clearly show that efficient immunotherapy
against T-ALL can be obtained by the disruption of immunoregulatory
mechanisms that synergize with DC-based immunotherapy.
Long-lasting, specific resistance of mice, cured of leukemia by
combined treatment, to high leukemia burden: generation of specific
anti-leukemia memory
We have shown in a previous study that mice, surviving after
immunotherapy, could be challenged with a high burden of live tumor
cells, and reject them, if they had developed a specific T or B
cell-mediated response [10]. In the present study, a selected group
of 6/12 mice, previously cured of leukemia following combined
treatment, and 5 control mice, were challenged with a high dose of
T-ALL H535 T cells (8 million) at 32 weeks. The group of
combined treatment-cured mice was followed for signs of leukemia
for about 60 additional weeks. We observed that, 6/6 of those mice
survived the leukemia challenge, while no control mice survived,
euthanasia or death occurring by 42 weeks in the 5 control mice.
Our results show that high numbers of leukemia cells could not
establish a tumor in mice treated with the combined treatment,
which strongly suggests a T cell-mediated immune memory. Since
T-ALL H535 and H39 cell lines were from distinct, individual mice,
these data indicate that the immune response was not
clonotype-specific and did not involve antigens other than TCR. The
fact that DC-based immunotherapy, together with CD25 and CTLA-4
depletion, induced the eradication of leukemia with a memory
response, strongly suggests the induction of a specific,
T cell-cytotoxic response.
Injection of high dose leukemia cells to mice cured of leukemia
by combined treatment, induced TNFα secretion in vivo
To measure cytokine production after tumor challenge with T-ALL
cells, 6/12 mice previously cured of leukemia following combined
treatment, and 5 untreated control mice, were challenged with the
same, high dose of T-ALL H535 T cells at 32 weeks. One week
after T-ALL challenge, Th1/Th2 cytokine protein levels were
measured in the blood serum of injected mice. We measured
interleukin-2 (IL-2), IL-4, IL-5, interferon-γ (IFN-γ) and tumor
necrosis factor-α (TNFα). In the serum of treated mice, TNFα
protein levels reached 26.3 - 93. 9 pg/mL, while it stayed below
detection level (< 20 pg/mL) in serum of control mice
(p = 0.002). The increase of TNFα protein in the serum was probably
due to secretion by CD8-expressing T lymphocytes [28]. In contrast,
the other cytokines were not differentially produced (not shown).
We further investigated the effect of TNFα on the mitochondrial
membrane of H535 cells, by measuring the mitochondrial damage with
the potentiometric dye Dioc6. We observed a partial
depolarization after 24h of culture, indicating that the tumor
cells were sensitive to TNFα apoptosis induction (not shown).
To analyze the immunogenic properties of our H535, H72C18 and
H39 tumor cell lines in CTL assays, we used allogeneic T cells
as effectors and syngeneic or allogeneic conA blasts as negative
and positive control targets respectively. While allogeneic blasts
could be lysed, syngeneic and tumor cells were resistant to
allogeneic, T cell -mediated lysis (not shown). Furthermore,
we observed that tumor cell lysis could be induced by
perforin-granzyme purified from IL2-activated LAK cells indicating
that their recognition by CTL could be the limiting reaction, and
not their lysis (not shown).
Auto-immune manifestations in T-ALL therapy
Since LMO1 is expressed in the brain [30], and both TAL1 and LMO1
are necessary for primary hematopoiesis [31, 32], autoimmune
responses were a potential risk of the combined treatment. We
analyzed red blood cell numbers for the anemia, and the behavior of
mice in their cage for neurological disorders. We did not observe
any evidence of auto-immune manifestations in this model as has
been reported with anti-CTLA-4-based therapy in both prostate tumor
and melanoma treatment [14, 33].
Discussion
Immune control of tumor regression is dependent on specific
interactions between T lymphocytes and host tumor. One limitation
to these immune responses is that tumor cells might express mainly
self antigen, but very low levels of tumor-specific antigens,
except when malignant transformation is due to viruses. Thus,
according to the self-non-self model [34], the thymic selection
would generate self-tolerance, with peripheral T cells having
a low avidity for these self-antigens. Nevertheless, studies in
bone marrow-transplanted patients have shown that leukemia can be
eliminated by T cells (graft-versus-leukemia effect, GVL).
Based upon this GVL effect, it is possible that a vaccine inducing
autologous T cells to kill leukemia cells, might have
therapeutic potential. Several publications report vaccine
induction of Wilm’s tumor antigen (WT1)-specific CTL associated
with clinical effects [35, 36]. However, WT1 is expressed in acute
myelocytic leukemia and chronic myelocytic leukemia, but not in
T-ALL. Thus, when cancer antigens are unknown, it is necessary to
develop efficient immunotherapy by using bulk sources of antigens.
