|
Luigi Vigorè1, Fernando Brivio2, Luca
Fumagalli2, Roberto Vezzo1, Giusy Messina6,
Franco Rovelli6, Massimo Colciago3,
Giovanna D’Amico4, Giuseppe Di Fede5,
Paolo Lissoni6
Summary
Several clinical investigations showed that the immune
status is a prognostic variable in cancer patients, even tough
the evaluation of the anticancer immunity is not generally
considered in the medical oncology. Several immune parameters,
including lymphocyte subsets and cytokine blood concentration,
had been proposed to quantify the functional status of the
anticancer immunity, but recent discoveries would suggest that
the end-result of the various immune interactions is represented
by a subtype of CD4 lymphocytes capable of suppressing the
antitumor immune reaction, the so called T-regulatory
lymphocytes (T-reg). This study was performed to detect T-reg
count and percentage in solid tumor patients, in relation to
tumor histotype, disease extension, lymphocyte sub-populations
and cortisol circadian secretion. The study included 114
consecutive cancer patients affected by the most frequent tumor
histotypes, 69 of whom showed a metastatic disease. In each
patient we evaluated T-reg cells, identified as CD4+CD25+, in
relation to T helper (CD4), T cytotoxic (CD8) and NK (CD16CD56)
cells. Abnormally high values of T-reg cells were seen in 52/114
(46%) patients, and the percentage of high values of T-reg was
significantly higher in metastatic patients than in
non-metastatic ones. In contrast, no significant difference was
seen in relation to tumor histotype. Patients with increased
T-reg count had a significantly lower NK cell number. Finally no
significant difference in T-reg number was seen between patients
with altered or normal rithm of cortisol. The study confirmed
that, irrespectively of tumor histotype the metastatic disease
is associated with a progressive and increased T-reg generation,
with a following suppression of anticancer immunity.
I. Introduction
At present, there is no doubt about the existence of a sub-type
of T lymphocytes, the so-called T regulatory lymphocytes (T-reg),
capable of suppressing the cellular immune responses,including
the anticancer immunity (Thomton and
Shevach, 2000; Shevach, 2002; von Herrath and Harrison, 2003;
Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006).
However, the exact definition
of T-reg cells in terms of cell surface marker expression still
remains controversial, particularly from a clinical point of
view. All authors are in agreement to consider Treg lymphocytes
as CD4+CD25+ cells, but at present it is still unknown whether
the expression of CD4 and CD25 antigens may be sufficient to
identify T-reg cells (Thomton and Shevach,
2000; Shevach, 2002; von Herrath and Harrison, 2003; Schwartz,
2005; von Boehmer, 2005; Ziegler, 2006; Zou, 2006), since
several authors retain that the intracytoplasmatic expression of
the FOX p3 protein is essential for the differentiation into
T-reg cells (Ziegler, 2006; Zou, 2006).Recently,
however, some preliminary observations would suggest that the
cytoplasmatic expression of FOX p3 by CD4+CD25+ cells may be
associated at least in some experimental conditions with a
diminished, rather than with an enhanced immunosuppressive
activity of T-reg cells (Siddiqui et al,
2007). In contrast, all authors agree that the expression
of CD152 antigen, also called cytotoxic T lymphocyteassociated
antigen-4 (CTLA-4) (Vasu et al, 2004),
is fundamental for the immunosuppressive activity of T-reg cells
(Takahashi et al, 2000), since the
block of its expression by using anti-CTLA-4 monoclonal
antibodies may abolish the suppressive activity of T-reg cells,
with a following stimulation of the anticancer immunity in
cancer patients (Knutson and Disis, 2007)
and an enhanced incidence of autoimmune diseases in the healthy
subjects (Lan et al, 2005).
Therefore, the addition of a third marker, such as CD152 antigen,
may allow to define a more homogeneous cell population provided
by a regulatory activity with respect to the simple CD4+CD25+
expression (Dieckmann and Plottner, 2001).
