Role of IRF-4 in the development and maintenance of CLL

Chronic lymphocytic leukemia (CLL) develops in a complex network of immunological interactions. Disease progression is dependent on an interplay between antigen receptor stimulation and T cell support that has relevant parallels with normal immune reactions. The transcription factor IRF-4 has been shown to be a central regulator of B-cell fate in shaped immune environments such as the germinal center (GC). Single nucleotide polymorphism (SNP) analyses point to a role of IRF-4 in the establishment of CLL disease, but the mechanisms involved and the exact role of the gene in modulating the CLL phenotype is elusive. Our preliminary data from human CLL show a potentially relevant role of IRF-4 in modulating proliferative responses within the CLL microenvironment, as well as a relation to prognostic outcome. This proposal aims to define the role of IRF-4 in CLL pathogenesis further by exploiting a well-characterized murine model of CLL, namely the Tcl-1 transgenic (tg) mouse. The role of IRF-4 expression in B-cells during the development and establishment of a CLL clone will be examined by the targeted deletion of IRF-4 in B-cells of Tcl-1 tg mice using a CD19-restricted Cre recombinase system. In parallel, we will also generate an inducible CD19-restricted IRF-4 knockout model to specifically delete IRF-4 in mice with already established CLL disease. Additionally, we propose to introduce an inducible IRF-4 transgene into Tcl-1 tg mice in order to study the influence of IRF-4 overexpression during CLL establishment as well as in already established clonal CLL disease. Ex vivo analysis of cell differentiation, cell death and proliferation will complete the findings in vivo. Finally, the significance of our findings in the murine system will be placed in the context of human CLL where we aim to characterize the relationship between IRF-4 expression and treatment-free survival in patients, study a potential relation to therapy resistance, cell survival and proliferation, and investigate a potential modulation of the immunophenotype.

In this project, we describe a novel mechanism of immune evasion in Chronic Lymphocytic Leukemia (CLL) and provide molecular insights for this regulation. CLL is a malignant disease involving the abnormal accumulation of mature B-cells in the peripheral blood and the lymphoid organs. The disease is characterized by a high clinical and biological heterogeneity which is dependent on a crosstalk of CLL cells with the microenvironment and the immmuesystem, in particular with T-cells. Under nomal conditions, T-cells are able to recognize foreign antigens to clear infections as well as tumor antigens to prevent cancer development. In fact, T-cells from CLL patients show features of antigen experienced T-cells, however, these T-cells are not fully functional (exhausted) which prevents an effective anti- tumor immune response, leading to tumor outgrowth in the longterm. In earlier studies we could demonstrate that the dysfunction of T-cells observed in CLL patients, was directly induced by CLL tumor cells, however, the mechanisms responsible for this tumor immune evasion, were largely unknown. In this project we suggested the transcription factor IRF4 (interferone regulatory factor 4) as cricial regulator of CLL tumor immune evasion. We applied a mouse model which develops a murine CLL highly similar to the human disease and was genetically modified to delete IRF4 expression specifically in CLL tumor cells but not in T-cells or other cells of the microenvironment. This strategy ensures that all effects studied in this project were specific for IRF4 loss in CLL cells and not biased by IRF4 loss in other cells of the microenvironment. Using this model we could show that CLL developed faster in the absence of IRF4 due to enhanced tumor immune evasion. The lack of IRF4 in CLL cells caused a severe downregulation of proteins ususally required for the recognition and clearence of tumor cells by T-cells. We could show that T-cells in IRF4 deleted leukemic mice did not show features of anti-tumor recogniton and were not exhausted, indicating that IRF4 deletion specifically in CLL cells rendered tumor cells less visible to the immune system contributing to a more aggressive course of disease. These data could be verified also in the human disease. CLL patients with low IRF4 expression showed enhanced disease progression in independent CLL patient cohorts, downregulation of proteins involved in the CLL / T cell crosstalk and T-cells lacked phenotypic markers usually indicative for anti-tumor recognition. In summary, we could show to the first time that IRF4 was critically involved in CLL disease progression and that lack or low expression of IRF4 enhanced CLL immune evasion in the human and murine disease. These findings may help to improve immuno therapeutic approaches with the goal to cure CLL.