ARTICLE
Auteur(s) : Gaëlle Quereux1,3, Marie-Christine
Pandolfino1,3, Anne-Chantal Knol1,3, Amir
Khammari1,3, Christelle Volteau1, Jean-Michel
Nguyen2, Brigitte
Dreno1,2,3
1Unit of skin oncology, CHU, Place A. Ricordeau 44035
Nantes, France
2Clinical Research Department, Methodological Unit, CHU
Hôtel Dieu, Nantes, France
3Unit INSERM U601
accepté le 6 Mars 2007
Standard therapies for metastatic melanoma such as
chemotherapy are often unsuccessful. Rather good evidence for the
efficacy of immunotherapy has been obtained during recent years in
humans [1-3]. The goal of such a therapy is to reverse the immune
tolerance state that enables the uncontrolled tumour growth.
Adoptive immunotherapy is an immunotherapy based on the infusion of
autologous tumour antigen specific T lymphocytes, after ex vivo
expansion.Steven Rosenberg’s group initially proposed adoptive
immunotherapy protocols in humans, based on the infusion of tumour
infiltrating lymphocytes (TIL) [1]. It has since been demonstrated
that the adoptive transferred T cell clones prefer to localize at
tumour sites [4, 5] and survive in vivo in response to low dose
IL-2 [5].The reasons behind the limited success of the
immunotherapeutic approach are still partially unknown. One
hypothesis is related to the involvement of CD4+CD25 high
regulatory T cells (Treg) [6]. Thus, by depleting the Treg cell
subset, self-reactive CD8+ T cells could mediate a more efficient
anti-tumour immunity against tumours expressing self-antigens and
enable longer overall survival in treated cancer patients [7].
Another hypothesis is that the tumour itself may develop immune
escape strategies, including impairment of antigen presentation
(tumour antigens, MHC molecules or adhesion molecules) or secretion
of immunosuppressive molecules such as α-MSH, IL-10 or TGF-β
[8].Our study was based on the aforementioned strategies. We used
metastatic lymph node samples collected from melanoma patients with
metastatic lymph nodes stage IIIb and IIIc AJCC (6th
edition), who were included in a therapeutic protocol based on the
transfer of autologous TIL [9, 10]. In this protocol, half of the
88 patients (44) received TIL combined with IL-2
Proleukin® (Chiron) and the other half of the patients
received IL-2 alone. This study demonstrated that only patients
with one invaded lymph node responded to TIL immunotherapy. In
those patients the overall survival was increased (p = 0.026) and
the estimated relapse rate was significantly lower (p = 0.019) in
the TIL+IL-2 group compared to the IL-2 group [9]. In patients with
more than one invaded lymph node, no difference was noted between
the two groups, neither in the relapse free survival nor in the
overall survival. In order to determine whether the efficacy of TIL
as adjuvant therapy in patients with lymph node invasion (stage III
AJCC) was related to the expression level of some antigens by
tumour cells or to the secretion of immunosuppressive cytokines by
these same tumour cells, we evaluated the potential correlation
between expression levels of these antigens or cytokines and the
relapse free survival and overall survival of patients who received
TIL. We focused on the following antigens: melanocyte
differentiation antigens (Melan-A and gp100), MHC molecules (class
I and II), adhesion molecules (ICAM-1, LFA-3) and suppressive
cytokines (IL-10, TGF-β and α-MSH). The main objective was to
assess how each marker expression correlated with the relapse free
survival and with the overall survival. The primary endpoint was
disease-free interval, and the secondary endpoint was overall
survival. Furthermore, in order to understand the different
clinical response obtained with TIL, we also compared the
expression levels of these markers in lymph node biopsies from
patients with one invaded lymph node versus patients having more
than one.
