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Development of diffuse large B-cell lymphoma in chronic lymphocytic leukemia, so-called Richter transformation (RT), occurs in 2–5% of patients and is associated with poor outcome. The clinical features of RT are fairly non-specific and unable to discriminate transformation from other mimics. In case of clinically suspected RT, a CT/MRT is recommended, and FDG-PET/CT may help to select the site of biopsy. Radiological features suggestive of RT have been defined, but there are only limited data about their predictive value, and histological confirmation is still considered the gold standard for RT diagnosis. We retrospectively analyzed 34 patients with clinically suspected RT and available radiological and histological data. A histopathological diagnosis of RT with concordant clinical and radiological findings was obtained in 13 patients. In 18 patients, CT did not show features of transformation, concordant with lack of RT in the biopsy. Of interest, a distinct lymphoma other than DLBCL was identified in two of these cases. A false-positive radiological diagnosis of RT was rendered in two patients, including a case of Herpes simplex virus lymphadenitis. In conclusion, our findings confirm the central role of tissue biopsy in the diagnostic work up in case of clinically suspected RT.

Chronic lymphocytic leukemia (CLL) may be defined as a clonal expansion of mature B cells with stereotypic B cell receptors (BCR). Somatic hypermutation of the BCR heavy chains (IGVH) defines subgroups with different prognosis. In up to 10% of patients Richter's transformation to a high grade lymphoma with a mostly dismal prognosis is observed. The TCL1 transgenic mouse is a well accepted model of human CLL. Upon B cell-specific knock out of NFAT2, TCL1 mice develop a disease resembling Richter's syndrome with a significantly more aggressive disease phenotype. While TCL1 B cells exhibit tonic anergic BCR signaling characteristic of human CLL, loss of NFAT2 leads to readily activated BCRs indicating different BCR usage with altered downstream signaling. Here, we analyzed BCR usage in C57BL/6 wildtype, TCL1, and TCL1-NFAT2-/-mice. Splenocyte cDNA of the respective animals at an age of 7 months was amplified by multiplex PCR covering known heavy chain VDJ alleles. The PCR products were subsequently cloned into standard bacterial plasmids and subjected to conventional DNA sequencing. In addition, PCR products were analyzed by next-generation-sequencing (NGS). For data analysis, the IMGT/HightV-QUEST online tool was applied. A very diverse and polyclonal BCR usage was found in wild type mice with more than 4000 different clones identified. Although TCL1 mice at 7 months of age exhibit all features of CLL, their BCR usage as analyzed by unambiguously identified VDJ recombination was still polyclonal. Only with respect to VH usage, TCL1 mice were found to use a more limited set of V alleles compared to wild type mice. Loss of NFAT2 by conditional knockout on the contrary, lead to an oligoclonal usage of VDJ recombination, to a further limitation of V alleles, and also to the usage of identical VDJ recombination in different mice with high frequency - indicating BCR selection either by antigen or self-signaling in these mice. Further analyses by sequencing of hypervariable regions showed preferential usage of mutated BCRs in TCL1 mice and of unmutated BCRs in NFAT2 ko mice reflecting benign and aggressive disease in humans. In summary, the loss of NFAT2 signaling in CLL prefers the selection of unmutated BCRs as well as the preferential usage of certain VDJ recombinations which results in the accelerated development of true oligoclonal disease. Disclosures No relevant conflicts of interest to declare.

NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation. We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice. While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells. In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration Disclosures No relevant conflicts of interest to declare.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. While some patients exhibit enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggressive disease, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype referring to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Up to 10% of CLL patients transform to an aggressive form of lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here, we analyzed the role of NFAT2 in CLL with respect to maintenance of the anergic phenotype and the development of Richter´s syndrome. For this purpose, we generated conditional NFAT2fl/fl CD19-Cre knock out mice on the Eµ-TCL1 transgenic background. These mice exhibit NFAT2 deletion limited to the B cell lineage and develop a human-like CLL due to the B cell specific expression of the TCL1 oncogene. Conditional TCL1+NFAT2fl/fl CD19-Cre mice with B cell specific deletion of NFAT2 served as the experimental cohort while TCL1 transgenic mice without an NFAT2 deletion served as controls. To extend our observations do the human disease, we further analyzed primary blood samples from patients with CLL (n=30) and Richter´s syndrome (n=5). Mice with genetic loss of NFAT2 in their B cell compartment showed a significantly more aggressive disease course with accelerated accumulation of CD5+ CD19+ CLL cells in different organs, significantly higher proliferation rates and a dramatically reduced life expectancy (200 vs. 325 days) as compared to TCL1 control animals. Immunohistochemical analysis of spleen and lymph nodes upon necropsy showed a transformed disease phenotype in the NFAT2 ko cohort with diffuse distortion of lymph node and spleen architecture by large B cells with blastic chromatin while animals with intact NFAT2 expression showed small lymphocytic infiltrates with conserved B and T cell areas as expected. Using calcium imaging technology, we were able to show that CLL cells with regular NFAT2 expression were essentially unable to mobilize calcium upon IgM ligation and thus exhibited the typical anergic phenotype. CLL cells with NFAT2 deletion, on the contrary, showed a highly IgM-responsive phenotype with inducible activation of downstream signaling via AKT and ERK. We subsequently defined a genetic signature of anergy in CLL cells employing Affymetrix microarrays consisting of Lck, Pacsin1 and the E3 ligases Cbl and Grail, which were highly expressed in anergic CLL cells with normal NFAT2 expression and virtually absent upon NFAT2 ablation. We could further show by western blotting with phospho-specific antibodies that Lck was constitutively activated in anergic CLL cells while it was essentially inactive in NFAT2 ko cells suggesting an important role for Lck in mediating the anergic phenotype. Primary human CLL samples (n=30) showed a significant overexpression of NFAT2 and Lck, predominantly in its active conformation, as assessed by western blotting and RT-PCR. Tissue samples (lymph nodes, bone marrow) from patients with Richter's Syndrome (n=4) on the other hand demonstrated a significantly reduced expression of NFAT2 and complete loss of Lck expression. Calcium imaging revealed that a subset of human CLL cells showed an anergic phenotype and were unable to flux calcium while tumor cells from Richter patients exhibited an intensive spontaneous calcium flux as well as hyperresponsiveness upon IgM ligation. In summary, our data provide strong evidence that NFAT2 and its target gene Lck are important mediators of anergy induction in CLL and that their loss leads to disease transformation to aggressive B cell lymphoma (Richter´s syndrome). Taken together, our results demonstrate that the NFAT2-LCK axis plays an important role in the pathogenesis of CLL and implicate it as a potential target in its treatment. Disclosures No relevant conflicts of interest to declare.

Megakaryopoiesis is controlled by a variety of hematopoietic growth factors and cytokines in order to maintain physiological levels of circulating platelets. Thrombopoietin (TPO) signalling via its receptor c-Mpl is a key regulator of megakaryopoiesis driving megakaryocyte differentiation, promoting endomitosis and proplatelet formation. Therefore TPO/c-Mpl signalling needs to be tightly regulated to maintain physiological megakaryopoiesis. One of the most effective mechanisms to permanently disable activated signalling proteins is by targeted degradation via lysosomes or proteasomes. Previous studies have identified c-Cbl as an E3 ligase responsible for the ubiquitination of c-Mpl in cell lines. In this study, we investigated the mechanisms of TPO-mediated c-Mpl degradation in primary mouse cells. In order to determine the potential role of c-Cbl in murine megakaryopoiesis we used a conditional PF4-Cre c-Cbl knockout (ko) mouse model to specifically delete c-Cbl in the megakaryocytic lineage. Megakaryocytes were generated in vitro by culturing bone marrow from WT and PF4-Cre/c-Cbl-floxed (c-Cbl ko) lines for 72 hrs in the presence of rmTPO. C-Cbl ko mice showed significant bone marrow megakaryocyte hyperplasia, however megakaryocyte numbers in the spleen remained unchanged. Platelets counts were significantly elevated as compared to control mice (1.2 x106 vs. 1.7x106 p=0.0001) and in addition, the platelets from the c-Cbl ko mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). Using a method of in vivo double labelling of platelets, we were able to simultaneously follow the survival of both the entire population of platelets and new platelets which were generated during the last 24 hours. There were more new platelets produced within a 24 h period in the c-Cbl ko mice although the half-life of platelets was similar in the both cohorts. Although c-Cbl ko mice exhibited thrombocytosis, they showed a severe defect in thrombus formation using an in vivo thrombus formation model with Fe3Cl. TPO plasma levels, known to be inversely regulated by circulating platelet numbers, were surprisingly increased (250 vs. 420 pg/ml, p=0.005) in the c-Cbl ko mice. There was no difference in liver mRNA levels in the two cohorts. We therefore looked at c-Mpl protein and mRNA expression in megakaryocytes and found c-Cbl ko mice to express more c-Mpl compared with wild type controls. Surprisingly, we found c-Mpl surface expression to be reduced and internalization of the receptor significantly impaired following TPO stimulation in c-Cbl ko mice. Incubating platelets in vitro with TPO for 2 hours to evaluate the TPO uptake capacity of platelets, we found c-Cbl ko platelets to show a severe uptake defect compared with wild type control platelets. Taken together, we have successfully ablated c-Cbl specifically from the megakaryocyte lineage and demonstrated that this has profound effects on platelet counts and size. In addition, we showed that c-Cbl ablation leads to reduced c-Mpl surface expression and impaired internalization, which culminates in increased TPO plasma levels causing increased megakaryopoiesis in the c-Cbl ko mice. In summary, our data enhance our understanding of the regulation of TPO signalling and the physiological role of c-Cbl in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

