TOSO Expression Research Articles

Ibrutinib Therapy Downregulates Toso, the Fcr for IgM, Expression in CLL Patients

Background: Toso, the FcR receptor (FcmR) for IgM, is a type I transmembrane protein belonging to the immunoglobulin gene superfamily. Toso is expressed in lymphocytes, and plays an important role in B cell development and survival. In normal B cells, Toso expression increases after B cell receptor (BCR) stimulation, and Toso enhances BCR signaling induced cell survival, NK-kB pathway activation, and BCL-xL expression. CLL B cells express high levels of Toso, especially in IgHV unmutated patients. Upon binding of IgM to Toso on CLL cells, the Toso-IgM complex is internalized and undergoes lysosomal degradation. Given the importance of BCR signaling in CLL pathogenesis and treatment, we performed a series of preclinical and correlative studies to examine the cross talk between Toso and BCR signaling, effects of Toso on CLL cell survival and how Toso is affected by BCR signaling inhibition with ibrutinib. Methods and Results: Toso gene expression (FAIM3) was analyzed in serial samples from eight CLL patients treated with ibrutinib plus rituximab using Affymetrics HG U133 plus 2.0 oligonucleotide arrays at baseline, and after 1 and 4 weeks of continuous ibrutinib-based therapy. We noted that the relative mean Toso gene expression declined during ibrutinib therapy (-0.26 ± 0.12 after 2 weeks and -0.3 ± 0.18 after 4 weeks). Accordingly, Toso surface expression on CLL cells, assessed by flow cytometry, also significantly decreased after 1 and 4 weeks of ibrutinib therapy (Figure 1). We next measured plasma levels of soluble Toso (sToso), which is encoded by an alternative spliced Toso transcript, in serial samples from 35 CLL ibrutinib treated patients using an enzyme-linked immunosorbent assay (ELISA). We noted that baseline sToso levels in CLL patients were significantly higher than in 6 normal controls, i.e. 70 ± 12.1 arbitrary units (AU, n=35) versus 18 ± 8 AU (n=6, p<0.01). sToso levels significantly declined during ibrutinib therapy to 51.9 ± 7.5 AU (n=35, p<0.01) after 1 week of treatment and further decrease in the subsequent time points to 11.9 ± 2.8 AU (p<0.01) after 12 months of ibrutinib therapy. In addition, we found that patients with unmutated IgHV had higher sToso levels at baseline compared to patients with mutated IgHV (76.9 ± 10.8 AU (n=19) versus 45.4 ± 6.7 AU (mean ± SEM, n=11, p<0.05)(Figure 1). Furthermore, time to normalization of sToso levels was longer in unmutated IgHV patients. We then interrogated effects of in vitro stimulation of Toso on CLL viability and BCR signaling-related chemokines CCL3 and CCL4, using mouse monoclonal antibodies (clone 2A5) or artificial human Fc Toso (hToso-Fc) for Toso triggering. After stimulation of CLL cells with 1 or 10 mg/mL of 2A5 antibody or hToso-FC for 24 hours, we noted significantly improved CLL cell survival and increased levels of CCL3 and CCL4 chemokine secretion, particularly in samples from IgHV unmutated patients. The mean relative CLL cell viability after Toso stimulation was 154.2 ± 23.6% compared to unstimulated controls for 2A5 and 140.9 ± 17.3% after hToso-Fc treatment (n=6, p<0.05). Toso triggering induced very high levels of CCL3 and CCL4 secretion using 2A5 (3901 ± 464% of controls) or hToso-FC (6712 ± 1717% of controls, n=6, p<0.05)(Figure 1). Conclusions: These studies demonstrate that ibrutinib therapy downregulates Toso (FAIM3) gene expression, as well as cell surface and soluble Toso expression. IgHV unmutated patients have higher levels of soluble Toso and the effect on Toso activation on viability and CCL3 and CCL4 secretion is more pronounced in this group of patients. These findings corroborate the close relation between BCR signaling and Toso in CLL. Disclosures Wierda: Cyclcel: Research Funding; Miragen: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Loxo Oncology Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Acerta Pharma Inc: Research Funding; Pharmacyclics LLC: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; GSK/Novartis: Research Funding; Gilead Sciences: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Sunesis: Research Funding. Burger:Janssen Pharmaceuticals: Consultancy, Honoraria; Aptose Biosciences, Inc: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics, an AbbVie company: Research Funding; AstraZeneca: Honoraria; BeiGene: Research Funding.