T-ALL represent a particularly difficult target for immunotherapy
protocols because they are poorly immunogenic, and they share many
molecules with normal effector T cells since they result from
the malignant transformation of their thymic precursors.
Previous studies have shown that DC are essential for the
initiation of tumor immunity since they are capable of activating
“low avidity” autoreactive T cells [24]. When DC are resting,
they have high endocytosis capacities, which allow them to
internalize antigens. Activation stimuli are then crucial for the
production of more effective adjuvants for use in vaccines against
pathogens. In contrast, immune responses to tumors can overcome
T cell tolerance in the presence of co-stimulatory signals
such as CD40 molecules [37]. Proof of efficiency of dendritic
cell-based immunotherapy has been shown in animal models and with
varying degrees of success in human clinical trials [38, 39]. In
this study, we show that therapeutic injections of dendritic cells
loaded with, and activated by, leukemia necrotic bodies induced an
immune response against poorly immunogenic T-ALL. Apoptotic bodies
and necrotic bodies can be endocytosed, but only the latter can
activate DC to become co-stimulatory. Our own experiments are
consistent with these observations since only leukemia necrotic
body internalization by DC (DC-LNB) was capable of inducing an
increased level of CD40 expression, while apoptotic body-loaded DC
did not induce tumor regression. Several factors could contribute
to the efficiency of DC-LNB, including the presence of heat shock
proteins that participate in the maturation of DC, and various
chaperone molecules bound to tumor-derived peptides which could
facilitate peptide transfer to immature DC by receptor-dependent
uptake [40-44].
Vaccination with DC loaded with leukemia-derived antigens alone
induced a partial regression of T-ALL as evaluated at 30 days.
Noteworthy is our observation that such a vaccination with loaded
DC only, led to a striking reduction of tumor cell numbers in the
bone marrow, but did not eradicate tumor cells from the kidneys.
This variation shows that cryptic organs are less easily accessible
to immune effectors than bone marrow, and might represent a hidden
source of leukemia cells for relapse. The tumor regression that we
observed in this setting after one month did not necessarily
predict long-term survival since it could be mediated by NK cells
which would not generate a memory response. This option was
examined by testing NK depletion in vivo with anti-NK1.1 mAb PK136,
but the time course of leukaemia progression was not modified (not
shown). Therefore, mechanisms, in addition to dendritic cell-based
therapy, are greatly needed to enhance tumor elimination.
In the present paper, we tested the idea that inhibition of
regulatory T cell activity would increase the efficiency of
such a protocol. This DC+ anti-CD25 + anti-CTLA-4 combined
treatment has not been used in mice or in human so far. It is
therefore possible that therapy using DC and CTLA-4 blockage might
have a synergistic effect, improving the anti-tumor activity of the
innate response. Human data in melanoma are promising, with
response rates of up to 30% when given with anti-melanoma vaccines
[33, 45]. Interestingly, CTLA-4 blockade seems to participate in an
increased inflammatory response, leading to inhibition of
angiogenesis, and to tumor growth inhibition [46, 47]. Regarding
anti-CD25 mAb, it has been shown that treatment with anti-CD25 mAb
alone was not enough to induce an immune response against several,
poorly immunogenic leukemias [12]. in addition, it has also been
shown to be insufficient for inducing autoimmunity in mice because
second signals (TCR activation, nonspecific proliferation,
inflammation) are needed to induce autoimmune diseases [48]. Thus
anti-CD25 antibodies might be used to augment vaccine-related
immunity in the clinical setting
Although one must be cautious in extrapolating mouse studies to
human trials, we have shown here that the combination of
LNB-DC-based therapy with CTLA-4 blockade and elimination of CD25+
suppressive T cells resulted in a remarkably effective
anti-T-ALL therapy, which could be correlated with TNFα secretion.
TNFα is known to be secreted by CTL against target cells during
anti-viral and anti-tumor immune responses [28]. Furthermore,
circulating TNFα could also play an important role in enhancing the
number and functional capacities of circulating DC, and their
survival [49-51].
In conclusion, this is a preliminary report presenting a
promising strategy for the induction of T cell immunity
against tumor-associated antigens that should be tested as
immunotherapy for many more tumor types.
Acknowledgements
We thank Dr M. Pla for helpful discussions and I. Ferreira for
technical assistance. We thank the co-workers of the flow cytometry
and the animal experimental facilities (Institut Universitaire
d’HÈmatologie) for their cooperation.
This study was supported by the French Association de Recherche
contre le Cancer (ARC grant 5561), and by INSERM (AR, SF, MC, MR,
HD, LL) and by the Université Denis Diderot Paris 7 (FS, AJ).
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