7019 Background: NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. CLL constitutes a heterogeneous disease with some...

10562 Background: Neurotrophins (NT) influence proliferation, survival, death, and differentiation by binding Tropomyosin-related kinase (Trk) receptors and p75NTR(CD271). While the three Trk-receptors TrkA, TrkB, and TrkC display specific binding to their ligands, i.e. TrkA to NGF (nerve growth factor), TrkB to BDNF (brain-derived neurotrophic factor), NT4/5 (neurotrophin 4/5) and TrkC to NT3 (neurotrophin 3), CD271 shows low affinity to all neurotrophins. Notably, CD271 has been suggested as marker for the prospective isolation of bone marrow mesenchymal stromal cells (MSC), and rhabdomyosarcomas have previously been found to express neurotrophins and NT receptors. Methods: RNA was isolated from sarcoma cell lines and freshly obtained sarcoma (src) cells from patients after surgical resection. RNA was reverse transcribed and cDNA products underwent PCR analysis with specific primers. Furthermore, cell lines from the same primary tissues were established and treated with doxorubicin. Effects of K252a (Trk receptor inhibitor), Pep5 (CD271-Inhibitor), and neurotrophins on sarcoma cell proliferation and survival were analyzed. Results: An array of freshly obtained clinical src samples (n=10 each subtype) was screened for expression of NT3, NT4/5, BDNF, NGF as well as TrkA, TrkB, TrkC, and CD271. Interestingly, src subtype-specific expression patterns were observed. In the established cell lines, K252a increased doxorubicin-induced tumor cell death, while CD271-Inhibitor Pep5 enhanced proliferation in doxorubicin treated cells, especially under serum free conditions and when combined with BDNF and NGF. Conclusions: NT and NT receptors are expressed on all adult src samples screened in this study. Chemotherapy resistant liposrc as well as myxofibrosrc samples displayed the strongest expression of NT3 as well as TrkB, TrkC, and CD271. The concomitant expression of NT and their receptors suggests autocrine self-stimulation, which may propagate survival, proliferation, and treatment resistance. NT receptor tyrosine kinases may represent an interesting therapeutic target in adult src.

7019 Background: CLL is a malignancy of mature B cells and constiututes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the significance of NFAT signaling in B-CLL. Methods: NFAT2 expression and aberrant nuclear translocation in CLL cells (n=30) was assessed by Western Blotting and immunofluorescence. In addition, NFAT2 mRNA levels were measured by qRT-PCR and its DNA binding capacity was assessed using an electrophoretic mobility shift assay. Transcriptional activity of NFAT2 proteins in CLL cells was further analyzed by determining the expression of several well characterized NFAT target genes. Results: We detected a profound overexpression of NFAT2 mRNA and protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited a significant overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors (8-200fold). This overexpression of NFAT2 in CLL cells could also be confirmed on the protein level. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in non-malignant B cells. Nuclear NFAT2 in CLL cells was able to bind DNA but its transcriptional activity with respect to several apoptosis-regulating genes (i.e. Spp1, Pdcd1) was severely compromised. Conclusions: These results provide strong evidence that the Ca2+/NFAT signalling axis is constitutively activated in CD5+CD19+ CLL cells. Reduced expression of several apoptosis regulators which are known target genes of NFAT2 links deregulation of this signaling cascade to CLL progression. Further investigation is warranted to investigate the therapeutic potential of modulating Ca2+/Calcineurin/ NFAT signaling in CLL.

