mesothelioma cancer

Although there is a considerable body of published literature concern¬ing the
putative role of immune status in the pathogenesis and pro¬gression of common
malignancies such as lung and breast cancers, this area of research previously
had been relatively neglected with respect to malignant mesothelioma, a
comparatively uncommon tumor. Over the past decade, however, the development of
animal mesothelioma models and the widespread availability of mesothelioma cell
lines to researchers has focused increasing interest in this area. Furthermore,
occupational and environmental asbestos exposure hitherto had been regarded as
the most important global causes of mesothelioma and, since inhaled asbestos
fibers have been shown to suppress innate cel¬lular immunity, studies of
asbestos-exposed individuals and of ex-perimental asbestos exposure have
provided valuable insight into how altered immune status may allow mesothelial
tumors to escape immune surveillance. It is also conceivable that variability in
host immune status, coupled with individual differences in genetic
suscep¬tibility to mesothelioma among similarly exposed subjects (1,2), may
account for the considerable variation in incidence of mesothelioma in different
exposure settings, which can span two orders of magnitude (3–5). Given the now
well-recognized association of simian virus 40 (SV40) with malignant
mesothelioma (6), opportunities now exist to study the immune status and to
develop vaccination protocols of seropositive subjects at risk.

Innate Immunity Against Mesothelioma Cells

Non–major histocompatibility complex (MHC)-restricted cytotoxic lym-phocytes
have the capacity to lyse tumor cell targets of various origins and comprise
natural killer (NK) cells, NK T cells, and gd T cells (7). All are derived from
a common lymphoid precursor but differentiate along separate pathways. Whereas
NK cells are CD56+ but lack the CD3 and T-cell receptor markers, NK T cells and
gd T cells coexpress CD3 as well as differing forms of the T-cell receptor.
Unlike conventional T cells,

gd T cells do not express CD4 or CD8. It is of interest that established gd T
-cell clones have been shown to demonstrate varying degrees of cytotox-icity
against individual mesothelioma cell lines (8). The NK cells can be stimulated
to proliferate ex vivo by the addition of interleukin-2 (IL-2) to produce
lymphokine-activated killer (LAK) cells (9), which have been shown to exhibit a
broader spectrum of tumoricidal activity than do NK cells (10). Similarly, NK T
cells can be greatly expanded ex vivo by the timed addition of interferon-g
(IFN-g), IL-2, and anti-CD3 to generate cytokine-induced killer (CIK) cells (7).
All these cell types are involved in innate immune responses to tumors, but they
lack the capacity for immunologic memory.

In cancer-bearing hosts, NK cells have been considered to be the major component
of antitumor immunity responsible for rapid elimi¬nation of malignant cells, and
there is evidence that adoptively trans¬ferred LAK cells can selectively
localize in solid tumors tissue and eliminate established tumors (11). However,
there have been few clin¬ical studies of NK or LAK cell function in mesothelioma
patients. In one study, 70% of patients demonstrated significantly depressed NK
cell activity, a finding that was improved by the addition of IFN-g to the
patients’ peripheral blood lymphocytes in vitro (12). In this regard, it should
be noted that NK cells have inhibitory receptors whose ligands are MHC class I
molecules (7,13). This could provide one expla¬nation as to why mesothelioma
patients have impaired NK cell killing, since mesothelioma cell lines are known
to express human leukocyte antigen (HLA) class I molecules constitutively
(14,15). In another study, patients demonstrated significant reduction of LAK
cell activity against mesothelioma cell targets, a phenomenon that appeared to
be due to prostaglandin-induced immunosuppression, since LAK functional activity
was restored by the addition of 10mg/mL of indomethacin to the patients’
lymphocytes (16). Nevertheless, LAK cell killing of both freshly explanted
mesothelioma cells and established mesothelioma cell lines has been shown to be
significantly greater than that exhibited by NK cells (17).

