Fc receptors specifically bind to the Fc region of Igs to mediate the unique functions to each class of Igs. To identify a novel Fc receptor for IgM, we searched expressed sequence tag database for molecules containing Ig domains with homology to those of known Fc receptors for IgM, Fcalpha/muR and polymeric Ig receptor. As a result, we identified TOSO/Fas apoptotic inhibitory molecule 3 (FAIM3) as a possible Fc receptor for IgM. HeLa cells transfected with a TOSO/FAIM3-expression vector bound to IgM but not IgG and were able to internalize IgM-conjugated beads but not IgG-conjugated beads, suggesting that TOSO/FAIM3 is indeed a receptor for IgM (FcmuR). FcmuR protein was expressed predominantly on B-lineage cells; expression of the Fcmr transcripts was observed from the pre-B-cell stage and maintained thereafter during B-cell development. These results identify TOSO/FAIM3 as a receptor for IgM and suggest that FcmuR may serve as an uptake receptor for IgM-opsonized antigens by B cells.

Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

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.

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 IgV(H) 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 IgV(H) 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.

MicroRNAs play a key role in cellular regulation and if deregulated in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). RNAs from primary cells of 50 treatment-naive CLL patients and peripheral B-cells of 14 healthy donors were applied to miRNA-expression profiling using bead chip technology. In CLL cells a set of 7 up- and 19 down-regulated miRNAs was identified. Among the miRNAs being downregulated in CLL cells, 6 out of 10 miRNA promoters being examined showed gain of methylation as compared to normal B cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3'UTR of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107 and miR-424. While expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells as compared to the levels in healthy donor B cells. In summary, we could demonstrate disruption of miRNA-mediated translational control, partly due to epigenetic transcriptional silencing of miRNAs, with subsequent overexpression of the oncogenic transcription factor PLAG1 as a putative novel mechanism of CLL pathogenesis.

The alkylphosphocholine (APC) erufosine is a synthetic phospholipid analogue with antineoplastic activity. APC are known to interact with lipid metabolism and modulate cellular signaling pathways, particularly the phosphorylation of Akt. Here, in primary CLL cells induction of apoptosis was detected with an IC50 of 22muM whereas healthy donor PBMC were less sensitive towards erufosine. Treatment with erufosine caused dose-dependent cleavage of PARP, co-incubation with caspase inhibitor z-VAD almost completely abrogated the cytotoxic effect of erufosine indicating a caspase-dependent mechanism of erufosine. Erufosine was shown to induce apoptosis in primary CLL cells and merits further investigation regarding therapeutic options in CLL.

Src family kinases (SFKs) were described to be overexpressed in chronic lymphocytic leukemia (CLL). Therefore, we wished to examine the effects of the Src and Abl kinase inhibitor dasatinib on the intracellular signaling and survival of CLL cells. Dasatinib showed a dose and time dependent reduction of global tyrosine phosphorylation and of activating phosphotyrosine levels of SFKs. Treatment with 100 nM dasatinib led to decreased levels of the activated, phosphorylated forms of Akt, Erk1/2 and p38, and induced PARP cleavage through caspase activity. In Mec1 and JVM-3 cell lines, dasatinib increased p53 protein levels and inhibited proliferation. In freshly isolated CLL cells, dasatinib reduced the expression of Mcl-1 and Bcl-xL. Combination of 5 microM dasatinib and fludarabine increased the apoptosis induction of each by ~50 %. In 15 primary CLL samples, cells with unmutated immunoglobulin variable heavy chain (IgVH) genes were more sensitive to dasatinib than those with mutated IgVH genes (p=0.002). In summary, dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro most prominently in samples obtained from patients with unfavorable prognostic features.

Inappropriate Nuclear Factor kappa B (NFkappaB) activity is one major hallmark of B cell-malignancies as well as chronic lymphocytic leukemia (CLL). NFkappaB-dependent genes are involved in anti-apoptosis, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFkappaB activity in CLL still need to be elucidated. Previously we identified translocations in a region on chromosome 6q that encodes tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20), which is a key player in negative feedback loop regulation of NFkappaB. Inactivation of this ubiquitin-editing enzyme is involved in immunopathologies and in tumorigenesis. Frequent mutations in the A20 locus - leading to sustained NFkappaB activity - could be shown to play a dominant role in development of different B-cell malignancies. In order to check if A20 is involved in upregulation of NF-kappa B activity in CLL, we sequenced exons 2-9 of the A20 gene in 55 CLL DNA samples. Furthermore, we determined the methylation status of the promoter region in 63 CLL DNA samples and compared to 10 control DNAs of B-cells from healthy donors. Contrary to reports from other B cell-malignancies the A20 region showed neither mutations nor aberrant DNA methylation. Moreover its expression could be confirmed by immunoblotting and showing comparable results to healthy B cells. These results indicate that malignant development in CLL differs from most of other B-cell malignancies, which show frequent inactivation of A20.

Although fludarabine-based regimens and monoclonal antibodies are able to induce high overall and complete remission rates in patients with chronic lymphocytic leukemia (CLL), a major impact on overall survival or even potential cure by these treatments is still lacking. Increased sensitivity and specificity of flow cytometry and polymerase chain reaction techniques enable us to detect few CLL cells in peripheral blood and bone morrow. This has brought significant advancement into the evaluation of response quality of CLL treatment. The eradication of minimal residual disease (MRD) below measurable levels seems to be critical to overcome recurring clonal expansion resulting in disease progression or relapse. Several studies suggest that achieving MRD negativity in patients with CLL provokes prolonged response duration and survival. Thus, elimination of MRD should become a surrogate end point of modern clinical trials and a goal in CLL. This review summarizes the current status of and future strategies for MRD eradication in CLL.