In fact, the suppressive regulatory action of CD4+CD25+CD152+
has appeared to be clearly higher than that played by the simple
CD4+CD25+ T lymphocytes (Leong et al, 2006).This
finding is not surprising, since the simple expression of CD25
marker, corresponding to the !-chain of IL-2 receptor, is not an
exclusive characteristic of T-reg lymphocytes, but it is a
non-specific property of the overall activated T lymphocytes (Thomton
and Shevach, 2000; Shevach, 2002; von Herrath and Harrison,
2003; Schwartz, 2005; von Boehmer, 2005; Ziegler, 2006; Zou,
2006). At present, preliminary clinical studies would
show that the percent of circulating CD4+CD25+ cells may be
about 10% of the all CD4+ lymphocytes, and that of
CD4+CD25+CD152+ cells may be about 40% of the total CD4+CD25+
cells, then the expected percent of CD4+CD25+CD152+ in the
healthy subjects would be less than 5% of the total circulating
CD4+ lymphocytes (Jago et al, 2004).
Finally, the expression of glucocorticoidinduced TNF-α receptor
(GITR) is also associated with an evident suppressive activity
by T-reg lymphocytes (Kanamaru et al, 2004),
which in fact are stimulated by
cortisol (Sthephens et al, 2004),
that in contrast may inhibit the activity of the most other T
lymphocytes, namely that of T helper lymphocytes, with a
following diminished production of IL-2 (Claman,
1998). As far as the mechanisms responsible for
T-reg-induced suppression of the anticancer immunity are
concerned, several experimental observations have shown that
T-reg cells may suppress the antitumor immune response through
the release of immunosuppressive cytokines, namely IL-10 and
TGF-β (Dieckmann et al, 2002), even
though other authors would suggest that the suppressive activity
of Treg cells on CD4+ and CD8+ lymphocyte activation
may be relatively independent from the action of cytokines, by
mainly requiring cell surface contact (Birebent
et al, 2004). IL-2 has been proven to be essential for
T-reg generation and some authors consider IL-2 as the main
growth factor of T-reg lymphocytes (Antony
and Restito, 2005), but more adequate studies have
demonstrated that IL-2 may induce both stimulation and
inhibition of T-reg generation and activation (Malek
and Bayer, 2004). In fact, IL-2 has appeared to induce
and promote T-reg differentiation only in the presence of TGF-β
(Chen et al, 2003). Therefore, IL-2
would constitute the main human cytokine in influencing the
characteristics of the anticancer immunity, since it may be
responsible for both activation and suppression of an effective
immune response against cancer cell proliferation and
dissemination (Wang et al, 2001),
namely depending on the whole status of the cytokine network, in
particular on the presence or in the absence of adequate
concentrations of TGF-β. In the absence of TGF-β, IL-2
stimulates the anticancer immunity, whereas it counteracts the
generation of an effective antitumor immunity in the presence of
TGF-β. In other words, IL-2 would physiologically control both
tolerance and immunity, depending on the presence of TGF-β and
other less known factors (Annunziato et
al, 2002). In fact, under cancer immunotherapy with IL-2
the percent of T-reg cells has been shown to decrease in
responding patients and to enhance in those with disease
progression (Cesana et al, 2006).
However, the regulation of T-reg functions does not depend only
on immune factors, since it is also under a neuroendocrine
control (Ji et al, 2004). In
particular, cortisol has appeared to stimulate T-reg cell
generation (Ji et al, 2004), with a
following enhanced release of IL-10, by representing the main
mechanism responsible for cortisol-induced immunosuppression.
From a clinical oncological point of view, preliminary
observations showed an enhanced percent of circulating CD4+CD25+
lymphocytes in cancer patients, namely in those with advanced
disease (Sasada et al, 2003). The
present study was performed to better establish which is T-reg
behaviour in cancer patients in relation to both tumor histotype
and disease extension.