Anti-TOSO antibody treatment promotes T cell activation-induced cell death (AICD) in vitro and in vivo

T cell activation-induced cell death (AICD), that involves the induction of Fas-mediated apoptosis, is very important for the maintenance of immune homeostasis. TOSO was firstly described as an inhibitor of Fas-mediated apoptosis and overexpressed in chronic lymphocytic leukemia. Recently, TOSO was identified as IgM FcR. In this study, we produced anti-TOSO monoclonal antibody (mAb) that could block the binding of IgM to TOSO and found that T cell apoptosis is negatively correlated with TOSO expression during T cell activation. Treatment of activated T cells with anti-TOSO blocking mAb promoted T cell AICD in in vitro AICD model, and treatment of xenogeneic-GVHD mice with the antibody also increased the sensitivity of activated T cells to Fas-induced apoptosis, which was accompanied by reduction of c-FLIPL expression and up-regulation of AP-1 complex. In summary, our data indicate the anti-apoptotic effect of TOSO in T cell AICD and open up new therapeutic prospects for the treatment of hematologic malignancies and immune disorders.

Reply to Honjo et al.: Functional relevant expression of Toso on granulocytes

Functional relevant expression of Toso on granulocytes. (A) Toso expression. Splenocytes from WT and Toso−/− mice were immunostained with anti-CD19 (B cells) and anti-Gr1 (granulocytes) antibody in combination with DAPI and anti-Toso antibody after pretreatment with Fc receptor block. One representative staining of cells gated on Gr1hi DAPI− cells (Left) or CD19+ DAPI− cells is shown (Right). (B) Quantification of FACS stainings derived from A (n = 4/group). (C–E) WT recipient mice were irradiated and reconstituted with BM from WT mice (CD45.1; group 1), Toso−/− mice (CD45.2; group 2), or a 1:1 mixture of WT and Toso−/− BM (group 3). At 30 d posttransplantation, peripheral blood cells were stimulated with different concentrations of fMLP (Formyl-Methionyl-Leucyl-Phenylalanine) and granulocytes (Gr1+ cells) were analyzed after 30 min for CD45.1 expression (anti-CD45.1), CD45.2 expression (anti-CD45.2), reactive oxygen species production (dihydrorhodamine staining), and degranulation (side scatter). Representative FACS …

TOSO Deficiency Exerts Impact On BAFF-R Expression, Thereby Causing Impaired B Cell Development in Vivo

