Supplemental Figure 1: ILK protein expression in viable CD5+/CD19+ primary CLL cells was assessed by flow cytometry; Supplemental Figure 2: ILK levels are upregulated upon activated T cell and alternative IL2/CpG induced CLL cell activation; Supplemental Figure 3: Control experiments undermine that ILK transcription is regulated in a CD40L independent but highly specific NF-kB dependent manner; Supplemental Table 1: Patient characteristics; Supplemental Table 2: List of used antibodies.

PDF file - 245K, Supplementary Figure S1: Activated, autologous T cells induce CLL cell proliferation. Supplementary Figure S2: CD40L activated CLL cells fully retain their chemotactic response towards CCL21. Supplementary Figure S3: PMA/ionomycin and IgM cross-linking induces FL-HA binding. Supplementary Figure S4: CD44 surface expression on CLL patients does not differ between clinical risk groups. Supplementary Figure S5: The CLL cell subpopulation remaining CD44v6 negative after CD40L-activation displays low activation levels. Supplementary Figure S6: The mAbs used for immunoblotting specifically recognize panCD44 and CD44v6

Supplementary Figures S1-S5

Peripheral immune cell infiltration into the brain is a prominent feature in aging and various neurodegenerative diseases such as Alzheimer’s disease (AD). As AD progresses, CD8+ T cells infiltrate into the brain parenchyma, where they tightly associate with neurons and microglia. The functional properties of CD8+ T cells in the brain are largely unknown. To gain further insights into the putative functions of CD8+ T cells in the brain, we explored and compared the transcriptomic profile of CD8+ T cells isolated from the brain and blood of transgenic AD (APPswe/PSEN1dE9, line 85 [APP-PS1]) and age-matched wild-type (WT) mice. Brain CD8+ T cells of APP-PS1 and WT animals had similar transcriptomic profiles and substantially differed from blood circulating CD8+ T cells. The gene signature of brain CD8+ T cells identified them as tissue-resident memory (Trm) T cells. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis on the significantly upregulated genes revealed overrepresentation of biological processes involved in IFN-β signaling and the response to viral infections. Furthermore, brain CD8+ T cells of APP-PS1 and aged WT mice showed similar differentially regulated genes as brain Trm CD8+ T cells in mouse models with acute virus infection, chronic parasite infection, and tumor growth. In conclusion, our profiling of brain CD8+ T cells suggests that in AD, these cells exhibit similar adaptive immune responses as in other inflammatory diseases of the CNS, potentially opening the door for immunotherapy in AD.

Introduction: Chronic lymphocytic leukemia (CLL) is a disease of inhibited cell death, increased proliferation and high importance of interactions with the microenvironment e.g. with T-cells or stromal cells. A central effector in this concert is the activation of B-cell receptor (BCR) signalling pathway, associated with selection of specific BCR qualities (either autonomous signalling or reactivity to chronic (auto)-antigenic stimuli) and these signals play an essential role in CLL disease development and progression, also evidenced by the clinical success of kinase inbibitors directed at this signal. Mechanisms modulating selection of specific BCR types are ill understood, but since checkpoints during BCR selection in B cells are guarded by the Bcl2 family of proteins it is likely that cell death proteins are also influencing these decisions in CLL. To study the importance of microenvironmental interactions and antigen stimulation Tcl1 tg mice, which develop a murine CLL highly similar to the human disease 1,2, were frequently applied in the past, aiming to overcome the limitations of more or less artificial CLL in vitro culture systems. During clonal evolution of CLL, predominantly unmutated and stereotyped IgVH-11 and IgVH-12 BCRs are selected in Tcl1 tg mice which were shown to be specific for the autoantigen phosphatidylcholine (PtC) 3, however, the detailed mechanisms of clonal selection in CLL are still unclear. Here we propose a role of the BH3-only and pro-apoptotic protein Bmf in clonal selection by eliminating CLL cells expressing highly responsive BCRs.

