Eva Zebedin

Eva Eckelhart; Wolfgang Warsch; Eva Zebedin; Olivia Simma; Dagmar Stoiber; Thomas Kolbe; Thomas Rülicke; Mathias Mueller; Emilio Casanova; Veronika Sexl

We generated a transgenic mouse line that expresses the Cre recombinase under the control of the Ncr1 (p46) promoter. Cre-mediated recombination was tightly restricted to natural killer (NK) cells, as revealed by crossing Ncr1-iCreTg mice to the eGFP-LSLTg reporter strain. Ncr1-iCreTg mice were further used to study NK cell-specific functions of Stat5 (signal transducers and activators of transcription 5) by generating Stat5(f/f) Ncr1-iCreTg animals. Stat5(f/f) Ncr1-iCreTg mice were largely devoid of NK cells in peripheral lymphoid organs. In the bone marrow, NK-cell maturation was abrogated at the NK cell-precursor stage. Moreover, we found that in vitro deletion of Stat5 in interleukin 2-expanded NK cells was incompatible with NK-cell viability. In vivo assays confirmed the complete abrogation of NK cell-mediated tumor control against B16F10-melanoma cells. In contrast, T cell-mediated tumor surveillance against MC38-adenocarcinoma cells was undisturbed. In summary, the results of our study show that STAT5 has a cell-intrinsic role in NK-cell development and that Ncr1-iCreTg mice are a powerful novel tool with which to study NK-cell development, biology, and function.

Eva Zebedin; Olivia Simma; Christian Schuster; Eva M Putz; Sabine Fajmann; Wolfgang Warsch; Eva Eckelhart; Dagmar Stoiber; Eva Weisz; Johannes A. Schmid; Winfried F. Pickl; Christian Baumgartner; Peter Valent; Roland P. Piekorz; Michael Freissmuth; Veronika Sexl

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL(+) human leukemic cells express PI3Kdelta and therefore explored its impact on leukemia development. Using PI3Kdelta-deficient mice, we define a dual role of PI3Kdelta in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell-mediated tumor surveillance: Abelson-transformed PI3Kdelta-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kdelta accelerated leukemia progression in vivo. However, the absence of PI3Kdelta also affected NK cell-mediated tumor surveillance. PI3Kdelta-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3Kdelta-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3Kdelta. Other tumor models confirmed that PI3Kdelta-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kdelta in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3Kdelta inhibitors as antileukemic agents in clinical trials.

Olivia Simma; Eva Zebedin; Nina Neugebauer; Carola Schellack; Andreas Pilz; Souyet Chang-Rodriguez; Karen Lingnau; Eva Weisz; Eva Maria Putz; Winfried F. Pickl; Thomas Felzmann; Mathias Müller; Thomas Decker; Veronika Sexl; Dagmar Stoiber

We showed previously that Tyk2(-/-) natural killer cells lack the ability to lyse leukemic cells. As a consequence, the animals are leukemia prone. Here, we show that the impaired tumor surveillance extends to T cells. Challenging Tyk2(-/-) mice with EL4 thymoma significantly decreased disease latency. The crucial role of Tyk2 for CTL function was further characterized using the ovalbumin-expressing EG7 cells. Tyk2(-/-) OT-1 mice developed EG7-induced tumors significantly faster compared with wild-type (wt) controls. In vivo assays confirmed the defect in CD8(+) cytotoxicity on Tyk2 deficiency and clearly linked it to type I IFN signaling. An impaired CTL activity was only observed in IFNAR1(-/-) animals but not on IFNgamma or IL12p35 deficiency. Accordingly, EG7-induced tumors grew faster in IFNAR1(-/-) and Tyk2(-/-) but not in IFNgamma(-/-) or IL12p35(-/-) mice. Adoptive transfer experiments defined a key role of Tyk2 in CTL-mediated tumor surveillance. In contrast to wt OT-1 cells, Tyk2(-/-) OT-1 T cells were incapable of controlling EG7-induced tumor growth.

