Received: February 7, 2014
Accepted: May 6, 2014
Meditterr J Hematol Infect Dis 2014, 6(1): e2014039, DOI 10.4084/MJHID.2014.039
This article is available on PDF format at:
Alessandra Romano1, Calogero Vetro1, Giovanni Caocci2, Marianna Greco2, Nunziatina Laura Parrinello1, Francesco Di Raimondo1 and Giorgio La Nasa2
1
Division of Haematology, Azienda Ospedaliera “Policlinico-Vittorio
Emanuele”, University of Catania. Via Citelli 6, 95124 Catania, Italy.
2 Hematology Unit, Department of MedicalSciences
“Mario Aresu,” University of Cagliari, Italy.
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Abstract Classic
Hodgkin Lymphoma (cHL) has a unique histology since only a few
neoplastic cells are surrounded by inflammatory accessory cells that in
the last years have emerged as crucial players in sustaining the course
of disease. In addition, recent studies suggest that the abnormal
activity of these inflammatory cells (such as deregulation in
regulatory T cells signaling, expansion of myeloid derived suppressor
cells, HLA-G signaling and natural killer cells dysfunction) may have
prognostic significance. This review is focused on summarizing recent
advanced in immunological defects in cHL with translational
implications.
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Introduction
In
the classic Hodgkin's lymphoma (cHL) microenvironment, a few neoplastic
cells - Hodgkin and Reed-Stemberg (HRS) cells- grow in a contest of a
tissue rich in immune system cells, including fibroblasts, eosinophils,
lymphocytes, histiocytes, neutrophils and monocytes.[1]
These immune system cells are unable of mounting effective anti-tumor
immune responses and, on the contrary, even stimulate and promote the
growth of HRS cells.
The strong correlation between classic HL (cHL) and Epstein-Barr virus
(EBV) infection strengthens the hypothesis that alterations in the
mechanisms involved in viral clearance (antigen presentation, innate
natural killer cell-dependent immune response) may influence the onset
of cHL.
For its peculiar histology cHLis an extremely interesting study model
for the assessment of immunogenetic factors that may confer
susceptibility to tumours or, alternatively, facilitate tumour immune
escape mechanisms.
After the demonstration of the prognostic significance of
Interim-2-[18F]Fluoro-2-deoxy-D-glucose Positron Emission Tomography
(PET-2) performed in the mid of chemotherapy, the role of accessory
cells in cHL has been evaluated with increased interest. In fact, it
has been demonstrated that PET-2 positivity is mainly due to the
Fluoro-2-deoxy-D-glucose uptake by the accessory cells rather than the
HRS cells.[2]
Inflammation-related accessory cells can be indirectly evaluated in the
peripheral blood as well: several reports investigated the prognostic
impact of the ALC/AMC-DX ratio, obtained by dividing the absolute
lymphocyte count (ALC) over the absolute monocytes count (AMC) from the
complete blood count, as a surrogate of host immune homeostasis and
tumour-associated macrophages (TAM) respectively, with contrasting
results.[3,4]
This review is focused on the novel advances about the role of myeloid
and lymphoid subsets involved in sustaining HRS and favouring
immune-escape.
NK Dysregulation
Natural killer (NK) cells represent a key component
of the innate immune system against cancer.