AbstractAbstract 3898Apoptosis resistance concomitant with aberrant upregulation of pro-survival pathways is a main pathogenic mechanism in development and maintenance of chronic lymphocytic leukemia (CLL). Our group recently identified TOSO to be significantly overexpressed in CLL compared to other B cell lymphomas or healthy B cells. Interestingly, TOSO is thought to exert pro-survival signaling, although it remains still enigmatic, how TOSO is regulated and why TOSO is expressed extremely heterogeneous on different B cells entities. Moreover, we previously detected elevated TOSO expression to be associated with progressive disease, including unmutated IgVH status of the B cell receptor (BCR). Since the BCR is a driving force in CLL, TOSO expression was investigated after BCR crosslinking and resulted in an increase of TOSO. To date, the TOSO promoter has not been described yet. Here, we firstly identified the TOSO proximal region to exert promoter activity. Moreover, in silico analysis and phylogenetic footprinting exhibited existence of transcription factor binding sites for NF-κB and BCL6. In luciferase reporter assays, including targeted mutagenesis, NF-κB was confirmed as novel inducer of TOSO expression. Whereas BCL6 binding, confirmed by ChIP and luciferase assays, was shown to exert repressing activity on the TOSO promoter. Although it can explained now how TOSO is regulated by the BCR, the reason for its distinct basal expression levels in normal B cells and other B cell malignancies still remained unclear. Our data illustrate for the first time that DNA hypomethylation of the TOSO promoter is a conspicuous characteristic in CLL patients compared to healthy donors. Indeed, the methylation status seems to play a major role, since the methylation level correlates with TOSO expression also in other B cell lymphomas.Moreover, it is indispensible to clarify the biologic significance of TOSO, particularly in the CLL relevant B cells. Therefore, we generated a B cell-specific knockout mouse model and identified impaired B cell development characterized by diminished B cell count. Gene expression analysis and flow cytometry revealed a decrease of the B-cell activating factor receptor (BAFF-R). BAFF-R ligation is known to promote B cell survival in particular via degradation of IκBα and translocation of NF-κB to the nucleus, thus activating the NF-κB pathway. Thus, BAFF-R decrease caused by TOSO depletion might lead to the detected reduction of B lymphocytes, which corresponds to the previous observations.Taken together, this work reveals a counteractive TOSO regulation by transcription activator NF-κB and transcription repressor BCL6 in a BCR-dependent manner in B cells. Moreover, we detected CLL-specific hypomethylation of the TOSO promoter, which is supposed to be causative for elevated TOSO level in CLL. Moreover, our results might reveal a new function of TOSO in pro-survival signaling and B cell homeostasis, supporting the anti-apoptotic feature of TOSO in B cells.Identifying the regulating mechanisms and biological function of the anti-apoptotic TOSO, is an essential step towards elucidation of the underlying molecular causes for the development of CLL. Disclosures:No relevant conflicts of interest to declare.

TOSO promotes β-cell proliferation and protects from apoptosis

Decreased β-cell mass reflects a shift from quiescence/proliferation into apoptosis, it plays a crucial role in the pathophysiology of diabetes. A major attempt to restore β-cell mass and normoglycemia is to improve β-cell survival. Here we show that switching off the Fas pathway using Fas apoptotic inhibitory protein (Faim/TOSO), which regulates apoptosis upstream of caspase 8, blocked β-cell apoptosis and increased proliferation in human islets. TOSO was clearly expressed in pancreatic β-cells and down-regulated in T2DM. TOSO expression correlated with β-cell turnover; at conditions of improved survival, TOSO was induced. In contrast, TOSO downregulation induced β-cell apoptosis. Although TOSO overexpression resulted in a 3-fold induction of proliferation, proliferating β-cells showed a very limited capacity to undergo multiple rounds of replication. Our data suggest that TOSO is an important regulator of β-cell turnover and switches β-cell apoptosis into proliferation.

Toso, a Functional IgM Receptor, Is Regulated by IL-2 in T and NK Cells

We find that the cell surface receptor Toso is dramatically downregulated by in vitro stimulation of human T and NK cells with IL-2 in a STAT5-dependent manner. The fact that IL-2 is known to prime NK and T cells for Fas/TNF-mediated activation-induced cell death (AICD) fits nicely with the original and recent descriptions of Toso as an inhibitor of Fas/TNF-induced apoptosis. In support of this possibility, effector memory T cells express markedly lower levels of Toso than those of naive T cells, indicating that activation in vivo correlates with the downregulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically downregulates Toso expression compared with that of naive CD4 T cells. However, overexpression of Toso in human NK cells and Jurkat T cells does not inhibit Fas-mediated apoptosis, and, in agreement with other recent reports, Toso clearly functions as an IgM receptor. Unlike CD16, Toso expression by NK cells does not convey cytotoxic potential, but its ligation does trigger intracellular signaling in NK cells. In summary, our data indicate that Toso is a functional IgM receptor that is capable of activating signaling molecules, is regulated by IL-2, and is not inherently an antiapoptotic molecule.