Asbestos Exposure and Innate Immunity

Since asbestos workers are at risk of developing mesothelioma and other cancers,
clinical studies of such individuals have provided insight into how tumors might
escape immune surveillance in susceptible individuals with disordered
immunoregulation. There is substantial evidence that systemic T-cell function is
depressed in asbestotic subjects. Several studies have shown that such
individuals manifest impaired peripheral blood T cell responsiveness to
mitogens, findings that were independent of age or smoking status (18–21).
Defective lymphokine generation also has been described in association with
asbestosis (22). When delayed-type hypersensitivity responses were assessed in
asbestos workers and in unexposed subjects, a disproportionate number of
individuals with radiologically detectable asbestosis demonstrated cutaneous
anergy to either de novo (2,4-dinitrochlorobenzene) or recall

(tuberculin, streptokinase-streptodornase, or Candida albicans) antigens
(18,23,24). Moreover, there was a good correlation between the presence of
cutaneous anergy and the duration of asbestos exposure. Likely explanations for
these findings include the presence of circulating “inhibitors” of lymphoid cell
activation (18,25) and the selective loss of circulating T-suppressor (TS) cells
that has been demonstrated in patients with asbestosis (20). The fact that
chrysotile asbestos fibers can induce the temporary downregulation of cell
surface CD4 and CD45RA expression in cultured human peripheral blood mononuclear
cells (26) provides additional support for the latter notion, since CD4+ CD45RA+
T cells have been shown to induce the activation of CD8+ TS cells (27). Whatever
mechanisms are involved, the impairment of innate immu¬nity that has been
detected in asbestos workers may be a consequence of the translocation of
inhaled asbestos fibers to lymph nodes and to the spleen (28–30).

The use of flow cytometry has demonstrated notable differences in the
proportions of T-cell subsets in the blood and bronchoalveolar lavage (BAL) of
asbestos-exposed subjects when compared with those in unexposed individuals.
Peripheral lymphopenia has been well described in association with asbestosis
(21,31–33), and in one study the intensity of the lymphopenia correlated with
the radiographic severity of asbestosis (33). The number of circulating T cells
(as defined by CD3 and other pan–T-cell markers) also has been shown to be
sig¬nificantly reduced in asbestotic subjects compared with that in unex-posed
controls (21,31–35). Analysis of T-cell subsets in the peripheral blood
generally has demonstrated a reduction in the proportions of T cells expressing
either the helper/inducer (CD4+) or suppressor/cyto-toxic (CD8+) phenotypes. A
correlation also has been shown between the intensity of asbestos exposure and a
decreased blood CD8+/CD4+ ratio when corrections were made for the confounding
effects of smoking (33,34).

Several studies have analyzed the composition of BAL lymphoid cell populations
in asbestos-exposed individuals (32,35–37). A notable finding in most of these
studies has been a lymphocytic alveolitis, char¬acterized by increased
percentages and numbers of BAL lymphocytes and CD4+ cells with reductions in the
proportions of CD8+ cells. These changes were most likely to occur in
asbestos-exposed subjects with radiographic stigmata of asbestosis or
asbestos-related pleural injury.

Asbestos exposure also is known to influence NK function. Asbestos fibers were
shown to impair peripheral blood and BAL NK cell activ¬ity in vitro, an effect
that was induced by all commercial types of asbestos and that was prevented by
pretreatment of the lymphoid cells with recombinant interleukin-2 (rIL-2) (38).
The same group subse¬quently showed that chrysotile (but not amphibole) fibers
also sig¬nificantly suppressed the in vitro activity of LAK cells from normal
subjects (39). Although one study has demonstrated normal NK func¬tional
activity in asbestos workers (38), others have shown that NK function declines
with increasing intensity or duration of asbestos exposure (40–42). Immune
abnormalities that are detectable in asbestos workers are summarized in Table
10.1.

Tumor-Infiltrating Lymphocytes (TIL) in Mesothelioma

Lymphocytic infiltrates are not usually prominent within the stroma of
mesothelioma biopsies and there are few reports of their possible sig¬nificance.
An early South African study of 58 cases noted that 94% of those having a
minimal or absent lymphocytic reaction had a mean sur¬vival of 9 months after
diagnosis, whereas two thirds of those having a significant lymphocytic response
survived longer than 18 months after diagnosis (43). The authors concluded that
the presence of a sig¬nificant lymphocytic infiltration was indicative of a
better prognosis. A recent British study of only 15 mesothelioma cases has,
however, disputed this assertion (44). Nevertheless, the earlier authors’
conclu¬sions are supported by an anecdotal report of transient, spontaneous
regression of a pleural mesothelioma characterized by a prominent lymphocytic
infiltrate in association with serologic reactivity against autologous
mesothelioma antigens, findings that essentially disap-peared when the patient
eventually succumbed to her tumor (45).