Together with NK cells, a subset of CD1d-restricted Natural Killer-T
cells (NKT) exhibits direct anti-tumour activity and enhances
cytotoxicity of NK and CD8+ T cells. NKT cells are distinct lymphocyte
population characterized by the expression of CD3 and CD56 and an
invariant T-cell receptor (TCR) formed by the Ja18-Va24 and Vb11
rearrangements specific for glycosphingolipids presented by the
non-classical MHC Class-I molecule CD1d.[5]
A common immune escape strategy of HRS cells is to down-regulate the
expression of human leukocyte antigen (HLA) -A,-B and -C (classic: MHC
Ia) and to modify the expression of HLA-G and E (no classical: MHC Ib),
as seen in about 20% and 80% of primary cases of EBV+ and EBV- cHL,
respectively. However, because communication through MHC Ia-specific
inhibitory receptors on NK cells is lacking, downregulation of MHC Ia
generally leads to the activation of NK cells.[6]
The paucity of NK cells in the reactive infiltrate of cHL and the
systemic NK cell deficiency observed in cHL patients prompted further
investigation into the immune-modulatory mechanisms of NK receptors
such as the NKG2D activating receptor of the C-type lectin superfamily,
killer immunoglobulin-like receptors (KIRs), immunoglobulin-like
transcript 2 (ILT2) inhibitory receptors, immunoglobulin-like
transcript 4 (ILT4) and the NKG2A inhibitory receptor. New evidences
continue to emerge that a reduced activity of NK cells may be related
to the prevalence of inhibitory over activating KIR genes.[7]
Therapeutic strategies aimed at interfering with the crosstalk between
HRS cells and their cellular partners have inspired the development of
new immunotherapies targeting different cellular components of the
microenvironment.
NKG2D receptor and the group of natural cytotoxicity receptors (NCRs)
(NKp46, NKp44, and NKp30) are regarded as the major NK cell receptors
in tumour defence. Immune surveillance via NKG2D and the corresponding
ligands seems to be particularly effective in the early stages of
tumour growth.[8]
However, tumour cells develop escape mechanisms to evade NK cell
surveillance and NKG2D-ligand interaction, which obviously results in
either immune activation (tumour clearance) or immune silencing (tumour
evasion).
Silencing of NKG2D during tumour progression results from the
persistent exposure of ligands expressed on the surface of target
cells. Moreover, tumour cells release ligands into the environment by
shedding. The soluble molecules not only block NKG2D, but also induce
the internalization and degradation of the receptor.[9]
Plasma levels of soluble ligands correlate with disease progression in
many haematological and solid tumours. Former studies on NK cell
function in HL have shown that peripheral NK cells from patients with
HL are functionally inactive. The observed NK cell dysfunction
correlates to elevated serum levels for ligands engaging NKG2D (MICA)
and NKp30 (BAG6/BAT3). Low levels of the membranous NKG2D-ligands, i.e.
MHC class I related chain-A (MIC-A) and UL16 binding protein 3 (ULBP3),
on HRS cells presents another way to escape from cytotoxic T-cell
responses. These low levels are the result of proteolytic cleavage of
the NKG2D-ligands by ERp5 and a disintegrin and metalloproteinase
domain-containing protein 10 (ADAM10) produced by HRS cells and
mesenchymal stromal cells. Additionally, T-cells in cHL tissue have
lower NKG2D receptor expression levels as compared to T-cells in normal
lymph nodes, due to TGF-β produced by the mesenchymal stromal and HRS
cells, which blocks IL-15-induced expression of NKG2D receptor on
cytotoxic T-cells. Thus, the anti-tumour activity of CD8+ T-cells is
blocked by lack of membrane NKG2D-ligands, release of soluble
NKG2D-ligands and reduced NKG2D receptor levels on effector T-cells.[10]
Immunotherapeutic strategies targeting NK cells are promising because
NK cell cytotoxicity could be restored in vitro and patients using a
novel human antibody construct specifically designed for the treatment
of cHL and other CD30-expressing malignancies. In a previous study, a
tetravalent bispecific antibody construct (AFM13) was used to target
CD30 on HRS cells with two of its binding sites, whereas the activating
receptor CD16A on NK cells (CD30xCD16A, AFM13) was targeted by the
other two binding sites, thereby selectively cross-linking tumour and
NK cells.[11]
Also, epigenetic modifications have been implicated in the malignant
phenotype of HRS cells. In this context, the histone-deacetylase (HDAC)
inhibitor LBH589 (panobinostat) was shown to be clinically effective.