TOSO-Deficient B Cells Show An Impaired NF-κB and Migration Signaling In Vivo

Abstract 1779 Background:Our group firstly demonstrated that TOSO (FAIM3) is over-expressed in CLL compared to healthy B cell subsets as well as other B cell lymphomas. Furthermore, we detected a significant correlation of high TOSO expression to high lymphocyte count, unmutated IgVH status and Binet C, which are all markers for poor prognosis. TOSO has been described as pro-survival gene also in other settings. However, its mode of action is discussed controversially.Therefore, we aimed to elucidate the role of TOSO in B-cell specific gene expression by creating a knockdown mouse model. CD40 ligation and B cell receptor (BCR) activation influences TOSO expression and the fact that transcriptional regulation of TOSO is still unknown, we were eager to determine transcriptional factors that are directly responsible for the alterable TOSO levels. Methods:Faim3-floxed C57BL/6 FLP deleter mice were crossbred with CD19 specific Cre recombinase expressing mice. B-cells from the TOSOCD19−/− (KO) mice were isolated and gene expression was analyzed via mRNA based Illumina microchip array. Convincing results were verified by flow cytometry and blood count was carried out in addition.To determine the promoter region of TOSO, three overlapping DNA fragments (containing either NF-κB, Bcl-6 or both binding sites) upstream of the transcription start site of the first TOSO exon were cloned into a luciferase reporter vector lacking a promoter. Those constructs were transfected into HeLa cells. After 24 hours luciferase assays were performed. The involvement of NF-κB in the regulation of TOSO transcription was measured by TNFα stimulation of transfected cells prior to luminescence measurement. Targeted mutagenesis of the NF-κB binding site was performed to confirm the data. In addition, Bcl-6 expression vector was co-transfected for evaluation of repressing influence on TOSO expression. Results:In order to cover the functional part of TOSO, we generated a B-cell specific TOSOCD19−/− mouse model. Downstream effects of TOSO were validated via microarray-based gene expression analysis. Results displayed a clear clustering of deregulated genes compared to control mice. Nearly 400 genes showed expression alterations; genes involved in the NF-κB pathway and migration processes were downregulated in TOSOCD19−/−. These results were confirmed by flow cytometry analysis. The TOSO KO displayed also relevant effects on the hematopoietic system. Lymphocyte (p=0,0048), neutrophil (p=0,0007) and red blood cell counts (p=0,0051) were significantly decreased in the TOSOCD19−/− mice. Most important, the B-cell count was significantly reduced in TOSO-deficient settings (n=9; p=0,032).Since TOSO level seems to be so important for such fundamental pathways, investigation of gene expression regulation is mandatory. In situ analysis of the TOSO promoter region revealed NF-κB and Bcl-6 as promising results. Luciferase reporter assays including targeted mutagenesis confirmed the positive regulation of NF-κB and the repressing influence of Bcl-6 on TOSO expression significantly. Conclusions:We reveal for the first time a TOSO-dependent expression profile. We identified TOSO-dependent deregulated genes, which were involved in NF-κB signaling and migration, suggesting that TOSO represents an important factor in these pathways. Additionally, TOSO KO caused a decrease of peripheral B-cells in vivo. Furthermore, we identified NF-κB and Bcl-6 to regulate the TOSO expression in an opposite manner. Disclosures:No relevant conflicts of interest to declare.

TOSO is overexpressed and correlated with disease progression in Chinese patients with chronic lymphocytic leukemia

Recently, a gene named TOSO was identified as being overexpressed and associated with the anti-apoptotic characteristic of chronic lymphocytic leukemia (CLL). However, the association of TOSO expression with clinical features of CLL has not been fully described, especially in Chinese patients. TOSO expression was detected by quantitative RT-PCR in CD19+ sorted cells in a cohort of 81 untreated patients with CLL. The results showed that the expression of TOSO in CLL was significantly higher than that in healthy controls (p = 0.027) and other B-cell lymphoproliferative diseases (p = 0.033). The expression level of TOSO was significantly correlated with Binet staging, IGVH mutation status, age, and time to treatment in CLL. A negative correlation was observed between age and TOSO expression (Spearman's, p = 0.025). No correlation was observed between the expression of TOSO and CD38 or ZAP-70. Cox regression analysis indicated that high expression of TOSO (more than 8.4) was an independent indicator for shorter treatment-free survival in CLL. We conclude that TOSO is specifically overexpressed and associated with progressive disease, and might be an important prognostic factor in CLL.

