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Abstract
Background
HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases.
Results
As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde.
Conclusion
The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G.
While TGF-β is able to regulate miRNA expression in numerous cell types, TGF-β-dependent changes in the miRNA profile of CD8+ T cells had not been studied before. Considering that TGF-β suppresses CD8+ T cell effector functions in numerous ways, we wondered whether induction of immune-regulatory miRNAs could add to the known transcriptional effects of TGF-β on immune effector molecules. In this study, we used miRNA arrays, deep sequencing and qRT-PCR to identify miRNAs that are modulated by TGF-β in human CD8+ T cells. Having found that the TGF-β-dependent downregulation of NKG2D surface expression in NK cells and CD8+ T cells does not go along with a corresponding reduction in mRNA levels, this pathway appeared to be a possible target of TGF-β-inducible miRNAs. However, this hypothesis could not be confirmed by miRNA reporter assays. Instead, we observed that DAP10 transcription is suppressed by TGF-β which in turn negatively affects NKG2D surface expression. In spite of promising preliminary experiments, technical difficulties associated with the transfection of primary NK cells and NK cell lines unfortunately precluded the final proof of this hypothesis.
Instead, we focused on the TGF-β-induced changes in the miRNome of CD8+ T cells and confirmed the induction of the miR-23a cluster members, namely miR-23a, miR-27a and miR-24 by three different techniques. Searching for potential targets of these miRNAs which could contribute to the immunosuppressive action of TGF-β in T cells, we identified and confirmed a previously unknown regulation of IFN-γ mRNA by miR-27a and miR-24. Newly generated miRNA reporter constructs further revealed that LAMP1 mRNA is a target of miR-23a. Upon modulation of the miR-23a cluster in CD8+ T cells by the respective miRNA antagomirs and mimics, significant changes in IFN-γ expression confirmed the functional relevance of our findings. Effects on CD107a/LAMP1 expression were, in contrast, rather minimal. Still, overexpression of the miR-23a cluster attenuated the cytotoxic activity of antigen-specific CD8+ T cells. Taken together, these functional data reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8+ T-cell effector functions, even in the absence of TGF-β signaling.
Ovarian cancer (OvCa) is the tumor with the most unfavourable prognosis among all gynaecological malignancies causing more than 6000 deaths per year in Germany alone. Patients with OvCa show symptoms at very advanced stages of tumor progression when the only available treatments consist on tumor debulking surgery and administration of platinum based chemotherapeutics and anthracyclins. There is an urgent need to develop new therapeutical strategies since the actual 5 year survival rate of OvCa patients does not exceed 20-40%.
Immunotherapy is a promising approach for treatment of ovarian cancer, since it has been observed that immunological parameters can influence the outcome of the patient. The aim of our research is to overcome tumor immune escape by counteracting the immunosuppressive mechanisms developed by the tumor. In particular, this work studies the influence of adenosine generated by the ectonucleotidases CD39 and CD73 in the tumor microenvironment. Cellular expression of CD39 and CD73 contributes to immunosupression as these ectonucleotidases convert immune-stimulatory extracellular ATP into immunosuppressive adenosine. This was primarily described as effector mechanism for regulatory T cells, but may also be important in the tumor microenvironment.
Having found that tumor cells from OvCa-patients express high levels of ATP-depleting ectonucleotidases CD39 and CD73 we set out to investigate a potential immunosuppressive mechanism via adenosine production in the tumor microenvironment. We could measure 30-60 times higher adenosine production by OvCa cell lines and ascites-derived cancer cells as compared to physiological normal conditions. To confirm this putative immune escape mechanism we investigated its effect on several immune cell populations. CFSE-based assays, for example, showed an inhibition of CD4+ T cell proliferation by OvCA cell-derived adenosine. In this context, we have further established an in-vitro assay, where OvCa cells modulate the function of macrophages towards a M2 or tumor associated (TAM) phenotype. Together with the phenotype modulation adenosine exerts chemotactic effects on human monocytes and is thus likely to attract myeloid precursor cells towards the tumor tissue. Moreover, in a microenvironment that is shaped by OvCa cells, human monocytes differentiate into M2 macrophages or TAMs which themselves express significant levels of the adenosine-generating ectonucleotidases CD39 and CD73.