Adaptive Immunity Against Mesothelioma

There is evidence that mesotheliomas sometimes can be immunogenic and that
affected patients are capable of mounting specific serologic responses to their
tumors. In one study of 29 mesothelioma patients, 28% manifested high-titer
immunoglobulin G (IgG) antibodies detect¬able by Western blot analysis against a
variety of different antigens, some of them nuclear, expressed by a range of
tumor cells of differing lineage (46). Moreover, antibody titer also increased
in tandem with disease progression. In contrast, normal sera displayed no such
reac¬tivity. In a follow-up study, the same group identified six
patient-specific nuclear antigens using the technique of serologic
identification by recombinant expression cloning (SEREX) and pooled patients’
sera as the probe against an expressed complementary DNA(cDNA) library

derived from a cloned mesothelioma cell line (47). However, none of the antigens
detected were uniquely expressed in mesothelioma cells, and it is not clear
whether the patients’ serologic responses necessar¬ily conferred immune
protection against their tumors.

Of central importance is the ability of cancer patients to mount an effective
cell-mediated immune response against their tumors by generating effector CD8+
cytotoxic T lymphocytes (CTL) against tumor-associated antigens. Efficient
activation of CTL requires tumor-associated peptides to be presented in the
context of membrane-bound MHC class I complexes, the presence of membrane
co-stimulatory molecules such as CD80 and CD86 on antigen-presenting cells, the
induction of IL-2 and other cytokines by CD4+ helper T cells, and the
differentiation of recruited CD8+ T cells into CTL (48). As is the case with
many other cancers, however, the milieu is not conducive for efficient
activation of CTL in patients with mesothelioma, allowing the tumor to evade the
host’s adaptive response. There are a number of possible explanations for this
state. These include altered MHC class I phenotypes on tumor cells (49),
inadequate expression of co-stimulatory molecules (50,51), insufficient numbers
of tumor-specific CD4+ helper T cells that may be needed to synergize with CTL
(52), secretion of transforming growth factor-b (TGF-b), generation of reac¬tive
nitrogen species (RNS), upregulated tumor cyclooxygenase-2 (COX-2) expression
and prostaglandin E2 secretion (53,54), and the gen¬eralized immunosuppression
associated with advanced cancer (48).

A daunting challenge to circumvent the possibility of tumor escape in
individuals at risk of developing mesothelioma (e.g., asbestos workers or
SV40-exposed subjects) would be to vaccinate them against putative
mesothelioma-associated antigens in order to generate CTL in immunologically
uncompromised hosts. One study took a step in that direction by identifying
seven candidate peptides within the Tag protein domain required for SV40
transformation that could bind and stabilize HLA-A*0201 molecules, the most
widely expressed human MHC allelic product (55). Two of those peptides were
shown to be immunogenic because they induced SV40 large-tumor antigen
(Tag)-specific CTL from healthy peripheral blood lymphocytes and one was found
to be endogenously processed by an SV40-transformed human mesothelial cell line.
Recently, other investigators have generated CTL from the peripheral blood of
one of three HLA-A2+ mesothelioma patients using one of the same candidate
peptides used in the earlier study (56). Activity of CTL was shown to be
directed against an HLA-A2.1+ mesothelioma cell line transfected with SV40 Tag
cDNA in an MHC class I–restricted manner.

Oxidant-Mediated Immunosuppression and Mesothelioma

A number of studies have shown that inhaled asbestos fibers can induce the
formation of reactive oxygen and nitrogen species in the lungs and pleura.
Iron-rich crocidolite asbestos fibers have been shown to gener-