II. Materials and methods
The study included 114 consecutive solid tumor patients with
locally limited or metastatic disease, whose clinical
characteristics are shown in Table 1. Lung cancer and
gastrointestinal tumors were the most frequent neoplasms in our
patients. For the immune detections, venous blood samples were
collected in the morning after an overnight fast. Operable
patients and metastatic patients were investigated before the
surgical operation and before the onset of chemotherapy,
respectively, in an attempt to exclude the possible influence of
the various anticancer therapies on the immune status of
patients.
In each sample, we have evaluated total lymphocyte count and
the various lymphocyte subpopulations by a flow cytometric assay
and monoclonal antibodies, including T helper lymphocytes (CD4),
T cytotoxic lymphocytes (CD8), NK cells (CD16CD56), and T
regulatory (T-reg) lymphocytes (CD4CD25). Normal values (95%
confidence limits) of T-reg observed in our laboratory were
below 240/mm3. Moreover, because of its importance in regulating
lymphocyte functions and proliferation (Claman,
1998; Sthephens et al, 2004), the circadian rhythm of
cortisol was also investigated by collecting blood samples at
8.00 A.M. and at 4.00 P.M., and cortisol serum concentrations
were measured in duplicate by using the ECLA method. Data were
reported as mean +/- SE, and statistically analyzed by the
Student’s t test, the analysis of variance and the chi-square
test, as appropriate.
III. Results
As reported in Table 2, an abnormally high number of T-reg was
seen in 52/114 (46%) patients. Moreover, the percentage of cases
with elevated number of T-reg observed in metastatic patients
was significantly higher with respect to that found in
non-metastatic patients (44/69 (64%) vs 8/45(18%), p < 0.01).
Table 3 shows the mean number of T-reg and the mean percentages
of T-reg with respect to both total lymphocytes and T helper
(CD4+) lymphocytes observed in cancer patients in relation to
their disease extension. The mean number of T-reg observed in
metastatic patients was higher with respect to that found in
patients with locally limited disease, without, however
statistically significant differences. In contrast, the mean
percentages of T-reg with respect to that of both lymphocytes
and CD4 cells were significantly higher in metastatic patients
than in the non-metastatic ones (p< 0.05 and p< 0.001,respectively).
Moreover, within the metastatic group, patients with a normal
lymphocyte count greater than 1500/mm3 showed a significantly
higher mean number of T-reg with respect to the non-metastatic
patients, whereas no difference was seen between nonmetastatic
patients and metastatic patients with lymphocytopenia,
consisting of lymphocyte count lower than 1500/mm3. In contrast,
the mean percentages of T-reg with respect to total lymphocytes
and CD4+ cells observed in both groups of metastatic patients
with normal or low total lymphocyte count were significantly
higher than in non-metastatic patients (lymphocytes: p< 0.025,
CD4+ cells: p< 0.001).
The mean counts of NK and CD8 cells in relation to that of T-reg
are reported in Table 4. As shown, no significant
difference in the mean number of CD8 lymphocytes was found
between patients with normal or abnormally elevated number of
T-reg. On the contrary, patients with elevated number of T-reg
showed a significantly lower number of NK cells with respect to
that found in those with normal T-reg count. Finally, Table 5
shows the circadian rhythm of cortisol in relation to total
lymphocytes, CD4+ cells and T-reg mean number. A normal cortisol
rhythm, with morning values greater at least than 50% with
respect to the values occurring during the afternoon, was found
in 85/114 (75%). Total lymphocyte and CD4+ cell mean numbers
observed in patients with altered cortisol rhythm were
significantly lower than those found in patients with normal
cortisol circadianicity (p<0.01), whereas no significant
difference was seen in the mean number of T reg. Figure 1
and Figure 2 illustrate T-reg mean numbers in relation to
tumor histoptypes in the overall patients and with respect to
their disease extension, respectively. No significant difference
was seen in relation to tumor histotype. The highest values of
T-reg were observed in pancreatic cancer patients, without
however significant differences with respect to the overall
other histotypes. The metastatic disease was associated with a
higher number of T-reg with respect to the non-metastatic group
in all tumor histotypes, even though a statistically significant
differences occurred for the only breast cancer (p<0.05) and
colorectal cancer (p< 0.01).