Investigating the regulation of ectonucleotidase expression, we also observed that approaches clinically used to treat OvCa (namely application of doxorubicine or irradiation) influence CD73 and CD39 levels of OvCa and immune cells in vitro. In this study we show how this treatment-induced change in the ATP/adenosine ratio modulates the effector function of different immune cells. Furthermore, we investigate the potential benefit of clinically available small molecule inhibitors for CD39 and CD73 that could relieve immunosuppression in the tumor microenvironment especially in combination with common treatment regimes.
DHEA is a precursor for the male and female sex hormones testosterone and estradiol, which are mainly secreted from the testes and the ovary, respectively. In addition, epidemiological studies showed that low serum levels of DHEA and DHEAS correlate with the incidence of autoimmune disease, cancer and cardiovascular disease. In vitro, DHEA and DHEAS influenced glucose metabolism in a favourable manner. However, positive effects of DHEA substitution were only significant adrenal insufficiency in women.
Steroid sulphotransferase 2A1 (SULT2A1) is the responsible enzyme for sulphonation of DHEA to DHEAS which is thought to be the inactive form of DHEA. In this role, SULT2A1 acts as a central regulator of steroid synthesis because sulphonation of DHEA withdraws the substrate for further downstream conversion. Another essential cofactor for sulphonation is PAPS, which is produced by the enzyme PAPS synthase (PAPSS) from ATP and anorganic sulphate. PAPSS exists in the different isoforms PAPSS1 and PAPSS2 and splice variants PAPSS2a and PAPSS2b. Changes in PAPSS activity are thought to influence sulphonation of DHEA significantly. However, neither regulation of PAPSS nor its influence on SULT2A1 have been investigated in human cell lines or humans.
The main goal of this thesis was to analyze the enzyme expression of the DHEA/DHEA shuttle, i.e. mRNA and protein of SULT2A1, PAPSS1 and PAPSS2, in various human cell lines. Furthermore, I investigated which cell line could serve as a suitable model for further research regarding regulation of SULT2A1, PAPSS1 and PAPSS2.
Here, I could show that the enzymes of the DHEA/DHEAS shuttle were expressed in the human adrenal cell line NCI-h295R as both mRNA and protein. In enzyme assays, I was able to prove conversion of DHEA to DHEAS as well as to different other steroids. However, applying Trilostane, a potent inhibitor of CYP3B, effectively directed conversion of DHEA to DHEAS. Using these findings, future experiments can investigate for example the influence of certain cytokines or endocrine disruptors on expression and activity of PAPSS1/2 and on sulphonation of DHEA. In particular, the relatively equal expression of PAPSS1 and PAPSS2 will enable us to do knock down experiments with siRNA to elucidate how the activity of one enzyme changes when the other one fails.
Sulphonation of DHEA by SULT2A1 is thought to happen in the cytoplasm or more precisely in the Golgi apparatus. However, experiments in transfected cells have shown both a cytoplasmatic and a nuclear localisation when both enzymes were expressed at the same time. Immunocytochemistry revealed the same results in the adrenal cell line NCI-h295R, where both enzymes were expressed strongly in the nucleus. The physiological role is not clear and requires further research. Presumably, sulphate is activated in the nucleus. However, one could also speculate that a shift of PAPSS to the nucleus could generate a reservoir, which can be activated by re-localisation to the cytoplasm when more PAPS is needed.