Microenvironment Influences Expression of TOSO – a Novel NF-Kappa B Target Gene In Chronic Lymphocytic Leukemia

AbstractAbstract 695We recently identified the transmembrane protein TOSO to be significantly over-expressed in chronic lymphocytic leukemia (CLL) compared to other B-cell lymphomas or healthy B-cells and T-cells. TOSO was initially characterized as inhibitor of Fas-mediator of apoptosis; however, it could be demonstrated to be the receptor for the IgM-specific Fc-domain in immune cells. TOSO is the only Fcμ receptor expressed on B-cells and is solely expressed in the lymphoid compartment. However, little is known on its regulation and the molecular background of over-expression in CLL. We investigated TOSO expression on mRNA and protein level in freshly isolated primary CLL cells (n=10) and healthy B-cells (n=4) after single treatment for 24 hours with a comprehensive panel of different cytokines or stimuli (interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, interferon (IFN)-γ, transforming growth factor (TGF)-ß, tumor necrosis factor (TNF)-α, lipopolysaccharide, CpG, CD40-ligand (CD40L) and B-cell receptor (BCR)) being involved in B- and T-cell interplay, by qRT-PCR, western blotting and flow cytometry. Furthermore, we determined the impact of nurse-like cells (NLC) to TOSO expression and co-incubated primary CLL cells for up to 14 days with NLCs. To better understand the intracellular regulation of TOSO, we inhibited BCR and/or CD40L pathways, which were shown by us to be either stimulatory (BCR) or inhibitory (CD40L) in regard to TOSO expression. Since expression might be finally also controlled on epigenetic level, we determined the methylation status of the putative TOSO promoter in 64 CLL samples and 10 healthy B-cells samples. Quantitative DNA methylation analysis was conducted using the EpiTyper application by Sequenom (San Diego, CA, USA). Our experiments reveal novel extra- and intracellular stimuli regulating TOSO expression. We identified CD40L, IL-4 and CpGs to have strong inhibitory effects on TOSO expression (P<0.001) in primary CLL cells and healthy B-cells. In contrast, we identified NLCs (MFIR 15,8 vs. 25,8; P=0.049; n=4) and BCR cross-linking to induce TOSO expression on the cell surface of CLL cells. Based on extracellular stimuli, we were able to hypothesize on shared downstream pathways in order to identify the key regulatory factors and transcription factors controlling TOSO expression. By using a panel of inhibitors in BCR and CD40L downstream signaling, NF-kappa B was shown to have the strongest effect on TOSO expression (P=0,0294). Applying the I-kappa B kinase (IKK) inhibitor Wedelolactone at non-toxic concentrations (10μM), TOSO expression was profoundly suppressed after 24 hours. Regarding epigenetic alterations, our analysis from genome-wide screening experiments in CLL patients compared to healthy B-cells did reveal significant aberrant DNA de-methylation events in the TOSO promoter-associated CpG island (P<0.001). In conclusion, we revealed IL-4, CpG and CD40L as BCR stimulus and NLCs as the key components in regulation of TOSO in the CLL cell microenvironment. Furthermore, over-expression of TOSO in CLL cells compared to normal B-cells could be demonstrated being associated with epigenetic changes at its promoter. We identified TOSO as a novel NF-kappa B regulated target gene. In ongoing studies we elucidate whether NF-kappa B acts directly or in-directly on TOSO expression. Disclosures:No relevant conflicts of interest to declare.

CD25 Blockade protects T Cells from Activation‐induced Cell Death (AICD) via Maintenance of TOSO Expression