LBH589 modulates the crosstalk of lymphocytes with HL cell lines. More
specifically, LBH589 induces cell death, autophagy, and an increase of
major histocompatibility complex (MHC) class I chain-related genes
molecules (MICA/B); that act as key ligands for NK cell receptors, and
also favourably modulates the cytokine network and lymphocyte activity
in the HL microenvironment.[12]
Studies of innovative
therapies based on the immune system of HL patients treated with
chemo/radiotherapy and targeting NK cells rather than T cells are,
therefore, extremely promising.
Lymphoid Impairment
Lymphoid anergy is well-known in the biology and pathogenesis of cHL
and T-cell homing is central in determining the immunological
regulation of HRS growth and survival.[13,14]
The
lymphoid infiltrate in HL is different from the aspecific one
detectable in reactive lymphoid hyperplasia (RLH), since the CD3+/CD20+
ratio is greater in HL than in RLH,15 with augmented CD4+CD25+
infiltrate.[15,16]
Whole-tissue RNA analysis evaluated the specific microenvironment
characteristics of HL, discovering that a great release of cytokines is
present alongside suppressed expression of apoptotic genes and
augmented expression of cell-cycle regulatory enzymes. HRS cells
genotyping analysis showed a global suppression of principal tumor
suppression pathways, including Rb-p16INK4, p27KIP1, p53 and an
increased expression of components of G1-CDK checkpoint.[17,18]
The neoplastic HRS themselves create a favorable microenvironment for
their survival and growth regulating inflammatory infiltrate. In vitro
studies on KM-H2 HRS cell-line demonstrated that HRS cells are able to
induce CD4+CD25+ regulatory T- cells (Treg),[19]
whose function is to inhibit the cytotoxic effects of CD8+ T-cells, the
so called cytotoxic lymphocytes (CTL).[16,19,20]
Main factors involved in lymphocyte migration into the tumor milieu
include CCL20,[21] CCL5/Rantes,
IL-7, CCL17 and CCL22.[22]
CXCR3, CXCR4, CXCL13 and CCR7, and adhesion molecules including CD62
ligand (CD62L),are greatly expressed on T-cells of cHL patients.[23]
HRS cells are also able to express chemokine receptors useful to T-cell
migration into tumor milieu, such as CXCL12 (receptor of CXCR4) and
CXCR5 (receptor of CXCL13).[24]
T-regs accumulate into the tumor milieu thanks to the surface
expression of CCR4, the receptor for “thymus and activation regulated
chemokine” (TARC/CCL17), a factor greatly secreted by HRS cells.[25] T cells stimulated with TARC
acquire a regulatory function, able to silence the cytotoxic activity
of CTL.[26] Conversely, CTL are
not influenced by TARC since they lack the surface expression of CCR4.[27]
Once migrated into the tumor mass, lymphocytes are addressed toward Th2
and T-reg differentiation (in particular, a Tr1 phenotype)[28]
acquiring the ability (together with HRS cells) to produce and secrete
TGF-β and IL-10, able to suppress CTL function. Thus, T-regs regulate
the production of IL-2 and limit CTL activation,[23]
while Th2 cells induce the expression of several cyclins and
cyclin-dependent kinases [29] and
of antiapoptotic markers, such as Bcl-Xl and Mcl1,[30]
with overexpression of STAT3 in HRS cells, activation of cyclin D1 and
Bclx expression, and a down-regulation of STAT1, a tumor suppressor
factor.[17,18,31]
CTL are further silenced through the CD95-CD95L and PD1-PD1L
cell-to-cell contact between HRS cells and CTL.[32-37]
Additionally, the production of galectin-1, tissue inhibitor of
metalloproteinase1 (TIMP1), and prostaglandin E2 (PGE2) by HRS cells
inhibits CTL function with impairment of the IFN-γ production and
induction of theTh2 and T-reg expansion.[29,38-40]
Myeloid Derived Suppressor Cells
Recent investigations suggest that a subset of myeloid cells, the so
called “myeloid-derived suppressor cells” (MDSCs) are the progenitors
of tumour associated macrophages,[41-43]
that are considered among the most important and emerging prognostic
factors in HL.[44]
MDSC have been identified in solid and haematological cancers as a
heterogeneous population of immature and mature cells of myeloid origin
able of leading the tumour escape from immune-surveillance, through
depletion of arginin and cystein due to the high expression level of
arginase (Arg-1), nytrosylation of T-cell receptor, reactive oxygen
species (ROS) release, thus being responsible of cancer progression as
recently reviewed.[41,45,46] The
term suppressive refers to the peculiar ability to elicit T-cell anergy
thanks to the above-mentioned biochemical pathways.
In mice two distinctive mononuclear (Ly6G-, low “side-scattered
light”-SSC) and polymorphonuclear (Ly6G+, high SSC) tumour-induced MDSC
have been identified, while the phenotype in humans is still
controversial.[47] Overall,
current evidence suggests
a complex alteration of myeloid cell differentiation and function in
human cancer patients that involves polymorphonuclear[48]
and monocytic cells.[49] A
frequently used combination of markers for human MDSC includes CD33+/CD11b+/HLA-DR− and CD14+/HLA-DRlow to define
monocytic MDSC (mo-MDSC), CD66b+/CD15+/CD11b+/CD14− or CD11b+/CD13+/CD15+/CD14-/HLA-DR-/Lin-for the
identification of granulocytic MDSC (N-MDSC) and CD13+/CD14-/CD34+/HLA-DR- for the
immature subset MDSC (im-MDSC).
T cell dysfunction induced by MDSC can reflect the recruitment of
inflammatory cells and favour the aberrant MDSC production, setting up
a pathological loop.[45]
Our group hypothesized that the amount of MDSC in peripheral blood of
cHL-patients may reflect the complexity of cytokine and cell-cell
contacts of the pathologic neoplastic microenvironment and that the
myeloid cellular impairment could represent a prognostic factor in cHL
at diagnosis. Preliminary data from our single-centre small series of
60 newly diagnosed cHL-patients identify an increase of the absolute
count of im-MDSC, N-MDSC and mo-MDSC in peripheral blood at diagnosis
(Romano, manuscript submitted).
Progression free survival of patients carrying high levels of MDSC at
baseline was poor. In multivariate analysis, im-MDSC high levels were
an independent predictor of inferior outcome despite a PET-2 based risk
adapted treatment (Romano, manuscript submitted).
Soluble Factors
TARC.
The CC chemokine ligand 17 (CCL17), also well-known as Thymus and
Activation-Regulated Chemokine (TARC), is a member of the CC chemokine
group constitutively expressed in the thymus. TARC is produced by
monocyte-derived dendritic cells and binds specifically to the CC
chemokine receptor 4 (CCR4), mainly expressed on T-regs and Th2 cells
of the reactive infiltrate.[50]
TARC is considered a Th2-type chemokine because CCR4-expressing T cells
mainly produce interleukin (IL)-4.
In more than 90% of cases HL lymph-nodes have a positive TARC staining
in HRS cells detected by immunohistochemistry, with high specificity
for HL since the low/absent expression in anaplastic large cell
lymphoma or T-cell-rich B-cell lymphoma.
In about 85% of patients, TARC is detectable and elevated in serum at
diagnosis before treatment.[51,52] Pre-treatment serum TARC levels
correlate with stage of disease, erythrocyte sedimentation rate,
leukocyte and lymphocyte counts,[51,52]
pre-treatment
metabolic tumor volume, as measured by quantification of
2-[18F]fluoro-2-deoxyglucose positron emission tomography images, and
to treatment response.[53]
HLA-G.
The expression of non-classical Human leukocyte antigen G (HLA-G) is
another strategy adopted by HRS cells to evade immune defence and to
create protected niches where they grow and expand. HLA-G is expressed
on HRS cells in more than 50% of HL patients and is associated with
lack of HLA class I expression and tumour cell EBV status. HLA-G is a
non-classical major histocompatibility complex (MHC) Class I product
with limited sequence variability. The HLA-G gene generates seven
isoforms by alternative splicing encoding HLA-G1, -G2, -G3, and -G4
membrane-bound protein isoforms and HLA-G5, -G6, and -G7 soluble
protein isoforms.