e13501 Background: Hearing loss can be the dose limiting toxicity of CDDP. In the treatment of germ cell tumors (GCT) or adjuvant therapy of non-small cell lung cancer (NSCLC), CDDP cannot be replaced without loss of efficacy. Inhibition of PARP, an enzyme of the base excision repair pathway, is a novel anti-neoplastic principle. PARP-activation has been identified to induce cell death following inner ear trauma. We evaluated PARP-inhibition by pirenzepin as a means to protect the ear from CDDP. Methods: In an in vitro culture model, inner ears were treated continuously with 1.4 μM CDDP. Ears were obtained from heterozygous and homozygous PARP-knock out mice and wild-type controls at postnatal day 7. The number of preserved hair cell stereocilia bundles was counted by fluorescence microscopy after labeling with phalloidin. Cytotoxicity of CDDP and the effect of pirenzepin were assessed by MTS-assays and flow cytometry using the cell lines 2102EP, NT2 (both GCT) and NCI-H460 (NSCLC). Results: In wild-type ...

Abstract 711 Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells. NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases. We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls. To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells. To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease. In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures: No relevant conflicts of interest to declare.

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT translocates to the nucleus, where it orchestrates developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least three different kinases: CK1, GSK-3 and DYRK. NFAT signalling is further implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. Previous studies have primarily concentrated on NFAT2, which was found to be overexpressed and constitutively activated in a majority of biopsies from Diffuse Large B-Cell lymphomas and Burkitt’s lymphomas, presumably reflecting activation of the Ca/NFAT signalling pathway as part of their pathogenesis. Other recent studies have demonstrated that NFAT2 activation can lead to increased expression of different cell survival factors (CD154, BLyS) in several NHL subtypes. Here, we analyzed the role of NFAT1 in malignant transformation and in the pathogenesis of T-ALL using a transgenic mouse model for the disease. We show that sustained activity of NFAT1 and NFAT2 induces opposite phenotypes in NIH 3T3 cells. While NIH 3T3 cells infected with empty retrovirus showed normal viability and stopped growing upon reaching confluence, cells infected with a constitutively active version of NFAT2 overgrew the monolayer and continued to proliferate beyond confluence. NIH 3T3 cells expressing a constitutively active form of NFAT1 on the contrary exhibited a remarkable reduction in cell proliferation and never reached confluence, suggesting opposite roles for NFAT1 and NFAT2 in the control of cell growth and proliferation. To directly test whether NFAT1 has the capability to suppress tumor growth and to subvert a transformed cell type, we generated NIH 3T3 cell clones harboring the oncogene H-rasV12, which showed a typical transformed phenotype. Expression of constitutively active NFAT1 in the transformed NIH 3T3-HrasV12 cells leads to a significant reduction in cell proliferation which was accomanied by a large decrease in DNA replication and an accumulation in the G1 phase of the cell cycle. To test if NFAT1 has tumor suppressor characteristics in vivo, we generated transgenic mice conditionally expressing a hyperactivable form of NFAT1 from the ROSA26 locus. These mice were subsequently bred to Tel-Jak2 transgenic mice, which develop T-ALL at 8–20 wks of age. Mice that expressed hyperactivable NFAT1 in their T cells in addition to the disease inducing Tel-Jak2 transgene exhibited a significantly attenuated phenotype of the disease. Infiltration of vital organs such as lung, liver and bone marrow was significantly delayed and overall survival was almost twice as long in the animals that expressed hyperactivable NFAT1 in their T cell compartment (88 days vs. 159 days). To summarize, our data identify NFAT1 as a novel tumor suppressor gene in T-ALL and emphasize the importance of the Ca/NFAT signalling pathway in the pathogenesis of hematologic malignancies.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT translocates to the nucleus, where it orchestrates several developmental programs, including those of the immune, cardiovascular and central nervous systems. NFAT is rephosphorylated and inactivated by different kinases (CK1, GSK-3, DYRK). The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (SPRIEIT in NFAT1). Substitution of the SPRIEIT sequence with HPVIVIT increases the basal calcineurin sensitivity of the protein significantly. Similarly, mutation of the CK1 docking site from FSILF to ASILA leads to partial nuclear localization by decreasing rephosphorylation and nuclear export. To assess the impact of hyperactivable NFAT mutants on activation kinetics and signal responsiveness, we retrovirally transduced T cells from NFAT1−/− mice with contructs expressing wild type NFAT1, NFAT1-ASILA, NFAT1-HPVIVIT or NFAT1-ASILA-HPVIVIT. Analysis by western blotting and immunocytochemistry revealed, that the wild type protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, whereas the hyperactivable mutants exhibited an increasing degree of nuclear translocation and dephosphorylation (ASILA 20%, HPVIVIT 30–40%, ASILA-HPVIVIT 50–70%). Upon stimulation with PMA and ionomycin, the hyperactivable mutants exhibited an incremental acceleration of nuclear translocation and delay of nuclear export as compared to the wild type protein. Furthermore, T cells expressing hyperactivable NFAT proteins exhibited significantly higher expression rates of different cytokines (IL-2, TNF-α, IFN-γ) upon stimulation with low doses of ionomycin documenting their hyperresponsiveness and biological activity. To provide a new tool for the analysis of the calcineurin-NFAT pathway in vivo and to assess the effect of modulating the affinity of signaling molecules for their upstream regulators, which are consistently kept at low to moderate affinity during evolution, we subsequently generated transgenic mice conditionally expressing different hyperactivable NFAT1 mutants from the ROSA26 locus (NFAT1-HPVIVIT, NFAT1-ASILA-HPVIVIT). Transgene expression in the T cell lineage was achieved by breeding the ROSA26 transgenic mice to CD4-Cre mice, which express the Cre recombinase under the control of the CD4 promoter. CD4 and CD8 T cells from these mice showed significant hyperactivability as assessed by accelerated nuclear translocation and delayed nuclear export of NFAT and substantially increased cytokine expression upon stimulation. Expression of the hyperactivable NFAT proteins early in the germline was achieved by breeding the ROSA26 mice to Cre-Del mice, which express the Cre recombinase under the control of the ACE promoter. While breeding of ROSA26-YFP control mice to Cre-Del mice resulted in transgene expression in all T and B cells, expression of hyperactivable NFAT proteins early in the germline resulted in incremental mosaicism in T and B cells (70% transgene expression in NFAT1-HPVIVIT and 20–30% transgene expression in NFAT1-ASILA-HPVIVIT). This data demonstrate that hyperactivable NFAT1 proteins result in a selective disadvantage for the expressing cells in embryonic development and provide a potential explanation why evolution chose to keep the respective docking sites at moderate affinity.