Eva Zebedin; Michael Freissmuth; Veronika Sexl

Imatinib was a major breakthrough in the treatment of Bcr/abl-positive leukemias. The effectiveness and value of this drug is limited by the emergence of resistance. Alternative drug targets may be identified by analyzing the downstream signaling network including the Jak/Stat-pathway, Ras-dependent signaling, PI3-kinases (PI3K), or the nuclear transcription factors onto which these pathways impinge. However, several factors limit the possible suitability of a drug target: (i) tissue-specific versus ubiquitous expression of the target; (ii) redundancy within the signaling network; and (iii) off-target effects on the immune system. Although the former two aspects are well appreciated as limiting factors, the latter has not been addressed so far. The advent of genetically engineered mice provides a sophisticated target validation in vivo as well as analysis of interactions between the immune system and tumor cells. Based on studies in such mouse models, we predict that many targeted compounds including PI3Kδ-inhibitors, could act as double-edged swords because their beneficial action on tumor cells may be neutralized or even overwhelmed by their additional immunosuppressive effects.

Eva Maria Putz; Eva Zebedin; Veronika Sexl

Besides B and T cells, Natural Killer (NK) cells constitute the third lymphocytic population with a broad spectrum of skills and functions. For several decades NK cells have been portrayed as first line defense against virally infected and malignant cells. But recent reports unraveled far more diverse properties of NK cells, e.g. their involvement in reproductive immunology and in mucosal defense of pathogens in the gut, and especially their ability to retain memory over several months. This chapter combines well established paradigms of NK cell biology with recent findings and special emphasis on the JAK/STAT signaling pathway. NK cell development, activation and cytotoxic function are tightly regulated by a plethora of cytokines – prominent inducers of the JAK/STAT signaling cascade. The availability and detailed analysis of gene-targeted mice underscores the importance of STATs controlling all aspects of NK cell biology.

Eva M Putz; Eva Zebedin; Agnes Csiszar; Michaela Prchal; Angelika Berger; Thomas Decker; Pavel Kovarik; Veronika Sexl

Background Natural killer (NK) cells are important key players of the innate immune system and provide immediate defense against viral infection and tumor transformation. Therefore, their potential in cancer immunotherapy has grown prominently in the last years [1]. As is known from the literature, the Signal Transducer and Activator of Transcription 1 (STAT1) plays an important role for NK cell function, since STAT1-deficient mice display impaired basal NK cytolytic activity in vitro and are unable to reject transplanted tumors in vivo [2]. STAT1 mediates signals downstream of interferons and gets activated by phosphorylation of several tyrosine and serine residues. In particular phosphorylation of S727 is considered a prerequisite for the full-fledged activation of STAT1 [3]. The aim of this project is to investigate the role of STAT1-S727 in NK cell-mediated cytotoxicity and tumor surveillance. Methods The analysis of STAT1 −/− , STAT1-S727A and wild-type mice includes the preparation of primary splenic NK cells and their investigation regarding proliferation ([ 3 H]thymidineincorporation), cytotoxicity (standard [ 51 Cr]-release) and cytokine production (ELISA, multi-plex bead arrays). In vivo tumor models are employed using NK-sensitive tumor cell lines (v-abl + leukemia, B16 melanoma, 4T1 breast cancer). Results

Johannes Werzowa; Stefan Koehrer; Sabine Strommer; Daniel Cejka; Thorsten Fuereder; Eva Zebedin; Volker Wacheck

Walter Sandtner; Julia Szendroedi; Touran Zarrabi; Eva Zebedin; Karlheinz Hilber; Ian W. Glaaser; Harry A. Fozzard; Samuel C. Dudley; Hannes Todt

Eva Zebedin; Walter Sandtner; Stefan Galler; Julia Szendroedi; Herwig Just; Hannes Todt; Karlheinz Hilber

Karlheinz Hilber; Walter Sandtner; Touran Zarrabi; Eva Zebedin; Oliver Kudlacek; Harry A. Fozzard; Hannes Todt