ALBERT

Description: Key Points IRF4 deletion in Tcl-1 tg mice and IRF4low CLL patients enhances disease progression due to increased tumor immune evasion. This is caused by a downregulation of the antigen processing and presentation machinery and reduced T-cell costimulation.

Description: The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signalling via the B-cell receptor (BCR). Protein kinase C (PKC) beta is an essential signalling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKC beta in the development of murine CLL. While loss of PKC beta in wild type mice leads to a complete loss of CD5+ B cells in these mice, the targeted disruption of the PKC beta gene in Tcl1 transgenic mice did not abrogate the accumulation of CD5 positive B cells in the pretumor phase in these mice. This suggests that the basic survival signal transmitted by PKC beta in B1 cells can be substituted by the augmented Akt signalling in the Tcl1 transgenic cells. Yet despite this, PKC beta deficient TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKC beta in the establishment of the malignant clone. In fact PKC beta is likely to be essential for transformation in limiting the BCR signal via a negative feedback loop, suggesting that BCR signalling strength is an important component of the transformation process. In order to provide support for the potential of PKC beta as a drug target in CLL we targeted PKC beta with the specific inhibitor Enzastaurin. This led to killing of human CLL samples in vitro and this was independent of risk factors in the samples analysed. We thus propose that PKC beta may be a relevant target for the treatment of CLL.

Description: The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signaling via the B-cell receptor (BCR). Protein kinase C β (PKCβ) is an essential signaling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKCβ in the development of murine CLL. TCL1 overexpression did restore the CD5+ B-cell population that is absent in PKCβ-deficient mice. However, PKCβ-deleted TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKCβ in the establishment of the malignant clone. Moreover, targeting of PKCβ with the specific inhibitor enzastaurin led to killing of human CLL samples in vitro. We thus propose that PKCβ may be a relevant target for the treatment of CLL.

Description: Chronic lymphocytic leukaemia of B cells (B-CLL) is characterised by a clonal expansion of CD5-expressing B cells. However, the absolute number of T cells in patients with B-CLL is also increased, primarily due to an enlarged CD8+ population. Despite the enhanced number of T cells, they appear to be dysfunctional as they are largely devoid of anti-tumor activity and may even support the growth and maintenance of the malignant B-CLL clone. To further investigate the contribution this T cell dysfunction has on the establishment and progression of B-CLL, we monitored the changes in the T cell compartment in Eμ-TCL1 transgenic mice of various ages, during the progression of B-CLL-like disease. The deregulated expression of TCL1 in the B cell compartment of these mice leads to a hyperplasia of CD5+ CD19+ B cells early in life that is first detectable in the peritoneal cavity and later spleen and bone marrow. Later, this develops into a clonal disease, and onset of frank leukaemia, with infiltration of the CD5+ CD19+ cells into other organs (Bichi et al, 2003). Similar to human B-CLL, leukaemic TCL1 transgenic mice have increased absolute numbers of T cells, owing mainly to an expanded CD8+ population. Analyses of the cell surface expression of CD25, CD44, and CD62L, revealed a marked increase the number of memory and effector T cells compared to naïve T cells in the CD8+ subset that is also observed in the CD4+ subset, although to a lesser degree. Importantly, a decrease in the number of naïve T cells along with a corresponding increase in T memory and effector cells has been observed in the CD4+ T cell pool from patients with unmutated B-CLL (Tinhofer et al, unpublished), and the TCL1 mouse has been shown to be a model for the unmutated form of B-CLL. The relative shift from naïve to central memory T cells occurs alongside the CD5+ CD19+ hyperplasia in the TCL1 mice. In younger mice that exhibit the CD5+ CD19+ hyperplasia only in the peritoneal cavity, the changes in the T cell compartment are also observed solely within this organ. At later stages, when the CD5+ CD19+ hyperplasia has spread to other organs, the shift to memory T cells is also detectable in all infiltrated organs. Because memory and effector T cells represent antigen-experienced cells, we analysed the complementarity determining region 3 (CDR3) of the T cell receptor (TCR) of purified CD4+ and CD8+ populations by spectratyping in order to determine the degree of clonality (mono-, oligo-, or polyclonal) of the different T cell families within the T cell pool. More clonal T cells were observed in the CD8+ T cell subset in the infiltrated organs of leukaemic mice, though we could not observe a skewing towards any particular BV gene family. Interestingly, more mono- and oligoclonal CD4+ T cells have also been observed in the peripheral blood of patients with unmutated B-CLL. Thus, in many aspects, the TCL1 mouse recapitulates many of the T cell abnormalities observed in patients with B-CLL. It should be noted that, although driven from a B-cell specific promoter, the hTCL1 transgene is also expressed in the T cells of TCL1 mice. Interestingly, we have also found that TCL1 is overexpressed in the CD4+ and CD8+ T cells of patients with unmutated B-CLL compared to mutated CLL or healthy controls. Thus the Tcl1 transgenic murine CLL model seems suitable as a model for the immune aberrations found in human unmutated CLL.