IV. Discussion
According to previous preliminary clinical investigations (Sasada
et al, 2003; Cesana et al, 2006), this study confirms in
a greater number of cancer patients that the metastatic disease
is characterized by the evidence of an abnormally increased
percentage of T-reg lymphocytes with respect to both total
circulating lymphocytes and CD4+ lymphocytes. This finding does
not seem to represent a specific characteristic of some tumor
histotypes, then it could constitute a general alteration
occurring during the progression of the neoplastic disease, by
representing a fundamental immune parameter of cancer-related
immunosuppression.
Several immune molecules have appeared to suppress the
anticancer immunity, namely IL-6, IL-10, IL-1, TNF-α and TGF-β,
but it seems that the common end result of their mechanisms of
action may be represented by the stimulation of T-reg
generation, with a consequent inhibition of the activation of an
effective anticancer immune reaction. On the same way, several
immune cells are able to suppress the anticancer immunity,
including macrophages, T helper-2 lymphocytes and some
myeloidderived suppressor cells, but also in this case they
would act in a suppressive way by promoting the generation of
Treg.
Then, the detection of T-reg amounts in terms of both
absolute number and percentages with respect to total
lymphocytes and CD4+ cells could constitute a simple and
adequate clinical immune parameter to quantify the whole status
of the anticancer immunity in the single cancer patient.
Moreover, future clinical studies will be required to establish
the possible prognostic significance of changes in T-reg
percentage and number in relation to the anticancer efficacy of
the various standard antitumor therapies. Moreover, it has to be
remarked that T-reg lymphocytes would not represent the only
immune cells involved in the suppression of the anticancer
immunity. In fact, there is at least another fundamental
immunosuppressive system, consisting of the monocytemacrophage
cell lineage (Sica and Bronte, 2007).
In more detail, it has been observed that the bone marrow may
release myeloid precursors provided by suppressive activity on
the antitumor immune response and defined as myeloid-derived
suppressor cells (MDSC) (Kusmartsev and
Gabrilovich, 2005). These cells have appeared to be
characterized by the cell surface expression of GR-1, CD11b and
CD80 antigens (Anderson et al, 2002; van
Ginderachter et al, 2006). The myeloid suppressor cells
would promote the generation and activation of T-reg lymphocytes,
which at the other side would stimulate MDSC release from the
bone marrow and M2 macrophage differentiation (Terabe
et al, 2003; Wie et al, 2006).
Moreover, the myeloid suppressive cells would inhibit the
anticancer immunity by promoting macrophage differentiation into
the M2 sub-type (Ikemoto et al, 2003),
which plays a clear inhibitory effect on the anticancer immunity,
namely through the release of IL-6 (Ueno
et al, 2000), whereas the M1 macrophage sub-type may
either stimulate or suppress the antitumor immunity (Mantovani
et al, 2004). M1 and M2 macrophage sub-types have
appeared to be characterized by a high production of IL-12 or
IL-10, respectively (Ueno et al, 2000).
Then, further studies by concomitantly evaluating T reg and
MDSC count, will contribute to better define the immune
mechanism responsible for the suppression of the anticancer
immunity.
References
Anderson CF, Gerber JS, Mosser DM (2002) Modulating macrophage
function with IgG immune complexes. J Endotoxin Res 8,
477-81.
Annunziato F, Cosmi L, Liotta F, Lazzeri E, Manetti R, Vanini
V, Romagnani P, Maggi E, Romagnani S (2002) Phenotype,localization
and mechanism of suppression of CD4+CD25+ human thymocytes. J
Exp Med 196, 379-87.
Antony PA, Restito NP (2005) CD4+CD25+ T regulatory cells,immunotherapy
of cancer,and interleukin-2. J Immunother 28, 120-8.
Birebent B, Lorho R, Lechartier H, de Guibert S, Alizadeh M,
Vu N, Beauplet A, Robillard N, Semana G (2004)
Suppressive properties of human CD4+CD25+regulatory T cells are
dependent on CTLA-4 expression. Eur J Immunol 34,
3485-96.