ate hydroxyl radical (◊OH) formation via superoxide (◊O2-)-driven,
iron-catalyzed Haber-Weiss (Fenton) reactions, which have been implicated in
asbestos-induced injury, as evidenced by catalase-mediated inhi¬bition of
asbestos-induced mesothelial cell apoptosis (57) and lung inflammation and
fibrogenesis (58). The use of a rat asbestos inhalation model also has
demonstrated that both crocidolite and chrysotile asbestos fibers stimulate the
formation of reactive nitrogen species in vivo as a consequence of persistent
pleuropulmonary inflammation, macrophage recruitment, and cytokine secretion
(59–61). Notably, inhaled asbestos fibers induced upregulated inducible nitric
oxide syn-thase (iNOS) expression and nitric oxide radical (◊NO) production by
pleural macrophages (59,60) as well as nitrotyrosine formation in vis¬ceral and
parietal pleural mesothelial cells (60). Nitrotyrosine is a sur¬rogate marker
for peroxynitrite (ONOO-), a highly reactive oxidizing and nitrating species
that has been shown to activate the extracellular signal-regulated kinase (ERK)
signaling pathway by targeting the epi¬dermal growth factor (EGF) receptor,
Raf-1 and MEK independently (62). In this regard, it is significant that
crocidolite and chrysotile expo¬sure recently were shown to induce protracted in
vivo ERK activation in association with tyrosine nitration in the rat lung (61).
Since ERK acti¬vation can induce phosphorylation and stimulate the DNA-binding
activity of c-Fos, Fra-1, and other activator protein-1 (AP-1) transcrip¬tion
factors, it is conceivable that prolonged induction of targeted AP-1 family
members may lead to deregulation of cell proliferation and dif¬ferentiation that
may play a role in asbestos-mediated oncogenesis (63). Support for this notion
is provided by the recent demonstration that inhibition of MEK1 in the ERK
signaling pathway by the inhibitor PD98059 resulted in reversion of the
transformed phenotype in rat mesothelioma cell lines (64).

Several studies indicate that the generation of RNS can induce or potentiate an
immunosuppressive state by promoting apoptosis in T cells. Murine models of
trypanosomiasis (65) and histoplasmosis (66) are associated with
immunosuppression of antigen-specific lympho-proliferative responses and
elevated levels of apoptosis in splenocytes, findings that are significantly
reversible by treatment of infected mice with NG-monomethyl-L-arginine (L-NMMA),
an inhibitor of ◊NO production. Moreover, the in vitro addition of L-NMMA to
peripheral blood mononuclear cells from AIDS patients reduced lymphocyte
apoptosis and facilitated recovery of lymphoproliferative responses, whereas
co-incubation of the patients’ lymphocytes in vitro with ◊NO donors
significantly increased the severity of apoptosis (67). The addi¬tion of ONOO-
to normal human peripheral blood T cells also has been shown to suppress
mitogen- and CD3-mediated lymphocyte activation and proliferation by promoting
impaired tyrosine phosphorylation and apoptosis (68). It is noteworthy that
overexpression of iNOS has been detected in 74% to 100% of mesothelioma biopsies
but rarely or not at all in nonmalignant or nonreactive pleural tissues (54,69).
Moreover, culture supernatants of cytokine-treated human colon carcinoma cells
that contained high levels of ◊NO significantly suppressed human lym¬phocyte
mitogen-induced proliferation (70).

Transforming Growth Factor-b–Mediated Immunosuppression and Mesothelioma

Transforming growth factor-b (TGF-b) is a pleiotropic cytokine that exists in
three isoforms, TGF-b1, TGF-b2, and TGF-b3, and, when secreted, exists
predominantly in a latent form bound to a latency-binding peptide. When
activated by protease action, TGF-b mediates its effector functions via its
cognate receptors (TbR-I and TbR-II) and the Smad protein signaling pathway
(71). Tumor-specific mutations of TbR-I and especially of TbR-II have been
described in several different types of malignancies (72) as have mutations of
smad2 (73) and smad4 (74). Transforming growth factor-b appears to have a
biphasic role in tumorigenesis: in the early phases, it acts as a tumor
suppressor, whereas in the later stages, when tumor cells have escaped its
antipro-liferative effects and start to secrete high amounts of TGF-b, this
cytokine may act to promote tumor invasion and metastasis (75). Noteworthy in
this respect is the fact that both human and murine mesothelioma cells, as well
as cell lines derived from human, murine, and rat mesothe-liomas, have been
shown to express and secrete TGF-b (76–80), while antisense oligonucleotides
targeting TGF-b messenger RNA (mRNA) inhibited tumor cell proliferation (78) and
anchorage-independent growth (77). The importance of secreted TGF-b within the
local milieu of mesothelioma patients is underscored by the finding of TGF-b1
and TGF-b2 levels in pleural effusions caused by mesotheliomas that were three
to six times as high as those detected in effusions caused by primary lung
cancers (81). Genetic factors can influence the effects of TGF-b–mediated
signaling. Thus, in one study, TbR-I(6A), a polymorphic allele of TbR-I, was
identified as a tumor susceptibility allele in cancer patients (82), whereas
another study demonstrated that the 129J mouse strain was uniquely resistant to
the fibrogenic effects of asbestos and manifested a delayed response to the
fibroproliferative effects of TGF-b1 (83).