Summary Alemtuzumab has shown considerable activity in untreated and relapsed chronic lymphocytic leukaemia. We report our long-term experience in 21 patients within a randomized phase III trial investigating the role of alemtuzumab for consolidation therapy after first-line fludarabine +/- cyclophosphamide, which was stopped prematurely due to severe infections. However, after a median follow-up of 48 months, progression-free survival was significantly prolonged for patients receiving alemtuzumab consolidation compared to those with no further treatment (P = 0.004). Minimal residual disease (MRD) levels were persistently reduced after consolidation. Therefore, despite toxicity, MRD reduction by alemtuzumab consolidation translates into a significantly improved long-term clinical outcome.

CD200 plays a key role in regulating the immune system and has been shown to be upregulated on the surface of different tumors including chronic lymphocytic leukemia. In this study we addressed the effects of CD200 over-expression in CLL cells on autologous T cells in a mixed lymphocyte reaction system. We used native and CD40 ligand (CD40L)-stimulated CLL cells as antigen-presenting cells (APCs) to expand autologous T cells of 14 patients. T cell proliferation over 3 weeks of in vitro culture was significantly enhanced compared to control cells when using CD40L-stimulated APCs and the anti-CD200 antibody 1B9 (p=0.0004). CD200 blockade was further shown to stimulate antigen-specific T cell responses towards the CLL-associated antigen fibromodulin (p=0.04). Finally, the number of CD4+/CD25high/FOXP3+ T cells (T(reg)) was significantly decreased adding anti-CD200 antibody (p=0.04). In summary, CD200 blockade may provide therapeutic benefits in CLL by augmenting an antigen-specific T cell response with suppression of regulatory T cells.

Here, we report a rare coincidence of heterozygous hemoglobinopathy (Hb) Stanleyville II and severe pernicious anemia due to autoimmune gastritis. Hb Stanleyville II is characterized by a single base exchange (AAC-->AAA) resulting in a substitution Asn-->Lys at position 78 of hemoglobin alpha2-chain. Under normal conditions this hemoglobinopathy does not cause any symptoms even if present as homozygous variant. However, in our case diagnosis of pernicious anemia was hampered by the absence of typical erythrocytic macrocytosis and hyperchromasia. In addition, interpretation of bone marrow smears was difficult as characteristic findings for pernicious anemia were little pronounced. All known reasons for the absence of typical cytomorphologic signs in pernicious anemia as underlying iron deficiency and thalassemia could be excluded.

OBJECTIVE: CD23 is constitutively and atypically expressed on malignant B cells in patients with chronic lymphocytic leukemia (B-CLL). Here, we investigated whether CD23-derived peptides might function as B-CLL-specific tumor-associated antigen (TAA). PATIENTS AND METHODS: Using IFN-gamma-ELISPOT assays and HLA-A2/dimer-peptide staining we identified autologous, CD23-specific HLA-A0201-restricted T cells after 4 weeks of in vitro culture. RESULTS: We were able to expand autologous T cells from 8/11 B-CLL patients by using native and CD40L-activated B-CLL cells as antigen-presenting cells (APCs) in 5 cases whereas for 3 samples an autologous T cell response could only be evoked by use of CD40L-stimulated B-CLL cells as APCs. The number of CD8(+) T cells could be expanded during 4 weeks of in vitro culture with native or CD40L-activated B-CLL cells. We could demonstrate that the expanded T cells were also able to secrete IFN-gamma upon recognition of the antigen using IFN-gamma-ELISPOT assays. Furthermore, these T cells not only recognized HLA-A0201-binding CD23-derived peptides presented by T2 cells, but also CD23-overexpressing autologous B-CLL cells in an MHC-I-restricted manner. CONCLUSION: In sum, CD23-derived peptides were shown to be naturally processed and presented as TAA in primary B-CLL, enabling the expansion of autologous tumor-specific T cells.

OBJECTIVE: CD229, a cell-surface molecule being involved in cell adhesion, is overexpressed in B-CLL cells. In this study we wanted to explore whether CD229 might function as B-CLL-specific tumor-associated antigen (TAA). PATIENTS AND METHODS: Autologous, CD229-specific HLA-A2-restricted T cells were identified using IFN-gamma-ELISPOT assays and HLA-A2/dimer-peptide staining after 4 weeks of in vitro culture. RESULTS: We were able to expand autologous T cells from 9/11 B-CLL patients using native B-CLL cells as antigen presenting cells (APCs) in 5 cases, whereas for 4 samples an autologous T-cell response could only be evoked by use of CD40L-stimulated B-CLL cells as APCs. The number of CD8(+) T cells could be expanded during 4 weeks of in vitro culture with native or CD40L-activated B-CLL cells while the amount of specific T cells recognizing CD229 peptides bound to HLA-A2 dimers increased on average 12-fold (native CLL) and 13-fold (CD40L-activated CLL), respectively. Using IFN-gamma-ELISPOT assays we could demonstrate that the expanded T cells were able to secrete IFN-gamma upon recognition of the antigen. These T cells not only recognized HLA-A0201-binding CD229-derived peptides presented by T2 cells, but also CD229-overexpressing autologous B-CLL cells in an MHC-I-restricted manner. CONCLUSION: In summary, CD229 was shown to be naturally processed and presented as TAA in primary B-CLL cells, enabling the expansion of autologous tumor-specific T cells.