Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance.

Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNß-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1(f/f) CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1(-/-) NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1(f/f) animals), maturation was not altered. However, NK cells derived from Ifnar1(f/f) Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1(f/f )Ncr1-iCre compared with Ifnar1(f/f) control mice. Moreover, the ability of Ifnar1(f/f) Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.

PI3Kδ is essential for tumor clearance mediated by cytotoxic T lymphocytes.

BACKGROUND: PI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance. PRINCIPAL FINDINGS: PI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69(low)CD62L(high)). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ-/- CTLs. CONCLUSION: Our results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment.

The cooperating mutation or "second hit" determines the immunologic visibility toward MYC-induced murine lymphomas.

In Eµ-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the "second hit" affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2(-/-) mice or by Ab-mediated depletion of CD8(+) T or natural killer (NK) cells accelerated formation of BCL-2-overexpressing lymphomas but not of those lacking p53. Most strikingly, enforced expression of BCL-2 prolonged disease latency in the absence of p53, whereas blocking p53 function in BCL-2-overexpressing tumors failed to accelerate disease. This shows that blocking apoptosis in p53-deficient cells by enforcing BCL-2 expression can mitigate disease progression increasing the "immunologic visibility." In vitro cytotoxicity assays confirmed that high expression of BCL-2 protein facilitates NK and T cell-mediated killing. Moreover, we found that high BCL-2 expression is accompanied by significantly increased levels of the NKG2D ligand MULT1, which may account for the enhanced killing. Our findings provide first evidence that the nature of the second hit affects tumor immunosurveillance in c-MYC-driven lymphomas and define a potential shortcoming of antitumor therapies targeting BCL-2.

c-JUN promotes BCR-ABL-induced lymphoid leukemia by inhibiting methylation of the 5' region of Cdk6.

The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL-induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression, thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study, we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, because lack of c-JUN impairs proliferation of p185(BCR-ABL)-transformed cells without affecting their viability. The decreased proliferation of c-Jun(Δ/Δ) cells is associated with the loss of cyclin-dependent kinase 6 (CDK6) expression. In c-Jun(Δ/Δ) cells, CDK6 expression becomes down-regulated upon BCR-ABL-induced transformation, which correlates with CpG island methylation within the 5' region of Cdk6. We verified the impact of Cdk6 deficiency using Cdk6(-/-) mice that developed BCR-ABL-induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition, we show that reexpression of CDK6 in BCR-ABL-transformed c-Jun(Δ/Δ) cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the activating protein 1 (AP-1) transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1-regulated DNA methylation on BCR-ABL-induced transformation, thereby accelerating leukemogenesis.

A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK-cell survival and development.

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.

Type I interferons as mediators of immune adjuvants for T- and B cell-dependent acquired immunity.

Originally identified as antiviral substances produced by infected cells, type I interferons (IFN-I) are now known to have a wide range of additional activities within both the innate and adaptive immune response. Here we review properties of IFN-I contributing to their 'natural immune adjuvant' character, and their important role for the function of complete Freund's adjuvant (CFA) and the TLR9-dependent immune adjuvant IC31. We show data to demonstrate that treatment with IFN-I boosts the ability of vaccine/adjuvant combinations to induce peptide-specific CTL in both young and old mice. We view these findings in the perspective of previous clinical applications of IFN-I for vaccination.

Dendritic cells require STAT-1 phosphorylated at its transactivating domain for the induction of peptide-specific CTL.

Phosphorylation of transcription factor STAT-1 on Y701 regulates subcellular localization whereas phosphorylation of the transactivating domain at S727 enhances transcriptional activity. In this study, we investigate the impact of STAT-1 and the importance of transactivating domain phosphorylation on the induction of peptide-specific CTL in presence of the TLR9-dependent immune adjuvant IC31. STAT-1 deficiency completely abolished CTL induction upon immunization, which was strongly reduced in animals carrying the mutation of the S727 phospho-acceptor site. A comparable reduction of CTL was found in mice lacking the type I IFN (IFN-I) receptor, whereas IFN-gamma-deficient mice behaved like wild-type controls. This finding suggests that S727-phosphorylated STAT-1 supports IFN-I-dependent induction of CTL. In adoptive transfer experiments, IFN-I- and S727-phosphorylated STAT-1 were critical for the activation and function of dendritic cells. Mice with a T cell-specific IFN-I receptor ablation did not show impaired CTL responses. Unlike the situation observed for CTL development S727-phosphorylated STAT-1 restrained proliferation of naive CD8(+) T cells both in vitro and following transfer into Rag-deficient mice. In summary, our data reveal a dual role of S727-phosphorylated STAT-1 for dendritic cell maturation as a prerequisite for the induction of CTL activity and for T cell autonomous control of activation-induced or homeostatic proliferation.

The dark and the bright side of Stat3: proto-oncogene and tumor-suppressor.

Stat transcription factors have been implicated in tumorigenesis in mice and men. Stat3 and Stat5 are considered powerful proto-oncogenes, whereas Stat1 has been demonstrated to suppress tumor formation. We demonstrate here for the first time that a constitutive active version of Stat3alpha (Stat3alphaC) may also suppress transformation. Mouse embryonic fibroblasts (MEFs) deficient for p53 can be transformed with either c-myc or with rasV12 alone. Interestingly, transformation by c-myc is efficiently suppressed by co-expression of Stat3alphaC, but Stat3alphaC does not interfere with transformation by the rasV12-oncogene. In contrast, transplantation of bone marrow cells expressing Stat3alphaC induces the formation of a highly aggressive T cell leukemia in mice. The leukemic cells invaded multiple organs including lung, heart, salivary glands, liver and kidney. Interestingly, transplanted mice developed a similar leukemia when the bone marrow cells were transduced with Stat3beta, which is also constitutively active when expressed at significant levels. Our experiments demonstrate that Stat3 has both - tumor suppressing and tumor promoting properties.

Identification of an indispensable role for tyrosine kinase 2 in CTL-mediated tumor surveillance.

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.

Leukemic challenge unmasks a requirement for PI3Kdelta in NK cell-mediated tumor surveillance.

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.

Clarifying the role of Stat5 in lymphoid development and Abelson-induced transformation.

The Stat5 transcription factors Stat5a and Stat5b have been implicated in lymphoid development and transformation. Most studies have employed Stat5a/b-deficient mice where gene targeting disrupted the first protein-coding exon, resulting in the expression of N-terminally truncated forms of Stat5a/b (Stat5a/b(DeltaN/DeltaN) mice). We have now reanalyzed lymphoid development in Stat5a/b(null/null) mice having a complete deletion of the Stat5a/b gene locus. The few surviving Stat5a/b(null/null) mice lacked CD8(+) T lymphocytes. A massive reduction of CD8(+) T cells was also found in Stat5a/b(fl/fl) lck-cre transgenic animals. While gammadelta T-cell receptor-positive (gammadeltaTCR(+)) cells were expressed at normal levels in Stat5a/b(DeltaN/DeltaN) mice, they were completely absent in Stat5a/b(null/null) animals. Moreover, B-cell maturation was abrogated at the pre-pro-B-cell stage in Stat5a/b(null/null) mice, whereas Stat5a/b(DeltaN/DeltaN) B-lymphoid cells developed to the early pro-B-cell stage. In vitro assays using fetal liver-cell cultures confirmed this observation. Most strikingly, Stat5a/b(null/null) cells were resistant to transformation and leukemia development induced by Abelson oncogenes, whereas Stat5a/b(DeltaN/DeltaN)-derived cells readily transformed. These findings show distinct lymphoid defects for Stat5a/b(DeltaN/DeltaN) and Stat5a/b(null/null) mice and define a novel functional role for the N-termini of Stat5a/b in B-lymphoid transformation.