Immunosuppressive effects of TGF-b on innate and adaptive immu¬nity have been
noted in a number of model systems that have shown that this cytokine can
suppress lymphocyte activation, proliferation, and function both in vitro and in
vivo (84). Also, TGF-b has been shown to block LAK activity of human lymphocytes
in a dose-dependent fashion (85). Conversely, splenic LAK cell activity from
mice injected with a human glioma cell line was greatly enhanced when the glioma
cells were transfected with antisense TGF-b1 prior to being injected (86). In
another study, cloned NK T cells established from TIL in a B16 mel¬anoma that
were shown to secrete TGF-b were able to inhibit in vivo antitumor responses
(87). Furthermore, TGF-b–transduced dendritic cells in C57BL10 (H2b+) mice
showed marked impairment in allostim-ulatory activity of C3H/HeJ T cell (H2k+) T
cells in vitro and were able to prolong heart allografts’ survival in vivo (88).
Importantly, TGF-b also can inhibit the generation of CD8+ CTL both in vitro
(89) and in vivo (90), and T-cell–specific blockade of TbR-II–mediated signaling
has allowed mice to effectively overcome live tumor challenge via gen¬eration of
a tumor-specific immune response (91).

Asbestos-exposed individuals may especially be prone to the immunomodulatory
effects of TGF-b in the pleural microenvironment. All three TGF-b isoforms have
been detected in both lung parenchy-mal and pleural fibrotic lesions in lung
sections from Canadian asbestos miners and millers (92) and in the evolving lung
lesions of rats exposed to chrysotile asbestos by inhalation (93). Since pleural
plaques can coexist with mesothelioma (94), these could provide a ready source
of TGF-b production in some patients that might facilitate the pro¬gression of
their tumors. It is also possible that protracted asbestos-induced TGF-b
secretion in the lungs may diffuse across to the pleural space in a manner
analogous to that seen when rats transduced intra-tracheally with an adenovector
overexpressing the active form of TGF-b1 were shown to develop significant
pleural fibrosis (95).

Conclusion

Although malignant mesothelioma may not represent a classic immunogenic tumor,
there is abundant evidence suggesting that immune status may play an ancillary
role in determining host suscep¬tibility to the development and progression of
mesothelioma. As illus¬trated in Figure 10.1, it is conceivable that
cytokine-driven, persistent pleural inflammation and macrophage recruitment, in
association with locally generated reactive oxygen and nitrogen species and
TGF-b secretion, may provide a favorable milieu for the development and
progression of mesothelioma in genetically susceptible subjects who were
occupationally or environmentally exposed to asbestos. Similar considerations
possibly may pertain to erionite-exposed persons who developed mesothelioma in
the Cappadocian region of Turkey (2,96).

Attempts to boost the antitumor immune response in mesothelioma patients have
served as the basis for several immunotherapeutic clinical trials, which
generally have had disappointing results (97). These have included intrapleural
or intratumoral instillation of IL-2, IFN-g, or granulocyte macrophage
colony-stimulating factor (98–102), intrapleural infusion of activated
macrophages and IFN-g (103), and intrapleural gene therapy approaches using
replication-deficient adenovirus-mediated delivery of herpes simplex
virus–thymidine kinase (Ad.HSV-tk) to transduce the patients’ mesothelioma cells
(104,105). Some of the limitations in these approaches have been in the
selection of patients with advanced tumor stage or in whom there was inadequate
debulking of tumor prior to commencing immunotherapy.

Acknowledgments

This work was supported by grant HL-54196 from the National Insti¬tutes of
Health and grant MDA905-01-1-0001 from the U.S. Department of Defense.

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