Description: Abstract 90 Introduction: The p53 gene is non-functional in 〉50% of human tumors. In mice deletion of p53 leads to a high incidence of tumors and to a significant acceleration of tumorigenesis induced by repeated gamma-irradiation. While a large number of effects have been described for p53, current concepts of p53-mediated tumor suppression discuss the roles of p53 in regulation of cell cycle and apoptosis as being essential. Two main targets have been identified in this respect: p21Waf1 as an essential regulator of cell cycle arrest downstream of p53 and Puma as the largest single contribution towards p53 induced cell death. Methods: We have generated p21Waf1/Puma doubly deficient (i.e. double-knockout – DKO) mice on a pure C57BL/6 background to investigate the effects on tumorigenesis. Results: In ex vivo irradiation studies DKO thymocytes expectedly showed reduced cell death and loss of a G1/S arrest upon irradiation. When following a cohort of mice for spontaneous tumor development, the DKO mice did not differ from wild-type (WT) controls. Since this may be explained by additional p53 down-stream effectors essential for tumor suppression, we set out to challenge the mice with an established repeated irradiation protocol (4 × 1.75 Gy over 4 weeks) in order to increase the likelihood of uncovering a defect in tumor suppression not apparent in unchallenged mice. While irradiated WT mice developed thymic lymphomas at an expected rate and p53 deficiency accelerated the lymphoma formation as published, irradiated DKO mice did not develop any thymic lymphoma at all. During the irradiation protocol WT mice followed a series of depletion and regrowth cycles in thymic cellularity with a high rate of cell death early post irradiations in TUNEL assays and a surge of proliferation on day 5 after irradiations detected by in vivo BrdU labeling. By contrast in DKO mice thymic cellularity dropped only slightly during the first irradiation cycle. This was followed by a slow and steady decline in cellularity over the following 3 cycles of irradiation. No late apoptotic wave or loss of proliferative capacity of remaining thymocytes could explain the loss of cellularity, nor could senescence of thymocytes be detected by SA-β-Gal staining in situ, suggesting that thymic influx was defective. It had previously been reported for the repeat-irradiation lymphomagenesis model, that the irradiation of hemopoietic precursor cells was essential for tumorigenesis. In contrast to thymic cellularity, DKO LSK numbers stayed relatively stable over the course of the 4 irradiations. By comparison WT LSK numbers dropped to about 50% by the time 4 irradiations were completed. Indeed, short-term repopulating (ST) cells dropped significantly, while long-term repopulating (LT) and multipotent progenitor (MPP) cell populations stayed more stable. In DKO marrows the relative content of LT, ST and MPP cells proved very stable across the irradiation schedule. In vivo BrdU labelling showed that WT LSK had a higher fraction of labelled cells at baseline and a 〉100% increase in the proliferative fraction during irradiation, while in DKO LSK the proliferation index was lower and stayed stably low over time, compatible with the replenishment defect observed in the thymus. DKO stem cells were only slightly more efficient (1.6-fold) than WT in bone marrow reconstitution experiments without challenge. However, when mixed chimeras were then subjected to the irradiation protocol with 4 × 1.75 Gy a clear advantage of the DKO cells became apparent (28-fold). Moreover, when reconstituting lethally irradiated mice with a mixture of WT and DKO marrow taken from repeatedly irradiated donors the efficacy ratio was 1:152. Conclusion: Our data contrast observations made in cell lines, where loss of Puma and p21Waf1 led to a p53-resistant outgrowth of cells. We present in an animal model that loss of Puma and p21Waf1 is not tumorigenic and in fact protects mice from irradiation carcinogenesis. Together with our recently published findings in irradiated Puma singly-deficient mice (Labi G&D 2010), our data suggest that tumorigenesis in irradiated DKO mice is inhibited by effects on hemopoietic stem cell reactivity to DNA damage. A combination of lack of generation of free niche space through protection of hemopoietic stem cells from cell death and a stem cell quiescence state retained in DKO stem cells after irradiation seems responsible for the phenotype. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction The development and maturation of B cells is highly dependent on signals provided by the microenvironment of the lymphatic organs. As B cells move from one developmental stage and niche to the next, the integrin family of adhesion molecules provides important cues for their correct positioning and retention. The integrin adaptor protein Kindlin-3 (encoded by the Fermt3 gene) regulates integrin activity and function in a wide range of hematopoietic cell types. In this study, we aimed to define its precise role in the development and function of the different murine B cell subsets. Methods We crossed a Fermt3flox/flox mb1-cre mouse strain (hereforth called K3ΔB mice), harboring a B cell specific Kindlin-3 deletion. B cell subsets in the different lymphoid organs of these K3ΔB mice and control littermates were defined by multicolor flow cytometry. Adoptive transfer, microscopy and real-time flow cytometry were used to analyze the different steps of integrin activation. A co-culture system with OP9 stromal cells and BAFF was used to assess the in vitro differentiation potential of immature progenitors into the different mature B cell subsets. Transcriptional differences between follicular B cells isolated from spleens of K3ΔB- and control mice were assessed by transcriptome array. Results In vitro, we found that integrin activation on B cells was induced upon activation of the chemokine receptors CXCR4 and CXCR5 or the B cell receptor. This stimulation triggered adhesion of wild type B cells to integrin ligands under shear flow. The increase of VLA-4 integrin affinity to its ligand substrates during this process could also be calculated from real-time flow cytometrical analyses. In contrast, K3ΔB-derived B cells could not reach high affinity states of integrins and thus failed to adhere on the substrates upon stimulation, despite slight upregulation of chemokine receptors CXCR4 and CXCR5. B cell migration towards the respective chemokines also required Kindlin-3, even in an integrin ligand-free setting. In vivo, Kindlin-3 was required for homing of mature B cells to the bone marrow and to lymph nodes. When further characterizing K3ΔB mice by flow cytometry and immunohistochemistry we observed increased early B cell numbers in the bone marrow. Of note, marginal zone (MZ) B cells in the spleen were completely absent (Figure 1 A+B). We consequently assessed the potential of immature B cells to develop into B cells with high expression of CD21, a marker for MZ B cells, upon their co-culture with OP9 stromal cells in the presence of the B cell survival factor BAFF. While 18% of B cells differentiating from wild type bone marrow displayed high expression of CD21, the percentage of CD21 high cells recovered from Kindlin-3 deficient progenitors was significantly lower (~12%, Figure 1C). Pathways involved in these developmental differences were analyzed by a transcriptome array, revealing increased activity of the B cell receptor pathway in the knockout situation accompanied by higher, NFkappaB and Notch signaling. Conclusion/Outlook Whereas our results highlight the importance of Kindlin-3 dependent, integrin mediated cell retention and migration during B cell development they also indicate that Kindlin-3 functions in an integrin-independent manner when regulating cell motility and transcription. The complete lack of MZ B cells in the absence of Kindlin-3 is thus most likely a combination of defective retention in the MZ area and transcriptional alterations favoring the development of transitional B cells into follicular- rather than MZ B cells. Figure 1 : B-cell specific Kindlin-3 knockout leads to loss of splenic marginal zone B cells. The percentage of MZ B-cells among total splenic B cells was determined by flow cytometry in K3ΔB mice and wild type (wt) littermates (A). Immunohistochemistry staining of CD19 showed a loss of loosely packed marginal zone B cells (yellow arrows) in the absence of Kindlin-3 (B). B cells were enriched from the bone marrow of K3ΔB mice and wt littermates and cultured on a confluent layer of OP9 cells in the presence of 200 ng/ml BAFF for 72 h. Development of CD21 high/CD23 low B cells was then determined by flow cytometry (C). Figure Disclosures Greil: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Astra zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Daiichi Sankyo, Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; BMS/celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; MSD Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding.