Abstract:
Introduction: Cancer is one of the leading causes of death worldwide. While there are high hopes on immunotherapy to treat tumors, the roles of individual cell types contributing to the immunotherapeutic success are still incompletely understood. This dissertation is mainly concerned with the role of leucocytes – with a special focus on B cells. Understanding B cell function in anti-tumor immunity and their interaction with the microenvironment may help improve cancer therapy. Therefore, in the first part, leucocyte infiltration in the most common malignant primary brain tumor is assessed and associated with the immunological tumor milieu. In the second part, the role of cerebrospinal fluid (CSF) B cells is determined, the scope of their antibody dependent immunity is assessed and antigen targets are identified. Methods: In study 1, tumor tissues of 18 glioma patients (14 glioblastomas (GBM), 4 astrocytomas grade III (AA)) were stained for leucocyte infiltration by immunohisto-chemistry. Lymphocytes (CD45+), total macrophages (CD68+) and alternatively acti-vated pro-tumor macrophages (CD163+) were identified and their relative contribution to the immune cell composition was determined. Additionally, the levels of 30 cyto-kines were measured in the CSF of the respective patients, compared to non-inflam-matory controls and correlated with the immune cell infiltration pattern. In study 2, the B cell repertoire of three meningeosis neoplastica patients was determined from CSF B cells by single cell sequencing. Clonal expansion was analyzed and recombinant antibodies were produced for some clones. Circulating antibodies in blood and CSF were measured by mass spectrometry and resulting peptide sequences were com-pared to the CSF B cell repertoire. Moreover, a blood B cell repertoire was generated by next generation sequencing and screened for clonally related cells to the CSF B cell repertoire. Lastly, recombinant antibodies produced from CSF B cells were used to determine tumor specificity and antigen targets. Therefore, recombinant antibodies were used to stain primary fibroblasts of a healthy control subject and a malignant fibroblast cell line by flow cytometry and binding against target antigens was assessed by a protein microarray and enzyme linked immunosorbent assays (ELISAs). 9 Results: In study 1, macrophage infiltration was prominent in the glioma tissue, but lymphocyte infiltration was low. For CD45+ lymphocytes as well as for CD68+ macro-phages infiltration was higher in perivascular tissue than in the tumor core. CSF of glioblastoma patients showed a decreased level of interleukin-7 compared to controls and a tendency towards higher levels of interleukin-2, interleukin-6 and vascular en-dothelial growth factor. Additionally, CD68+ macrophages were negatively correlated with eotaxin, interferon-γ, interleukin-1β, interleukin-2, interleukin-10, interleukin-13, interleukin-16, vascular endothelial growth factor and the inflammatory cluster compo-nent derived by factor analysis. In study 2, CSF B cells were clonally expanded and overlapped with antibody sequences from CSF and serum. No clonal relation could be determined to the blood repertoire. Flow cytometric data showed similar staining pat-terns for healthy and malignant cells. Screening of recombinant antibodies against the protein microarray produced a large hit list with no clear cut results, but subsequent ELISAs uncovered relevant staining of various antibodies against DDX53, some mar-ginal staining of recombinant antibody F1-P3 against AKR1A1and MTUS2 and some marginal staining of recombinant antibody B8-P1 against AKR1A1, MTUS2 and ACY3 as well as F6-P1 against MTUS2. Conclusion: Lymphocyte levels in gliomas are low with macrophages being the major infiltrating leucocyte. Cytokine levels in the CSF of glioma patients appear to be dysregulated as in chronic inflammation. This may support tumor proliferation and growth by providing stimulating factors while protecting the tumor from immune attack. These cytokine levels are associated with immune cell infiltration and understanding the precise mechanisms of this interaction may help to induce an anti-tumor microen-vironment increasing the efficiency of immunotherapies. In contrast, study two shows that CSF B cells in metastatic melanoma patients are clonally expanded and produce functional antibodies which can be found in serum and CSF. DDX53, a cancer testis antigen, was identified as a promising target for immunotherapy as it is hardly ex-pressed in healthy somatic tissues. Additionally, since CSF B cell receptors bound this target, an immune response is likely to be triggered by this antigen. Identification of suitable targets for immunotherapy is critical for treatment efficiency in the future.