Chronic lymphocytic leukemia (CLL) is a heterogenous disease that is highly dependent on a cross talk of CLL cells with the microenvironment, in particular with T cells. T cells derived from CLL patients or murine CLL models are skewed to an antigen-experienced T-cell subset, indicating a certain degree of antitumor recognition, but they are also exhausted, preventing an effective antitumor immune response. Here we describe a novel mechanism of CLL tumor immune evasion that is independent of T-cell exhaustion, using B-cell-specific deletion of the transcription factor IRF4 (interferon regulatory factor 4) in Tcl-1 transgenic mice developing a murine CLL highly similar to the human disease. We show enhanced CLL disease progression in IRF4-deficient Tcl-1 tg mice, associated with a severe downregulation of genes involved in T-cell activation, including genes involved in antigen processing/presentation and T-cell costimulation, which massively reduced T-cell subset skewing and exhaustion. We found a strong analogy in the human disease, with inferior prognosis of CLL patients with low IRF4 expression in independent CLL patient cohorts, failed T-cell skewing to antigen-experienced subsets, decreased costimulation capacity, and downregulation of genes involved in T-cell activation. These results have therapeutic relevance because our findings on molecular mechanisms of immune privilege may be responsible for the failure of immune-therapeutic strategies in CLL and may lead to improved targeting in the future.

The clinical decisions made when treating patients with metastatic cancer require knowledge of the current tumor extent and response to therapy. For the majority of solid tumors, a response assessment, which is based on imaging, is used to guide these decisions. However, measuring serum protein biomarkers (i.e. tumor markers) may be of additional use. Furthermore, tumor markers exhibit variable specificity and sensitivity and cannot therefore be solely relied upon when making decisions regarding cancer treatment. Therefore, there is a clinical requirement for the identification of specific, sensitive and quantitative biomarkers. In recent years, circulating cell-free DNA (cfDNA) and mutation-specific circulating cell-free tumor DNA (cftDNA) have been identified as novel potential biomarkers. In the current study, cfDNA and cftDNA were compared using imaging-based staging and current tumor markers in 15 patients with metastatic colorectal, pancreatic or breast cancer. These patients were treated at the Third Medical Department of Paracelsus Medical University Salzburg (Austria). The results of the current study demonstrated a statistically significant correlation between the concentration changes of cfDNA and cftDNA and response to treatment, which was assessed by imaging. A correlation was not indicated with current clinically used tumor markers, including carcinoembryonic antigen, carcinoma antigen 15-3 and carcinoma antigen 19-9. The present study also indicated a correlation between cfDNA and cftDNA and the tumor volume of metastatic lesions, which was not observed with the current clinically used tumor markers. In conclusion, cfDNA and cftDNA exhibit the potential to become novel biomarkers for the response assessment following cancer treatment, and may serve as a tool for the estimation of tumor volume. The current study further supports the increasingly important role of cfDNA and cftDNA as new monitoring tools for use during cancer therapy.

AbstractPurpose: Chronic lymphocytic leukemia (CLL) pathophysiology is characterized by a complex crosstalk of tumor cells with the microenvironment. In this regard, NF-κB signaling is considered as important signaling axis, with a variety of key molecules aberrantly expressed or genetically altered in patients with CLL. One of these molecules is BIRC3 (cIAP2), a central regulator of noncanonical NF-κB signaling that serves as pathway brake in the absence of microenvironmental signals. However, the contribution of BIRC3 expression to CLL progression and potential therapeutic implications is unknown. Experimental Design: We analyzed the role of BIRC3 mRNA expression in primary CLL samples in correlation to clinical datasets and used ex vivo assays to investigate functional consequences on the level of NF-κB signaling and downstream target gene regulation. For proof-of-principle experiments, we used genetically modified cell lines. Results: We demonstrate that patients with CLL with low BIRC3 expression experience a more rapid disease progression, which coincides with an enhanced activation of canonical NF-κB target genes evidenced by an increased p65/Rel-B nuclear translocation ratio. As a consequence of enhanced canonical NF-κB target gene activation, both anti- and proapoptotic Bcl-2 family members were upregulated in BIRC3low primary CLL cells, which was associated with higher sensitivity to venetoclax treatment in vitro. Conclusions: Here we show the impact of BIRC3 expression in CLL disease progression in the absence of BIRC3 mutations and show altered canonical NF-κB target gene activation with therapeutic implications.

Chronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell-specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation.

The TCL1 mouse model is widely used to study pathophysiology, clonal evolution, and drug sensitivity or resistance of chronic lymphocytic leukemia (CLL). By performing whole exome sequencing, we present the genetic landscape of primary tumors from TCL1 mice and of TCL1 tumors serially transplanted into wild-type recipients to mimic clonal evolution. We show that similar to CLL patients, mutations in mice are frequently subclonal and heterogenous among different primary TCL1 mice. We further describe that this molecular heterogeneity mirrors heterogenous disease characteristics such as organ infiltration or CLL dependent T cell skewing. Similar to human CLL, we further observed the occurrence of novel mutations and structural variations during clonal evolution and found plasticity in the expansion of B cell receptor specific subclones. Thus, our results uncover that the genetic complexity, pathway dependence and clonal dynamics in mouse CLL are in relevant agreement to human CLL, and they are important to consider in future research using the TCL1 mouse for studying CLL.