CD25 monoclonal antibody binding to the alpha-chain of the Interleukin-2 (IL-2) receptor, blocks high-affinity IL-2 binding, thereby preventing complete T-cell activation and being of ample importance in transplantation medicine and potentially the treatment of autoimmune disease. However, CD25 antibodies do not only block T-cell activation but also prevent activation-induced cell death (AICD) attributing a dual function to IL-2. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of humanized anti-CD25 mAb was investigated. PBMC were stimulated with CD3 antibody OKT-3 together with recombinant IL-2 in the absence or presence of anti-CD25 mAb. RNA was extracted and subjected to microarray analysis on U133A microarrays (Affymetrix). Anti-CD25 treatment inhibited several genes typically expressed during T-cell activation including granzyme B, signalling lymphocyte activation molecule, family member 1 (SLAMF1), CD40-Ligand (CD40-L), IL-9 and interferon (IFN)-gamma. Interestingly, anti-CD25 mAb also blocked the expression of several genes important for susceptibility to apoptosis, such as death receptor 6 (DR6) or reversed IL-2-mediated repression of anti-apoptotic genes, such as Fas apoptotic inhibitory molecule 3 (FAIM3)/TOSO. Functional significance of DR6 and TOSO expression in IL-2-dependent T-cell activation was subsequently evaluated by RNA interference in AICD: While siRNA specifically directed against DR6 did not modulate FAS-L-mediated apoptosis induction in primary T cells, down-regulation of TOSO significantly increased susceptibility to apoptosis, emphasizing an important role for TOSO in IL-2-mediated AICD.

Toso expression during B cell development (82.3)

Abstract Regulated apoptosis is essential for both the development and homeostasis of the immune system. Fas-mediated cell death has been implicated in apoptosis during the development and homeostasis of lymphocytes, and this apoptosis pathway is regulated by the interplay of BCL-2-family members, as well as other factors, such as cytokine withdrawal. However, Toso (also known as Fas Apoptosis Inhibitory Molecule-3) expression has been reported to inhibit Fas-mediated cell death. We isolated immune cells from the spleen and found that B cells expressed high levels of Toso mRNA compared to T cells, NK cells, granulocytes, macrophages and monocytes. After stimulation by LPS, anti-CD40 or anti-IgM, Toso expression is dramatically down-regulated in splenic B cells. Further analysis showed that bone marrow B cells express lower levels of Toso mRNA than splenic B cells. We sorted the bone marrow B cells into development stages and found that only immature B cells expressed high levels of Toso mRNA compared with pro-, pre- and mature B cells. We hypothesize that Toso has a role in normal B cell development and maintenance. To examine this, we are using Toso knockout and transgenic mice. This work was supported by the NIAID intramural research program.

Role of TOSO in activation induced cell death of human T- and NK cells (82.2)

Abstract Homeostasis of both NK and T cells in the periphery is regulated by activation induced cell death (AICD) mediated by signaling through Fas, and other death receptors. It has been shown that IL-2 is required for priming cells for this type of apoptosis. Cytokine primed NK cells, in addition to killing their target cells, also undergo AICD upon activation by activating ligands expressed by the target cells. TOSO is an anti-apoptotic molecule known to inhibit apoptosis mediated by the death receptor pathway and we investigated the role of TOSO in regulating AICD in NK and T cells. We observed by realtime PCR that stimulation of human NK cells and T cells in vitro with IL-2 resulted in down regulation of TOSO. We further analyzed TOSO expression levels on sorted human CD4 T cells and found that cells with an effector/memory phenotype have markedly lower levels of TOSO than naïve cells, which suggests that activation in vivo correlates with down-regulation of TOSO. In agreement, we found that activation of CD45RO+ cells in vitro dramatically down regulated TOSO compared to naïve CD4 T cells. Further, overexpression of Toso in Jurkat cells was able to inhibit Fas-mediated cell death. We hypothesize that IL-2 has a dual action: first, it activates and induces the proliferation of NK and T-cells and second, it down-regulates TOSO, thereby sensitizing or priming them for apoptosis upon re-stimulation. Considering that IL-2 is being used as a therapeutic agent for cancer treatment and the recent study showing that HIV envelope protein can down regulate TOSO levels on CD4 T cells, unraveling how TOSO functions and is regulated might have potential for treating cancer and other diseases.