Expression of SULT2A1 in some foetal tissues has been investigated earlier. Whilst in adult human cartilage PAPSS1 is predominant, in newly born hamsters PAPSS2 is more abundantly expressed. The expression of PAPSS isoforms in highly sulphonating tissue has not been investigated in humans, so far. This work demonstrated a differential expression of SULT2A1, PAPSS1 and PAPSS2 in adult and foetal liver, adrenal and foetal cartilage tissue. In adult and foetal adrenal expression was similar. However, foetal and adult liver differed in the expression of SULT2A1, which was expressed much more in adult tissue. Most importantly, in foetal cartilage there was only a low expression of SULT2A1 and PAPS seems to mostly provided by PAPSS1, which was considerably higher expressed in cartilage than in other tissues. In contrast, PAPSS2 was mainly expressed in adult and foetal adrenal.
Additionally, we reported a case of a female patient who had been investigated for hyperandrogenism. Two mutations in the PAPSS2 gene had led to massively reduced serum levels of DHEAS. One heterozygous mutation in the domain of the APS kinase of the PAPSS2 protein leads to substitution of one amino acid at position 48 (T48R). In vitro experiments showed a residual activity of 6% for this mutation. A second mutation in the ATP sulphurylase domain of PAPSS2 was found. The introduction of thymidine instead of cytidine leads to a stop codon, which is presumed to truncate the protein at position 329 (R329X). In vitro, no residual activity was seen for this mutation. The lack of PAPS reduces sulphonation of DHEA but also sulphonation of proteoglycanes, which leads to skeletal abnormalities. The abundance of DHEA enables massive downstream conversion to androgens leading to clinical features of hyperandrogenism. Regarding the bone abnormalities, it is interesting and surprising that activity of PAPSS1 compensated to a great extent in cartilage but was not able to keep up a more considerable sulphonation of DHEA. Possibly, the subcellular localisation might play a role in this scenario.
Despite accounting for only a small proportion of all skin cancers, malignant melanoma
displays a serious health risk with increasing incidence and high mortality rate. Fortunately,
advances in the treatment of malignant melanoma now prolong survival and enhance response
and treatment efficacy. Established biomarkers help evaluate disease progression and
facilitate choosing appropriate and individual treatment options. However, the need for easily
accessible and reliable biomarkers is rising to predict patient-specific clinical outcome.
Eosinophil infiltration into the tumor and high peripheral eosinophil counts prior and during
treatment have been associated with better response in patients for various cancer entities,
including melanoma. An analysis of a heterogeneous study cohort reported high serum ECP
levels in non-responders. Hence, eosinophil frequency and serum ECP as a soluble
eosinophil-secreted mediator were suggested as prognostic biomarkers in melanoma. We
examined whether melanoma patients treated with first-line targeted therapy could also benefit
from the effects of eosinophils. In total, 243 blood and serum samples from patients with
advanced melanoma were prospectively and retrospectively collected before and after drug
initiation. To link eosinophil function to improved clinical outcome, soluble serum markers and
peripheral blood counts were used for correlative studies using a homogeneous study cohort.
In addition, functional and phenotypical characterizations provided insights into the expression
profile and activity of freshly isolated eosinophils, including comparisons between patients and
healthy donors.
Our data showed a significant correlation between high pre-treatment blood eosinophil counts
and improved response to targeted therapy and by trend to combinatorial immunotherapy in
patients with metastatic melanoma. In accordance with previous studies our results links
eosinophil blood counts to better response in melanoma patients. High pre-treatment ECP
serum concentration correlated with response to immunotherapy but not to targeted therapy.
Eosinophils from healthy donors and patients showed functional and phenotypical similarities.
Functional assays revealed a strong cytotoxic potential of blood eosinophils towards
melanoma cells in vitro, inducing apoptosis and necrosis. In addition, in vitro cytotoxicity was
an active process of peripheral eosinophils and melanoma cells with bidirectional features and
required close cell-cell interaction. The extent of cytotoxicity was dose-dependent and showed
susceptibility to changes in physical factors like adherence. Importantly, we provide evidence
of an additive tumoricidal function of eosinophils and combinatorial targeted therapy in vitro. In
summary, we give valuable insights into the complex and treatment-dependent role of
eosinophils in melanoma. As a result, our data support the suggestion of eosinophils and their
secreted mediators as potential prognostic biomarkers. It will take additional studies to
examine the molecular mechanisms that underlie our findings.