Material and methods
Material
This work was performed on lymph node samples that were used to
produce autologous TIL from 38 patients receiving TIL plus IL-2 as
part of a clinical research protocol [9]. Patients underwent a
sterile lymph node resection. The majority of the invaded lymph
node was used to produce TIL and for each patient a biopsy fragment
was collected for in situ immunochemistry and for the establishment
of the autologous tumour cell line. The remaining biopsy was
addressed to the pathology laboratory.
Patients
The study was conducted according to the Helsinki declaration and
submitted and approved by the French Health products Safety Agency
and local ethic committee. An informed consent was obtained from
each patient. The median age of these 38 patients was 51 years; the
gender ratio was 1.7:1. Seventy six percent of the patients
presented a Breslow thickness of more than 1.5 mm of their
primary melanoma and the Clark level median was III (table 1).
The patients were treated between February 1994 and December
1998. All the patients who are alive are still followed up, and the
median of follow up is 793 days.
Table 1 Relevant clinicopathological data of the 38
patients with lymph node metastasis
|
Patients with only one invaded lymph node (n: 13)
|
Patients with more than one invaded lymph node (n: 25)
|
|
Median age
|
52 years (34 to 74)
|
49 years (22 to 74)
|
|
Sex ratio
|
9 males/4 females
|
15 males/10 females
|
|
% of patients with Breslow index more than 1.5 mm
|
92% (12/13)
|
68% (17/25)
|
|
Median Clark level
|
III
|
III
|
|
Median number of invaded lymph nodes
|
1
|
2
|
|
% capsular breakthrough
|
38% (5/13)
|
56% (14/25)
|
|
Relapse rate
|
31% (4/13)
|
80% (20/25)
|
Methods
Immunohistochemistry
Immunohistochemistry was performed using the
streptavidin/peroxidase technique as previously described [11] on
deep-frozen sections obtained from metastatic lymph nodes. Cryostat
sections (5 μm) sections were incubated for 30 minutes in a
moist chamber at room temperature with the primary antibodies. The
following monoclonal antibodies were used: anti-Melan-A (Dako,
Glostrup, Denmark), anti-gp100 (Dako, Carpinteria USA), anti- CMH
class I (A, B, C) (BD Pharmingen San diego, USA), anti-CMH class II
(DR, DP, DQ) (BD Pharmingen San diego, USA), anti-CD54 (ICAM-1)
(Immunotech, Marseille, France), anti-CD58 (LFA-3) (Serotec,
Oxford, UK), anti-α-MSH (polyclonal)(TEBU bio, Santa Cruz
biotechnology), anti-IL-10 (Diaclone research, Besançon, France)
and anti-TGF-β (Serotec, Oxford, UK). Secondary biotinylated rabbit
anti-mouse antibody was added for 30 minutes and then
peroxydase-conjugated streptavidin (Dako detection kit
peroxydase/AEC, Rabit/mouse, Glostrup, Danmark). The sections were
then incubated with AEC for 2 minutes. Finally the sections were
counterstained with Mayers’s hemalun and mounted with aqueous
mounting medium. Between the different steps tissue sections were
washed in TBS (Tris buffered saline)(SIGMA)-BSA(bovine serum
albumin) (SIGMA) 0.1%. The primary antibodies were omitted in
negative controls.
For α-MSH and IL-10, the signal was weak so it was amplified
using the biotinyl tyramide (kit TSA indirect; Dupont NEN, Boston,
USA) [11].
Slides were read with a Leica microscope (magnification × 40).
The expression of the molecule in the tumour cell was assessed
using a visual semi-quantitative scale from 0 to 3 for each studied
lymph node biopsy (n = 38). All the slides were read by two
independent physicians. For each section, the protein level was
taken as the mean of three fields. A weak expression (0-25% of
positive cells), moderate (26-50%), intermediate (51-75%) and
strong (> 75%) were represented respectively by levels 0, 1, 2
and 3 (figures 1-4).