Overexpression of the Fas-inhibitory molecule TOSO–a novel antiapoptotic factor in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterised by resistance to apoptotic stimuli, mediated by overexpression of anti-apoptotic factors or extracellular survival signals. In this context, TOSO, also known as Fas-inhibitory molecule 3, was identified as a candidate gene over-expressed in CLL. TOSO is a transmembrane protein that inhibits Fas-mediated apoptosis by binding Fas-associated death domain via its C-terminal intracellular domain. In CLL, high levels of TOSO expression have been correlated with more aggressive disease, being associated with high leukocyte count, advanced Binet stage, need for chemotherapy and unmutated IgVH gene status. Also, the CD38+ CLL subset with proliferative activity showed enhanced TOSO expression. B-cell receptor-stimulation was identified as positive regulator of TOSO expression, potentially providing a functional mechanism for aberrant TOSO expression in CLL. In contrast, CD40-ligand signalling reduces expression of TOSO, possibly explaining previously observed Fas-sensitisation by CD40-ligand in gene therapy trials. Both the association with unmutated IgVH gene status and the specific induction of TOSO via the BCR suggest autoreactive BCR signalling involving TOSO as a mediator of resistance to apoptosis in CLL. Further studies will reveal the functional context of TOSO in CLL and B cell biology. Surface expression of TOSO will enable antibody-based targeting of this novel CLL-antigen.

Overexpression of TOSO in CLL Is Triggered by B-Cell Receptor Signaling and Associated with Progressive Disease.

Resistance towards apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals like B-cell receptor stimulation (BCR) are considered to be responsible for accumulation of malignant B cells in CLL . TOSO, also known as Fas-inhibitory molecule 3 (FAIM3), was identified as overexpressed candidate gene in CLL based on re-evaluation of publicly available microarray data sets. Based on primary CLL samples from 106 patients, TOSO expression was compared to healthy donor B cells using quantitative real-time PCR, western-blot, flow cytometry and immunohistochemistry. To reveal underlying mechanisms of TOSO overexpression, B-cell receptor (BCR) and CD40Ligand stimulation as well as bone marrow stroma cell co-incubation was performed. Apoptotic resistance was assessed by annexin V/7-AAD flow cytometry in context of CH11-Fas-agonistic antibody. TOSO was identified to exhibit elevated relative expression of 6.8 compared to healthy donor B cells using quantitative real-time PCR (p=0.004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy and unmutated IgVH status. CD38+ CLL subsets harboring proliferative activity showed significantly enhanced TOSO expression. Immunohistochemistry revealed upregulation of TOSO in lymph nodes of CLL patients. In lymph nodes derived from healthy donors TOSO was detected in single plasmocytoid cells within the germinal center and in the marginal zone. No specific staining was seen in follicular lymphomas. CLL-specific upregulation of TOSO was confirmed by RT-PCR, samples of follicular lymphomas, DLBCL, marginal zone lymphoma and Hodgkin cell lines did not reveal TOSO up-regulation. We evaluated functional mechanisms of aberrant TOSO expression in CLL cells and identified TOSO expression significantly being induced by BCR-stimulation compared to control cells (relative expression (RE) 8.25 vs. 4.86, p=0.013). In contrast, CD40L signaling significantly reduced TOSO expression (RE 2.60; p=0.007). Spontaneous apoptosis of CLL cells was significantly reduced by BCR-stimulus, in CD40Ligandstimulated CLL samples a slight sensitization towards Fas-mediated apoptosis was seen. In summary, we show that the anti-apoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38+ cell subset. Both association with unmutated IgVH and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL. Down-regulation of TOSO by CD40Ligand in the context of CD40Ligand-mediated Fas-sensitization of CLL reveal TOSO as a new anti-apoptotic factor in CLL. CLL-specific over-expression of the transmembrane protein might further offer new therapeutic strategies in CLL treatment.

Overexpression of TOSO in CLL is triggered by B-cell receptor signaling and associated with progressive disease

AbstractResistance toward apoptotic stimuli mediated by overexpression of antiapoptotic factors or extracellular survival signals is considered to be responsible for accumulation of malignant B cells in chronic lymphocytic leukemia (CLL). TOSO was identified as overexpressed candidate gene in CLL, applying unit-transformation assays of publicly available microarray datasets. Based on CLL samples from 106 patients, TOSO was identified to exhibit elevated relative expression (RE) of 6.8 compared with healthy donor B cells using quantitative real-time polymerase chain reaction (PCR; P = .004). High levels of TOSO expression in CLL correlated with high leukocyte count, advanced Binet stage, previous need for chemotherapy, and unmutated IgVH status. CD38+ CLL subsets harboring proliferative activity showed enhanced TOSO expression. We evaluated functional mechanisms of aberrant TOSO expression and identified TOSO expression significantly induced by B-cell receptor (BCR) stimulation compared with control cells (RE; 8.25 vs 4.86; P = .01). In contrast, CD40L signaling significantly reduced TOSO expression (RE, 2.60; P = .01). In summary, we show that the antiapoptotic factor TOSO is associated with progressive disease and enhanced in the proliferative CD38+ CLL subset. Both association with unmutated IgVH and the specific induction of TOSO via the BCR suggest autoreactive BCR signaling as a key mediator of apoptosis resistance in CLL.