Cesana GC, DeRaffele G, Cohen S, Moroziewicz D, Mitcham J,
Stoutenburg J, Cheung K, Hesdorffer C, Kim-Schulze S, Kaufman HL
(2006) Characterization of CD4+CD25+ regulatory T cells in
patients treated with high-dose interkleukin-2 for metastatic
melanoma or renal cell carcinoma. J Clin Oncol 24,
1169-77.
Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady
G, Wahl SM (2003) Conversion of peripheral CD4+CD25-
naïve T cells to CD4+CD25+ regulatory T cells by TGF-β induction
of transcription factor Foxp 3. J Exp Med 198, 1875-86.
Claman HN (1998) Corticosteroids and the immune
system. Adv Exp Med Biol 245, 203-10.
Dieckmann D, Bruett H, Ploettner H, Lutz MB, Schuler G (2002)
Human CD4+CD25+ regulatory contact-dependent T cell induce IL-10
producing,contact-independent type-1-regulatory T cells. J
Exp Med 196, 247-53.
Dieckmann D, Plottner H (2001) Ex vivo isolation and
characterizationof CD4+CD25+ T cells with regulatory properties
from human blood. J Exp Med 193, 1303-10.
Ehrke MJ, Mihich E, Berd D, Mastrangelo MJ (1989)
Effects of anticancer drugs on the immune system. Semin Oncol
16, 230-9.
Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A,
Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin
F (2004) CD4+ CD25+ regulatory T cells suppress tumor
immunity but are sensitive to cyclophosphamide which allows
Immunotherapy of established tumors to be curative. Eur J
Immunol 34, 336-44.
Ikemoto S, Yoshida N, Narita K, Wada S, Kishimoto T, Sugimura
K, Nakatani T (2003) Role of tumor-associated macrophages
in renal cell carcinoma. Oncol Rep 10, 1843-9.
Jago CB, Yates J, Camara NOS, Lechler RI, Lombardi AG (2004)
Differential expression of CTLA-4 among T cell subsets. Clin
Exp Immunol 136, 463-71.
Ji HB, Liao G, Faubion WA, Abadía-Molina AC, Cozzo C, Laroux
FS, Caton A, Terhorst C (2004) Cutting edge, the natural
ligand for glucocorticoid-induced TNF receptorrelated protein
abrogates regulatory T cell suppression. J Immunol 172,
5823-7.
Kanamaru F, Youngnak P, Hashiguchi M, Nishioka T, Takahashi
T, Sakaguchi S, Ishikawa I, Azuma M (2004) Costimulation
via glucocorticoid-induced TNF receptor in both conventional and
CD25+ regulatory CD4+ T cells. Immunol 172, 7306-14.
Knutson KL, Disis M, Salazar L (2007) CD4 regulatory T
cells in human cancer pathogenesis. Cancer Immunol Immunother
556, 271-85.
Kusmartsev S, Gabrilovich DI (2005) STAT1 signaling
regulates tumor-assolciated macrophage-mediate T cell deletion.
J Immunol 174, 4880-91.
Lan RY, Ansari AA, Lian ZX, Gershwin ME (2005)
Regulatory T cells, development,function,and role in
autoimmunity. Autoimmun Rev 4, 351-63.
Leong PP, Mohammad R, Ibrahim N, Ithnin H, Abdullah M, Davis
WC, Seow HF (2006) Phenotyping of lymphocytes expressing
regulatory and effector markers in infiltrating ductal carcinoma
of the breast. Immunol Lett 102, 229-36.
Malek TR, Bayer AL (2004) Tolerance not immunity
crucially depends on IL-2. Nat Rev Immunol 4, 665-74.
Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati
M (2004) The chemokine system in diverse forms of
macrophage activation and polarization. Trends Immunol
25, 677-86.
Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A (2003)
CD4+CD25+ regulatory T cells in patients with gastrointestinal
malignancies, possible involvement of regulatory T cells in
disease progression. Cancer 98, 1089-93.
Schwartz RH (2005) Natural regulatory T cells and
selftolerance. Nat Immunol 6, 327-30.
Shevach EM (2002) CD4+CD25+ suppressor T cells, more
questions than answers. Nat Rev Immunol 2, 389-400.
Sica A, Bronte V (2007) Altered macrophage
differentiation and immune dysfunction in tumor development.
J Clin Invest 117, 1155-66.
Siddiqui SA, Frigola X, Bonne-Annee S, et al (2007)
Tumorinfiltrating Foxp3 CD4+CD25+ T cells predict poor survuival
in renal cell carcinoma. Clin Cancer Res 13, 2075-81.
Stephens GL, McHugh RS, Whitters MJ, Young DA, Luxenberg D,
Carreno BM, Collins M, Shevach EM (2004) Engagement of
glucocorticoid-induced TNFR family-related receptor on effector
T cells by its ligand mediates resistance to suppression by
CD4+CD25+ T cells. J Immunol 173, 5008-20.
Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J,
Sakaguchi N, Mak TW, Sakaguchi S (2000) Immunologic and
tolerance maintained by CD25+CD4+ regulatory T cells
constitutively expressing cytotoxic T lymphocyte-associated
antigen. J Exp Med 192, 1285-94.
Terabe M, Matsui S, Park JM, Mamura M, Noben-Trauth N,
Donaldson DD, Chen W, Wahl SM, Ledbetter S, Pratt B, Letterio JJ,
Paul WE, Berzofsky JA (2003) Transforming growth factor-β
production and myeloid cells are an eggector mechanism trhough
which CD1d-restricted T cells block cytotoxic T
lymphocyte-mediated tumor immunosurveillance, abrogation
prevents tumor recurrence. J Exp Med 198, 1741-52.
Thomton AM, Shevach EM (2000) Suppressor effector
function of CD4+CD25+ immunoregulatory T cells is antigen
nonspecific. J Immunol 164, 183-90.
Ueno T, Toi M, Saji H, Muta M, Bando H, Kuroi K, Koike M,
Inadera H, Matsushima K (2000) Significance of macrophage
chemo-attractant protein-1 in macrophage recruitment,angiogenesis
and survival in human breast cancer. Clin Cancer Res 6,
3282-9.
Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G,
Meerschaut S, Beschin A, Raes G, De Baetselier P (2006)
Classical and alternative activation of mononuclear phagocytes,
picking the best of both worlds for tumor promotion.
Immunobiology 211, 487-501.
Vasu C, Prabhakar BS, Holterman MJ (2004) Targeted
CTLA-4 engagement induces CD4+CD25+CTLA-4 high T regulatory
cells with target alloantigens specificity. J Immunol
173, 2866-76.
von Boehmer H (2005) Mechanisms of suppression by
suppressor T cells. Nat Immunol 6, 338-44.
von Herrath MG, Harrison LC (2003) Regulatory
lymphocytes, antigen-induced regulatory T cells in autoimmunity.
Nat Rev Immunol 3, 223-32.
Wang XB, Zheng CY, Giscombe R, Lefvert AK (2001)
Regulation of surface and intracellularexpression of CTLA-4 on
human peripheral T cells. Scand J Immunol 54, 453-8.
Wie S, Kryczeck I, Zou W (2006) Regulatory T-cell
compartmentalization and trafficking. Blood 108, 426-31.
Yang R, Cai Z, Zhang Y, Yutzy WH 4th, Roby KF, Roden RB (2006)
CD80 in immune suppression by mouse ovarian carcinoma-associated
Gr-1+CD11b+ myeloid cells. Cancer Res 66, 6807-15.
Ziegler SF (2006) FOXP3 of mice and men. Annu Rev
Immunol 24, 209-26.
Zou W (2006) Regulatory T cells, tumour immunity and
immunotherapy. Nat Rev Immunol 6, 295-307.
|
