Chemokine microenvironment in primary central nervous system lymphoma

Primary central nervous system lymphomas (PCNSL) are aggressive extranodal malignancies confined to the central nervous system (CNS), mostly of diffuse large B cell histotype. Despite improved understanding of the malignant B cells, little is known on the tumor microenvironment and on the response of the adaptive immunity against PCNSL. The majority of B-cell lymphomas, including PCNSL originate from germinal center (GC) B cells. The GC is the main source of memory B cells and plasma cell generation, which produce high affinity antibodies and are necessary to protect us against invading microorganisms. However, the beneficial role of GC B cells in immunity is counterbalanced by their detrimental role in lymphomagenesis. Germinal center B cells express a distinct set of chemokine receptors, which regulate their migration and positioning during and after germinal center reaction. Similar to centrocytes and centroblasts, malignant B cells derived from germinal centers can retain a particular set of chemokine receptors, which allows them to respond to their cognate ligands expressed in the microenvironment. Therefore investigation of lymphocyte chemoattractants in secondary lymphoid organs as well as in extranodal lymphomas does not only improve our understanding of B cell trafficking within secondary lymphoid organs, but also helps us understanding tumor cell distribution and dissemination of malignant B cells as well as tumor infiltrating lymphocytes. Gene expression analysis has previously shown, that malignant B cells in PCNSL resemble late germinal center B cells and express classical B cell chemokine receptors. This work focuses on the chemokine microenvironment and the potential role of bystander cells in PCNSL and their effects on malignant B cells as well as tumor infiltrating lymphocytes. The project includes four major sections: 1. Analysis of the expression of B cell attracting chemokines under normal and inflammatory conditions in human secondary lymphoid organs. 2. Analysis of T and B cell attracting chemokines in extranodal CNS lymphomas, 3. Analysis of type, density and localization of tumor infiltrating lymphocytes in PCNSL, 4. Analysis of the effect of coexpressed chemokines in PCNSL on the migratory responses of tumor-infiltrating lymphocytes and malignant B cells. We show by immunhistochemistry and in-situ hybridization, that there is a specific expression pattern of homeostatically expressed chemokines CXCL12, CXCL13 and CCL21 in normal, non-inflamed human secondary lymphoid organs. Under inflammatory conditions, the expression pattern of macrophage- derived CXCL12 and follicular dendritic cell- derived CXCL13 within the germinal center changes significantly. Within the germinal center, macrophage-derived CXCL12 and follicular dendritic cell-derived CXCL13 build a meshwork in which germinal center B cells reside. While CXCL13 shows a clear gradient between the dark and the light zone of the germinal center in human secondary lymphoid tissue, we could not detect a clear gradient for CXCL12 between the two zones. Analysis of CXCL12 and CXCL13 in primary central nervous system lymphoma showed an expression pattern similar to the one in germinal centers of secondary lymphoid organs. In addition, we identified a fraction of CXCL13-expressing lymphocytes in PCNSL. CXCL13-expression is a hallmark of germinal center T cells known as follicular T helper cells (TFH), which provide help to germinal center B cells. Yet, the majority of tumor infiltrating lymphocytes in PCNSL are CD8+ T cells, which show Granzyme B activity and vigorous proliferation. Tumor infiltrating CD8+ T cells show a higher frequency in the perivascular areas of small and intermediate vessels in PCNSL. They accumulate in areas with high expression of the inflammatory chemokine CXCL9. Perivascular CXCL9 is upregulated by perivascular macrophages and pericytes under inflammatory conditions in the CNS, indicating an important role for pericytes and perivascular macrophages in the recruitment of tumor infiltrating lymphocytes. Moreover, CXCL9 and CXCL12 are coexpressed on the tumor vasculature within PCNSL and can form heterocomplexes. Our in-vitro experiments show, that in the presence of CXCL9, CXCL12-induced migration is enhanced not only on CXCR4+/CXCR3+/CD8+ T cells but also on CXCR4+/CXCR3- malignant B cells. Our findings indicate, that malignant B cells in PCNSL may encounter a germinal-center like chemokine environment, which traps malignant B cells within the CNS. In addition, our results reveal the presence of a strong chemoattractant stimulus in the perivascular microenvironment, which might serve as regulator for the recruitment of tumor infiltrating lymphocytes and for the angiocentric positioning of malignant B cells in the perivascular cuff.