The properties of soluble and membrane-bound HLA-G proteins are
different, but in general, both are regarded as being
immunosuppressive.[54]
HLA-G is a tolerogenic molecule which inhibits cytolysis mediated by NK
cells or T lymphocytes, induces T cell apoptosis and blocks
transendothelial migration of NK cells and these roles are performed
upon binding the KIR2DL4 and the ILT2 and ILT4 ligands.[55]
It is known that antigen-presenting cells expressing membranous HLA-G
can induce regulatory T cells in freshly isolated peripheral blood
mononuclear cells, in vitro and that soluble HLA-G induces regulatory T
cells in an antigen non-specific manner.[56]
The latter can inhibit CTL responses and is present in the cHL reactive
infiltrate.[57]
Alterations in HLA-G antigen expression and function are often induced
in tumours and are likely to be mediated by various microenvironmental
factors. Interestingly, immunohistochemistry and flow cytometry
evaluations have shown expression of HLA-G protein in a large number of
solid and some hematopoietic malignancies, e.g. cutaneous lymphomas,
chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma.
In CLL, some B-cell, T-cell non-Hodgkin’s lymphomas, and leukemia,
plasma levels of soluble HLA-G are increased. Soluble HLA-G serum and
plasma levels have been useful markers for the prediction of some of
these malignancies.[57]
A population-based study showed that protein expression of HLA-G by HRS
cells is common at primary cHL diagnosis and that this expression is
associated with lack of EBV and absence of cell surface expression of
MHC Ia on HRS cells. The consequence of HLA-G expression or sHLA-G is
an escape from T and NK cell-mediated recognition. Thus, alterations of
non-classical and classical HLA class I antigens and components of the
antigen-processing pathway provide tumour cells with different
mechanisms to inactivate immune responses resulting in tumour growth
and evasion from host immune surveillance.[7]
sCD163.
Recently, an increasing interest has been focused on the amount of
CD68+ tumor associated macrophages (TAM) infiltration.44 The amount of
TAM is strongly associated with shortened survival in cHL, correlated
with likelihood of relapse after autologous stem cell transplantation
and outperformed the current International Prognostic Score (IPS) for
disease-specific survival.[44]
The functional characterization of TAM is still to be performed and,
possibly, differences in survival among patients could be explained by
the macrophages M1/M2 binary on which these cells differentiate, or by
the histological signature of myeloid derived immunosuppression.
Increasing evidences in mammary tumor model suggests that the most
immunosuppressive activity is played by TAM derived from circulating
MDSC, but it is still an open question.
The antigen CD163 is physiologically expressed on the macrophage
surface, and it is currently investigated as an additional marker of
macrophage infiltration in HL microenvironment, since the lack of
reproducibility of CD68 staining. An increased infiltration of
CD163/CD68 (M2 macrophages) was associated to poor outcome, with a rise
in treatment-related deaths and poor event-free survival,
disease-specific survival and overall survival.[58]
Recently, the circulating fraction of CD163 in serum (s-CD163) has been
evaluated in patients at diagnosis and relapse, showing not being
inferior to TARC to identify patients with poor outcome.[59]
Conclusions
Despite high initial cure rate, almost 20% of cHL patients fails front
line therapy and have a median overall survival less than three years.
Increasing evidences suggest that failure to conventional therapy is
not only due to the intrinsic resistance of HRS cell but accessory
cells and the so called microenvironment play an important role. The
network and the relationship between the HRS and accessory cells are
not fully elucidated, but several studies have highlighted new pathways
that currently are under investigation as prognostic markers, including
HLA-G, s-CD163 and MDSC. In addition, new immunological target are
emerging in cHL microenvironment, including NK, NKT and MDSC that in
the future could be treated with specific drugs. Actually, the
introduction of targeted immunotherapy has induced an increasing
interest about the prognostic implication of the microenvironment and
its manipulation with drugs able to elicit an immune response.
References
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