Statistical analysis
Survival analyses were performed. Univariate and multivariate Cox
models were used for each variable studied. Each model was used on
the following baseline characteristics: number of invaded lymph
nodes (1 versus > 1), age at onset of treatment, gender, Breslow
thickness (< 1.5 mm versus > 1.5 mm), Clark
level (1/2/3 versus 4/5), ulceration and significant variables in
the univariate Cox model.
To compare each expression level for all the markers studied
according to the number of invaded lymph nodes (one versus more
than one) the chi square was used.
Interactions between the number of invaded lymph nodes and the
previously mentioned variables were also analysed. Survival models
were based on the assumption of proportional hazards along time.
This hypothesis was graphically checked with survival curves
(Kaplan-Meier model) and on multivariate models with Schoenfeld’s
remnants model.
Results
Expression levels
The expression levels of the different markers are summarized in
table 2. Eighty percent (32/38) of lymph
nodes expressed gp100 antigen and 82% (31/38) Melan-A antigen, with
a median expression level of 2 for both. MHC class I expression was
absent in 6 lymph nodes (16%) and MHC class II in 3 lymph nodes
(8%).
Table 3 summarizes results for
relapse-free survival and overall survival according to a
univariate model and table 4 according
to a multivariate model.
Table 2 Expression levels of the different markers in
the lymph node biopsies
|
|
Expression levels of the markers
|
|
Patient number
|
Number of invaded lymph nodes
|
GP-100
|
Melan-A
|
|
|
ICAM-1
|
LFA-3
|
α-MSH
|
Il-10
|
TGF-β
|
|
1
|
5
|
0
|
0
|
1
|
1
|
2
|
1
|
1
|
1
|
0
|
|
2
|
38
|
1
|
1
|
1
|
2
|
2
|
0
|
0
|
0
|
0
|
|
3
|
6
|
3
|
3
|
2
|
3
|
3
|
0
|
0
|
0
|
1
|
|
4
|
2
|
2
|
1
|
0
|
0
|
0
|
0
|
0
|
1
|
0
|
|
5
|
10
|
1
|
1
|
0
|
1
|
1
|
1
|
1
|
1
|
0
|
|
6
|
2
|
3
|
2
|
0
|
1
|
1
|
0
|
0
|
0
|
0
|
|
7
|
2
|
2
|
2
|
2
|
3
|
1
|
1
|
1
|
0
|
3
|
|
8
|
3
|
1
|
2
|
0
|
2
|
0
|
0
|
0
|
1
|
1
|
|
9
|
6
|
3
|
2
|
1
|
2
|
0
|
0
|
0
|
1
|
0
|
|
10
|
2
|
2
|
1
|
2
|
2
|
1
|
1
|
1
|
0
|
0
|
|
11
|
4
|
1
|
1
|
2
|
2
|
1
|
0
|
0
|
0
|
0
|
|
12
|
2
|
2
|
3
|
3
|
3
|
1
|
0
|
2
|
2
|
1
|
|
13
|
2
|
2
|
2
|
2
|
2
|
1
|
0
|
1
|
2
|
0
|
|
14
|
2
|
3
|
3
|
2
|
2
|
2
|
0
|
0
|
0
|
1
|
|
15
|
7
|
1
|
2
|
3
|
2
|
3
|
1
|
2
|
2
|
1
|
|
16
|
2
|
1
|
3
|
3
|
2
|
0
|
0
|
0
|
1
|
0
|
|
17
|
4
|
1
|
3
|
2
|
3
|
2
|
0
|
0
|
2
|
1
|
|
18
|
2
|
0
|
0
|
1
|
1
|
1
|
1
|
1
|
1
|
0
|
|
19
|
3
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
1
|
|
20
|
3
|
2
|
2
|
0
|
0
|
1
|
0
|
1
|
1
|
0
|
|
21
|
2
|
1
|
1
|
2
|
1
|
0
|
0
|
3
|
2
|
0
|
|
22
|
3
|
2
|
3
|
1
|
2
|
1
|
0
|
2
|
3
|
0
|
|
23
|
2
|
1
|
0
|
1
|
2
|
2
|
1
|
0
|
1
|
0
|
|
24
|
2
|
0
|
0
|
3
|
3
|
3
|
0
|
0
|
0
|
2
|
|
25
|
2
|
3
|
3
|
2
|
1
|
2
|
2
|
1
|
1
|
0
|
|
Median
|
|
1
|
2
|
2
|
2
|
1
|
0
|
0
|
1
|
0
|
|
ARRAY(0x387e18)
|
|
26
|
1
|
3
|
2
|
2
|
3
|
3
|
0
|
0
|
0
|
1
|
|
27
|
1
|
0
|
0
|
3
|
3
|
2
|
1
|
0
|
0
|
0
|
|
28
|
1
|
3
|
3
|
1
|
2
|
3
|
1
|
3
|
3
|
0
|
|
29
|
1
|
1
|
1
|
3
|
3
|
3
|
0
|
2
|
2
|
2
|
|
30
|
1
|
2
|
3
|
1
|
2
|
2
|
0
|
0
|
0
|
0
|
|
31
|
1
|
3
|
3
|
1
|
1
|
0
|
1
|
0
|
2
|
0
|
|
32
|
1
|
1
|
3
|
1
|
2
|
3
|
1
|
2
|
2
|
0
|
|
33
|
1
|
2
|
2
|
3
|
3
|
1
|
0
|
2
|
3
|
1
|
|
34
|
1
|
3
|
2
|
1
|
3
|
1
|
1
|
1
|
1
|
0
|
|
35
|
1
|
2
|
2
|
2
|
2
|
1
|
1
|
0
|
0
|
0
|
|
36
|
1
|
3
|
2
|
3
|
2
|
0
|
0
|
2
|
2
|
0
|
|
37
|
1
|
3
|
3
|
2
|
2
|
1
|
1
|
0
|
0
|
2
|
|
38
|
1
|
0
|
0
|
1
|
2
|
2
|
0
|
2
|
2
|
1
|
|
Median
|
|
2
|
2
|
2
|
2
|
2
|
1
|
1
|
2
|
0
|
Table 3 Relapse-free survival and overall survival
analysis according to an univariate model
|
Relapse free survival
|
|
β
|
S(β)
|
RR
|
ICRR95%
|
p-value
|
|
Invaded lymph node (1 vs > 1)
|
1.57
|
0.55
|
4.80
|
[1.63-14.1]
|
0.0043
|
|
Age
|
– 0.027
|
0.019
|
0.97
|
[0.94-1.01]
|
0.15
|
|
Sex
|
– 0.31
|
0.41
|
0.74
|
[0.33-1.64]
|
0.45
|
|
Breslow thickness (< 1.5 vs > 1.5)
|
0.043
|
0.47
|
1.04
|
[0.42-2.62]
|
0.93
|
|
Clark level (1-2-3 vs 4-5)
|
– 0.22
|
0.41
|
0.81
|
[0.36-1.80]
|
0.60
|
|
Ulceration (0 : no vs 1 : yes)
|
0.47
|
0.44
|
1.60
|
[0.68-3.77]
|
0.28
|
|
Capsular breaking (0: no vs 1: yes)
|
0.92
|
0.42
|
2.51
|
[1.11-5.71]
|
0.028
|
|
ARRAY(0x3a2630)
|
|
GP 100a
|
– 0.25
|
0.41
|
0.78
|
[0.35-1.73]
|
0.54
|
|
Melan-Aa
|
– 0.00
|
0.42
|
0.99
|
[0.44-2.05]
|
0.99
|
|
MHC class Ia
|
0.63
|
0.43
|
1.89
|
[0.82-4.32]
|
0.13
|
|
MHC class IIa
|
– 0.27
|
0.45
|
0.76
|
[0.32-1.85]
|
0.55
|
|
ICAM-1a
|
– 0.41
|
0.43
|
0.67
|
[0.29-1.53]
|
0.34
|
|
LFA-3a
|
1.28
|
1.05
|
3.59
|
[0.46-28.3]
|
0.22
|
|
α-MSHa
|
– 0.08
|
0.47
|
0.93
|
[0.37-2.34]
|
0.87
|
|
Il 10a
|
– 0.03
|
0.43
|
0.97
|
[0.41-2.26]
|
0.94
|
|
TGF-βa
|
0.13
|
0.62
|
1.14
|
[0.34-3.85]
|
0.83
|
|
Overall survival
|
|
Invaded lymph node (1 vs > 1)
|
1.66
|
0.62
|
5.28
|
[1.57-17.7]
|
0.0072
|
|
Age
|
– 0.010
|
0.018
|
0.99
|
[0.96-1.03]
|
0.60
|
|
Sex
|
– 0.25
|
0.42
|
0.78
|
[0.34-1.79]
|
0.57
|
|
Breslow thickness (< 1.5 vs > 1.5)
|
– 0.082
|
0.47
|
0.92
|
[0.37-2.32]
|
0.86
|
|
Clark level (1-2-3 vs 4-5)
|
– 0.36
|
0.42
|
0.70
|
[0.30-1.59]
|
0.39
|
|
Ulceration (0 : no vs 1 : yes)
|
– 0.59
|
0.46
|
1.80
|
[0.74-4.38]
|
0.20
|
|
Capsular breaking (0: no vs 1: yes)
|
1.04
|
0.44
|
2.83
|
[1.20-6.68]
|
0.017
|
|
ARRAY(0x3b0d10)
|
|
GP 100a
|
– 0.038
|
0.42
|
0.96
|
[0.42-2.19]
|
0.93
|
|
Melan-Aa
|
0.50
|
0.45
|
1.64
|
[0.67-4.00]
|
0.28
|
|
MHC class Ia
|
0.76
|
0.44
|
2.14
|
[0.90-5.06]
|
0.084
|
|
MHC class IIa
|
0.039
|
0.08
|
1.04
|
[0.41-2.65]
|
0.93
|
|
ICAM-1a
|
– 0.47
|
0.44
|
0.62
|
[0.26-1.48]
|
0.28
|
|
LFA-3a
|
1.41
|
1.06
|
4.10
|
[0.51-32.8]
|
0.18
|
|
α-MSHa
|
– 0.11
|
0.51
|
0.90
|
[0.33-2.43]
|
2.43
|
|
Il-10a
|
– 0.00
|
0.45
|
0.99
|
[0.41-2.42]
|
0.99
|
|
TGF-βa
|
0.32
|
0.63
|
1.38
|
[0.41-4.69]
|
0.61
|
a0 or 1 vs 2 or 3.