Abstract The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell-cycle progression, but is poorly characterized in hematologic malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high-risk markers such as unmutated IGHV genes, high Zap70, or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes from CLL patients. Using coculture conditions mimicking the proliferative lymph node microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein colocalized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B-cell–specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα–NF-κB–mediated mechanism by which ILK expression is induced in the lymph node microenvironment and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation. Cancer Res; 76(8); 2186–96. ©2016 AACR.

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vβ7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.

The mutation status of the B-cell receptor (BCR) is a strong prognostic factor in chronic lymphocytic leukemia (CLL), dividing patients into BCR mutated and unmutated CLL (CLL-Mut, CLL-UM), the latter predicting for worse prognosis.[1][1]–[3][2] Furthermore, the occurrence of highly similar BCRs

Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

CD4+ T cells, but not non-classical monocytes, are dispensable for the development of chronic lymphocytic leukemia in the TCL1-tg murine model

The pathophysiology of chronic lymphocytic leukaemia (CLL) is characterised by a dynamic equilibrium of resting and proliferative tumour cells. While CLL cells in the peripheral blood are mostly G0-arrested, those residing in lymphoid organs have an activated signature due to supportive signals from diverse immune and stromal cell types (Herishanu et al, 2011). The clinical success of novel small molecule inhibitors targeting Bruton tyrosine kinase, such as Ibrutinib, and phosphatidylinositol-3 kinase (PI3K), such as Idelalisib, strengthens the idea that signals downstream of the B cell receptor are critical for CLL development and progression. In this context, the protein kinase C (PKC) and PI3K pathways are indisputable chief players (for review see Woyach et al, 2012). We and others have shown that downstream of PI3K and PKC-beta, the serine/threonine kinase AKT, also known as protein kinase B (PKB), regulates various signalling cascades involved in survival (Hofbauer et al, 2010; Zhuang et al, 2010). AKT is encoded by three distinct genes, namely AKT1, AKT2 and AKT3 (also termed PKB-alpha, PKB-beta, PKB-gamma, respectively). AKT1 and AKT2 are ubiquitously expressed whereas AKT3 is mainly expressed in testes and brain (Yang et al, 2003). In this study, we aimed to gain insight into isoform-specific expression of AKT, and the contribution of AKT1 and AKT2 to stromal and activated T cell-mediated survival and chemoresistance in CLL. Peripheral blood samples from CLL patients were collected after informed consent was obtained in accordance with the Declaration of Helsinki and under the ethical approval of the Ethics Commission of the Province of Salzburg (415-E/1287/4–2011, 415-E/1287/8–2011). First, analysing basal AKT isoform expression in unstimulated purified CLL cells, we observed increased AKT2 mRNA and AKT2 protein expression as compared to AKT1 (Fig 1A and B). As we had previously noted increased AKT phosphorylation in CLL stromal cell co-cultures (Hofbauer et al, 2010), we evaluated AKT phosphorylation kinetics and isoform contribution to this phenomenon. AKT was rapidly phosphorylated at serine 473 (pS473), a phosphorylation site crucial for full AKT activation (Sarbassov et al, 2005), and remained in the activated form for at least 24 h (Fig 1Ci). To assess the relative activation of AKT1 versus AKT2 in CLL cells co-cultured with primary stromal cells, we analysed the phosphorylation at Ser473 (AKT1) and Ser474 (AKT2). We observed robust stromal cell-induced AKT activation of both isoforms in CLL cells cultured in direct cell-cell contact with stromal cells (Fig 1Cii), but not in CLL cells separated from the stromal layer by a transwell insert (Fig 1Di). AKT activation was associated with increased cell viability (Fig 1Dii). Next, we treated co-cultured CLL cells with several AKT inhibitors. As there is no AKT1-specific inhibitor available, we used the pan-AKT inhibitors MK2206 and AiX, and the AKT2-selective inhibitor, Akti-2. The specificities of these inhibitors were confirmed by an AKT isoform-specific pull-down and subsequent in vitro kinase assay to detect phosphorylation of the AKT substrate GSK3A/B after MK2206 or Akti-2 treatment of the Epstein-Barr virus-positive CLL patient-derived MEC1 cells (Fig 1E). Applying the inhibitors to primary CLL cells co-cultured with stromal cells indicated that the selective inhibition of AKT2 did not decrease cell viability, whereas pan-AKT inhibition