SAGE Analysis Demonstrates Increased Expression of TOSO Contributing to Fas Mediated Resistance in CLL.

The molecular pathogenesis of CLL remains unclear; therefore we sought to identify new genes involved in the pathogenesis or mechanisms of resistance in CLL and other B cell malignancies. To this end we performed SAGE in CLL cells and compared results with those from healthy B cells (nCD19+). Gene expression profiles were obtained by SAGE and individual gene expression was measured by real time PCR. Analysis revealed only 27 genes with significant (p< 0.001) differences between mutated and unmutated subtypes of CLL cells. In contrast to the high level of similarity between the CLL subtypes, comparison of the complete CLL SAGE libraries versus nCD19, revealed that 56 genes were over represented and 50 genes were down regulated in CLL (fold change higher than 20x, p<0.001 as a cut-off). A gene ontology analysis using the top 1,000 transcripts revealed some genes aberrantly expressed in CLL not previously reported. One of these is TOSO (regulator of Fas-induced apoptosis) of interest since CLL cells are resistant to Fas mediated killing. Real-time RT-PCR analysis of 78 cases of CLL and 12 nCD19+ confirmed that TOSO mRNA was over expressed (4.4 fold) in CLL (p<0.0001), and its expression was positively correlated with BCL-2 expression. There was no correlation between TOSO expression and prognostic factors, including IgVH mutational status, cytogenetics, evolution or previous treatment. In addition, using tissue microarray of 1mm cores arranged in triplicate followed by indirect immunohistochemistry we evaluated TOSO protein distribution and intensity in 44 CLL lymph nodes and 5 reactive tonsils and appendix. Scoring was performed by two histopathologists independently and demonstrated that more than 50% of CLL cases express TOSO strongly (2+, 3+) with a range of 10% to 95% of positive tumour cells (p=0.0057, Chi-Square test), whereas among control samples analyzed all were negative except the mantle cells from one tonsil and one appendix were scored as weakly positive (1+); the remainder were negative. Flow cytometry analysis in 12 CLL cases and 5 healthy subjects demonstrated a 5.6 fold increase of TOSO in peripheral CLL cells (p=0.013). SAGE results confirmed that both CLL subtypes share a similar expression profile and highlighted a novel gene, TOSO. This protein plays a functional role upstream of Fas in the extrinsic apoptosis pathway and ongoing studies are assessing the mechanisms whereby TOSO mediates resistance of CLL cells to anti-Fas treatment and ways to reverse this.

E-LDL upregulates TOSO expression and enhances the survival of human macrophages

Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes atherosclerosis. Atherogenic lipoproteins are cytotoxic and induce cell death under certain conditions but may also enhance macrophage survival. Macrophages treated with enzymatically modified LDL (E-LDL) were subjected to GeneChip analysis and the antiapoptotic gene TOSO was found induced. TOSO mRNA is upregulated and apoptosis is reduced in E-LDL but not in oxidized LDL (Ox-LDL) loaded macrophages. FLIPL abundance was suggested to mediate the antiapoptotic properties of TOSO; however, FLIPL was not changed. Ox-LDL is internalized predominantly by scavenger receptors such as CD36 while E-LDL particles are preferentially internalized by Fc- and complement-receptor dependent phagocytosis and internalization of phagobeads by macrophages upregulates TOSO. In COS-7 cells however, phagocytotic activity was not affected by TOSO. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a FLIPL independent mechanism.

Toso, a Cell Surface, Specific Regulator of Fas-Induced Apoptosis in T Cells

Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library–based functional cloning we identified a gene toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up-regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.