Table 4 Relapse-free survival and overall survival
analysis according to a multivariate model
|
Relapse free survival
|
|
|
|
|
|
|
β
|
S(β)
|
RR
|
ICRR95%
|
p-value
|
|
GP 100a
|
– 0.08
|
± 0.47
|
0.92
|
[0.37-2.31]
|
0.86
|
|
Melan-Aa
|
– 0.29
|
± 0.51
|
0.75
|
[0.27-2.05]
|
0.57
|
|
MHC class Ia
|
0.38
|
± 0.59
|
1.46
|
[0.46-4.61]
|
0.52
|
|
MHC class IIa
|
0.08
|
± 0.60
|
1.08
|
[0.33-3.53]
|
0.90
|
|
ICAM-1a
|
0.93
|
± 0.54
|
2.53
|
[0.88-7.29]
|
0.09
|
|
LFA-3a
|
1.71
|
± 1.24
|
5.51
|
[0.48-62.8]
|
0.17
|
|
α-MSHa
|
0.73
|
± 0.60
|
2.08
|
[0.64-6.75]
|
0.22
|
|
Il-10a
|
0.90
|
± 0.58
|
2.45
|
[0.79-7.58]
|
0.12
|
|
TGF-βa
|
1.62
|
± 0.79
|
5.05
|
[1.07-23.8]
|
0.041
|
|
Overall survival
|
|
|
|
|
|
|
GP 100a
|
0.30
|
± 0.56
|
1.35
|
[0.45-4.03]
|
0.59
|
|
Melan-Aa
|
0.50
|
± 0.52
|
1.65
|
[0.59-4.03]
|
0.34
|
|
MHC class Ia
|
0.94
|
± 0.61
|
2.55
|
[0.76-8.50]
|
0.13
|
|
MHC class IIa
|
0.18
|
± 0.62
|
1.19
|
[0.35-4.03]
|
0.78
|
|
ICAM-1a
|
0.63
|
± 0.54
|
1.87
|
[0.65-5.40]
|
0.00042
|
|
LFA-3a
|
2.60
|
± 1.34
|
13.42
|
[0.97-186]
|
0.053
|
|
α-MSHa
|
1.76
|
± 0.81
|
5.82
|
[1.20-28.3]
|
0.029
|
|
Il-10a
|
1.73
|
± 0.66
|
5.63
|
[1.55-20.4]
|
0.0086
|
|
TGF-βa
|
2.50
|
± 0.91
|
12.15
|
[2.04-72.4]
|
0.0061
|
a0 or 1 vs. 2 or 3.
Correlation between expression levels and relapse-free
survival
At the clinical level, the number of invaded lymph nodes and
capsular breakthrough were significantly correlated to the
relapse-free survival using univariate analysis (table 3). However none of the studied tissue
markers were correlated to the relapse-free survival.
Using multivariate model (table 4),
only the expression of TGF-β was significantly correlated to
relapse: weak expression of TGF-β was associated with a longer
relapse-free survival.
Correlation between expression levels and overall survival
The number of invaded lymph nodes and capsule breakthroughs were
significantly correlated to the overall survival using univariate
analysis (table 3), but none of the
studied tissue markers was correlated to the overall survival.
Using multivariate model (table 4),
expression of cytokines (TGF-β, IL-10, α-MSH) and ICAM-1 were
significantly correlated to overall survival with a weak expression
associated with a longer survival.
Comparison of expression levels according to the number of
invaded lymph nodes
There was no correlation between the expression level of melanocyte
differentiation antigens, MHC molecules, adhesion molecules and
cytokines and the number of invaded lymph nodes.
The 6 lymph nodes with loss of expression for MHC class I came
from patients with more than one invaded lymph node, but it was not
significant (p = 0.07). Similarly the lymph nodes with a loss of
expression for MHC class II came from patients with more than one
invaded lymph node.
Discussion
In order to determine whether the lack of efficiency of TIL was
related to tumour escape mechanisms to adoptive immunotherapy, we
studied the expression level of antigens and the secretion of
immunosuppressive cytokines by the tumour cells. This study was
performed on lymph node samples that were used to produce
autologous TIL extracted from 38 patients receiving TIL plus IL-2
as part of a clinical research protocol [9]. A weak point of this
study was due to the technique that was used. Immunohistochemistry
is an imprecise technique for quantification and largely depends on
the technical conditions. Furthermore, to avoid the subjectivity of
the reading all the slides were read by two independent physicians.
We differentiated tumour melanoma cells and other immunocompetent
cells by their morphologic aspect, which is quite different. We
focused on melanocyte differentiation antigens, MHC molecules,
adhesion molecules and suppressive cytokines. Concerning the number
of lymph nodes, there was no significant difference regarding
expression levels for these different markers according to the
number of invaded lymph nodes. However, all melanoma tumour cells
with loss of MHC class I or class II expression came from patients
with more than one invaded lymph node. This could explain at least
in part the differential therapeutic response obtained from
patients having one or several invaded lymph nodes.
Concerning the level of expression of melanocyte differentiation
antigens by tumour cells, we did not, in our study, identify an
antigen whose expression was correlated with the relapse free
survival or the overall survival of patients receiving TIL. These
results are in accordance with those noted in the literature, but
in those studies none of the patients had been treated by
immunotherapy [12, 13]. Only Berset et al. [14] reported
significantly lower overall survival from patients who did not
express Melan-A. Their study was conducted on 73 primary melanomas
with a 4.5 year follow-up but without adoptive immunotherapy.
Regarding expression of MHC class I and class II molecules,
their prognostic value on relapse or overall survival remains
debatable [15]. Hofbauer et al. [12] did not report any prognostic
value for MHC class I molecules on relapse-free survival and
overall survival, while Kageshita et al. [16] reported a
correlation between a decreased expression level for MHC class I
molecules within primary tumours and a decreased overall survival
or relapse-free survival. But none of these studies had been
carried out during an immunotherapy. Concerning MHC class II
molecules, it has been shown that melanoma cells were stained by
anti-class II CMH molecules [15, 17]. Furthermore Ruiter et al.
showed that the percentage of class II CMH-bearing melanoma cells
is higher in metastasis than in primary lesions [18]. In our study,
we found no prognostic value of MHC molecule expression in the
response to TIL immunotherapy.
Regarding adhesion molecules using a multivariate model, the
overall survival was significantly increased following
immunotherapy when the expression level for ICAM-1 was weak, (0 or
1) (p = 0.00042). Similarly, Natali et al. [19] reported that
ICAM-1 was correlated to a worse prognosis for 31 primary
melanomas. Patients in this study were treated by surgery and
received chemotherapy or isolated limb perfusion but no
immunotherapy. Regarding integrin LFA-3, its expression level was
weak (0 or 1) for almost all of the studied samples (97%: 37/38).
Therefore LFA-3 does not appear to play a significant role in the
interaction between TIL and the tumour cell. To our knowledge, the
relationship between LFA-3 expression and survival in patients with
melanoma has not yet been studied.
Regarding cytokine expression levels, our work focused on 3 main
immunosuppressive cytokines: α-MSH, TGF-β and IL-10. Using a
multivariate analysis, a significantly increased relapse-free
survival and overall survival for patients receiving TIL was
associated to a weak expression level for these cytokines in the
invaded lymph node.
α-MSH has been extensively studied for the last ten years. It
down-regulates pro-inflammatory cytokines (IL-1, IL-2, IL-6, TNF-α,
IFN-γ), and co-stimulation molecules (CD86, CD40) [20, 21]. In
parallel, α-MSH up-regulates the immunosuppressive IL-10 [22].
Nagahama et al. [22] reported an increased α-MSH production in
melanomas and in melanoma metastases compared to nævi, suggesting a
potential role for α-MSH in melanoma progression, probably related
to its suppressive activity. TGF-β also has a suppressive activity
on immunosurveillance. IL-10 inhibits IFN-γ production, and
disables antigen-presenting cells from processing the antigen by
decreasing MHC class II expression levels. Thus, our results
indicate that the production of immunosuppressive cytokines by the
tumour cells could be one of the mechanisms inhibiting the activity
of TIL at the tumour site.
Conclusion
This work suggests that immunosuppressive cytokine production
(IL-10, TGF-β and α-MSH) may represent a prognostic marker for
TIL-receiving patients. Their expression level is inversely
correlated to the overall survival of the patients. Moreover, the
differential efficacy of TIL according to the number of invaded
lymph nodes (one versus more than one) does not appear to be
related to the expression levels of melanocyte differentiation
antigens, adhesion molecules nor to the expression levels of
immunosuppressive cytokines. However, a loss of MHC class I or II
expression was found only in lymph node samples from patients with
more than one invaded lymph node.
Acknowledgement
This work has been supported by Ligue contre le cancer 2005 and
“Cancer Immunotherapy” 6th Framework Programme and
INSERM
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