resulted in significantly decreased survival (Fig 1F), suggesting a dominance of AKT1 or a cooperation of both isoforms in maintaining cell vitality. Genetic manipulations in primary CLL cells are hard to achieve, therefore, to address these alternatives, we used a siRNA approach to target AKT1 or AKT2 in MEC1 cells. Successful and similar knockdown efficiencies were achieved in both experimental settings (Fig 1G). However, the transient knockdown of AKT1, but not AKT2, resulted in loss of cell viability, establishing AKT1 as the dominant AKT isoform (Fig 1H). Consistently, simultaneous knockdown of both isoforms did not further reduce viability compared to the single AKT1 knockdown (Fig 1H). Chronic lymphocytic leukaemia cells co-cultured with activated T cells are rapidly activated, allowing us to mimic in vitro at least part of the proliferative processes that take place in lymph nodes (Asslaber et al, 2013). Under these co-culture conditions, we observed significant transcriptional upregulation of both AKT1 and AKT2 within 24 h (Fig 2A), which was accompanied by phosphorylation of both isoforms on the protein level (Fig 2B). We next studied AKT isoform activation in the context of the oncogene STAT3, a factor of clinical significance to CLL, which has been described to directly interact with the AKT1 promoter (Park et al, 2005; Hazan-Halevy et al, 2010). Activation of CLL cells by T cells resulted in pronounced STAT3 signalling (pY(705)-STAT3), which could be antagonised by treatment with the STAT3 inhibitor S3I-201 (Fig 2C). STAT3 inhibition significantly decreased AKT1 mRNA expression (Fig 2D), indicating an interaction of these pathways upon CLL cell activation. Notably, relative AKT isoform transcription and protein expression in activated CLL cells was not altered upon treatment with the novel small molecule inhibitors Ibrutinib or Idelalisib (data not shown). These observations prompted us to investigate potential synergistic effects of AKT- or STAT3-inhibition with conventional drugs used in the treatment of CLL. We recently observed that T cell activated-CLL cells gain resistance towards fludarabine (Hofbauer et al, 2014). Consistently, CLL cells co-cultured with activated T cells remained viable for up to 48 h even in the presence of fludarabine. However, exposure to the pan-AKT or STAT3 inhibitor induced CLL cell apoptosis within 24 h (data not shown), indicating that inhibition of AKT or STAT3 is able to overcome CLL activation-induced protection. Notably, after 48 h of culture, CLL cells exposed to the STAT3 inhibitor underwent strong apoptosis irrespective of the presence of fludarabine. Pan-AKT inhibition, but importantly not AKT2 inhibition, resulted in decreased cell viability levels, which were significantly pronounced when combined with fludarabine (Fig 2E). Taken together, our results indicate a dominant role of AKT1 in microenvironment-mediated CLL survival and chemoresistance. CLL patients could particularly benefit from targeting the predominant AKT1 isoform, which may also increase the response rate towards classical chemotherapeutics. We would like to thank all patients and their clinicians for their participation in this study. This work has been supported by the Austrian Science Fund FWF (SFB-P021 to R. Greil, P26421-B13 to T.N. Hartmann), the Paracelsus Medical University Salzburg (PMU-FFF E-10/11/058-HAR and E-12/15/074-HAH to T.N. Hartmann), the SCRI-LIMCR GmbH, and the Province of Salzburg. SWH, PWK, JHP, SP, DA performed research; SWH, PWK, TNH designed research and interpreted data; SWH, PWK, SP, JFP, DA analysed data; RG contributed reagents and analytical tools and interpreted data; SWH, PWK and TNH wrote the manuscript. All authors were involved in critical discussion. The authors declare no conflict of interest.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of CD19+ CD5+ CLL cells in the peripheral blood and the lymphoid compartments. The disease is still incurable despite recent advances in the development of novel therapy concepts (e.g. inhibitors of BCR signaling). Although it is not entirely clear how CLL develops, proliferation centers located in the lymph node and the bone marrow are thought to be the crucial niche that provides the surrounding in which CLL cells originate and re-establish the clone after therapy, leading to relapse in almost 100% of CLL patients. In analogous normal lymphoproliferative environments the transcription factor IRF4 plays an important role in controlling B cell maturation, differentiation, proliferation and survival. A SNP in the human IRF4 3’UTR was linked to CLL susceptibility (DiBernardo Nat.Gen. 2008) and in the mouse model germline deletion of IRF4 in all lineages contributed to CLL development in the New Zealand Black and in the VH11 mouse model (Shukla Blood 2013, Ma J.Biol.Chem. 2013).