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT translocates to the nucleus, where it induces the transcription of a large number of genes necessary for a productive immune response. NFAT signalling has also been implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. A recent immunohistochemical evaluation of approximately 300 Non-Hodgkin’s Lymphoma biopsy samples showed overexpression of NFAT2 in the majority of specimens with strong nuclear translocation in certain histologic subtypes (DLBCL, Burkitt’s Lymphoma) presumably reflecting activation of the NFAT pathway as part of their pathogenesis. Other recent studies have shown that NFAT activation leads to increased expression of different cell survival factors (CD154, BLyS) in several lymphoma subtypes (DLBCL, MCL). To study the role of constitutive activation of the NFAT pathway on lymphomagenesis, we generated several hyperactivable NFAT mutants which will be used to generate transgenic mice expressing the mutant proteins from the ROSA26 locus. Here, we present the in vitro characterization of these hyperactivable NFAT proteins in cell lines and primary lymphocytes. The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (e.g. SPRIEIT in NFAT1 and NFAT2). Substitution of the SPRIEIT sequence of NFAT with HPVIVIT, a higher-affinity version obtained by peptide selection, increases the basal calcineurin sensitivity of the protein significantly. Similarly, a major kinase for NFAT is CK1, and mutation of the CK1 docking site (FSILF to ASILA in NFAT1, FDFEF to ADAEA in NFAT2) also leads to partial nuclear localization of NFAT proteins by decreasing rephosphorylation and nuclear export. In the current analysis we compared the different hyperactivable NFAT proteins with respect to dephosphorylation status and nuclear translocation using western blotting and immunocytochemistry. Whereas the wildtype NFAT protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, we observed an increasing degree of nuclear translocation and dephosphorylation for the different mutant proteins (ASILA 20%, VIVIT 30–40%, ASILA-VIVIT 50–70%). This system will allow us to study the impact of different levels of NFAT activation on the pathogenesis of lymphomas in vivo.