scholarly journals B Cell Anergy Modulated by TLR1/2 and the miR-17∼92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4

2016 ◽  
Vol 196 (10) ◽  
pp. 4410-4417 ◽  
Author(s):  
Stavroula Ntoufa ◽  
Nikos Papakonstantinou ◽  
Benedetta Apollonio ◽  
Maria Gounari ◽  
Chrysi Galigalidou ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Clinical Course ◽  
Lymphocytic Leukemia ◽  
Cell Anergy ◽  
B Cell Anergy

Targeting B-cell anergy in chronic lymphocytic leukemia

Blood ◽  
2013 ◽  
Vol 121 (19) ◽  
pp. 3879-3888 ◽  
Author(s):  
Benedetta Apollonio ◽  
Cristina Scielzo ◽  
Maria Teresa Sabrina Bertilaccio ◽  
Elisa ten Hacken ◽  
Lydia Scarfò ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Key Points ◽  
Cell Anergy ◽  
B Cell Anergy ◽  
Sizable Fraction

Key Points A sizable fraction of CLL patients is characterized by the expansion of clonal B cells with anergic features. The constitutive biochemical signature of B-cell anergy can be efficiently targeted in CLL for therapeutic purposes.

Targeting B Cell Anergy in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3863-3863
Author(s):  
Benedetta Apollonio ◽  
Cristina Scielzo ◽  
Maria Teresa Sabrina Bertilaccio ◽  
Elisa ten Hacken ◽  
Lydia Scarfò ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Nuclear Translocation ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Cell Anergy ◽  
B Cell Anergy ◽  
Constitutive Phosphorylation

Abstract Abstract 3863 B-Cell Receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of Chronic Lymphocytic Leukemia (CLL) clones. Previous studies have suggested that in a sizable fraction of CLL patients the B cell clone is functionally anergic and characterized by constitutive phosphorylation of ERK1/2 kinases. We have analyzed all the described anergy-related features in CLL cells and we observed that constitutive ERK1/2 phosphorylation (used as surrogate marker for anergy) together with NF-ATc1 nuclear translocation correlates with the in vitro inability to mobilize intracellular calcium upon anti-IgM stimulation and with reduced expression of surface IgM. The latter features are reminiscent of B cells where chronic Ag-stimulation favours continuous recycling of B cell receptor. Of interest the cells of the pERK(+) subset of patients have higher expression levels of CD22 and CD5, two receptors that are required for the negative regulation of BCR signalling. The activation of the biochemical program that culminates in the induction of B-cell anergy is transient and requires chronic BCR triggering. We showed that Ag removal, obtained by culturing CLL cells in vitro, causes the loss of the anergic signature (reduction of pERK and nuclear NF-ATc1 levels) and, together with the re-expression of surface IgM, restores the ability of CLL cells to fully respond to BCR triggering. These results led us to explore the possibility to interfere with the molecular features of anergy in a therapeutic perspective, using two classes of MAPK inhibitors (targeting MEK1/2 or ERK1/2) and one specific peptide (VIVIT) that blocks NF-AT nuclear translocation. Constitutive ERK1/2 activation was efficiently inhibited after 1 hour treatment with two MEK inhibitors (U0126 and CI1040), and 48 hours treatment with both compounds induced selective apoptosis in the restricted group of pERK(+) patients. In details, pERK(+) samples (n=21) treated with 10μM of U0126 showed a mean survival of 36.58 ± 5.242 (compared to 60.63 ± 6.557 survival of the pERK(-) subset, p=0,01), while pERK(+) samples treated with 10μM of CI1040 (n=17) experienced a mean survival of 36.59 ± 4.145 (compared to 59.17 ± 6.313 survival of the pERK(-) subset; p=0,01). The same results were obtained when we directly inhibited ERK1/2 activity with a new generation compound. By screening 16 samples treated with 10μM of ERK inhibitor, we observed that pERK(+) patients are very sensitive to treatment (29.38 ± 8.064 mean survival) compared to pERK(-) samples (68.12 ± 8.81; p=0,03). Taken together, these data suggest that MEK1/2 can be efficiently targeted in CLL for therapeutic purpose and that the phosphorylation status of ERK1/2 represents a good biomarker to predict and monitor treatment response. In parallel, inhibition of NF-ATc1 activation with a cell permeable version of VIVIT peptide, reduced cell viability in the group of NF-AT(+) patients (29.47 ± 7.509 mean survival in the NF-AT(+) group and 53.63 ± 6.044 in the NF-AT(-) subset, p=0,02), As expected, the killing activity also correlated with ERK activation (21.30 ± 4.191 mean survival for the pERK(+) group and 42.95 ± 5.465 mean survival for the pERK(-) subset; p=0,01). The apoptotic effect observed in vitro after the use of ERK1/2 and NF-AT inhibitors is preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation (together with the reduction of surface levels of CD22), and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program is responsible for the survival of leukemic lymphocytes. Combination studies with different inhibitors revealed that the concomitant inhibition of MAPK and NF-AT signalling did not enhance apoptosis, thus confirming that both pathways are directly interconnected. A possible link between ERK and NF-AT is the activation of the SHIP1/CD5 axis whose phosphorylation is lost after inhibitors treatment and re-acquired upon BCR triggering. In conclusion, our results demonstrate that one subset of CLL patients is characterized by the abnormal expansion of B cells with anergic features. In these cells the constitutive phosphorylation of ERK and NF-ATc1 activation can be efficiently targeted for therapeutic purpose, thus opening new clinical perspectives. Disclosures: No relevant conflicts of interest to declare.

CD40-Activated B-Cell Chronic Lymphocytic Leukemia Cells for Tumor Immunotherapy: Stimulation of Allogeneic Versus Autologous T Cells Generates Different Types of Effector Cells

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 1992-2002 ◽  
Author(s):  
Raymund Buhmann ◽  
Annette Nolte ◽  
Doreen Westhaus ◽  
Bertold Emmerich ◽  
Michael Hallek
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Tumor Antigens ◽  
Lymphocytic Leukemia ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Stimulation Of

Although spontaneous remissions may rarely occur in B-cell chronic lymphocytic leukemia (B-CLL), T cells do generally not develop a clinically significant response against B-CLL cells. Because this T-cell anergy against B-CLL cells may be caused by the inability of B-CLL cells to present tumor-antigens efficiently, we examined the possibility of upregulating critical costimulatory (B7-1 and B7-2) and adhesion molecules (ICAM-1 and LFA-3) on B-CLL cells to improve antigen presentation. The stimulation of B-CLL cells via CD40 by culture on CD40L expressing feeder cells induced a strong upregulation of costimulatory and adhesion molecules and turned the B-CLL cells into efficient antigen-presenting cells (APCs). CD40-activated B-CLL (CD40-CLL) cells stimulated the proliferation of both CD4+ and CD8+ T cells. Interestingly, stimulation of allogeneic versus autologous T cells resulted in the expansion of different effector populations. Allogeneic CD40-CLL cells allowed for the expansion of specific CD8+cytolytic T cells (CTL). In marked contrast, autologous CD40-CLL cells did not induce a relevant CTL response, but rather stimulated a CD4+, Th1-like T-cell population that expressed high levels of CD40L and released interferon-γ in response to stimulation by CD40-CLL cells. Together, these results support the view that CD40 activation of B-CLL cells might reverse T-cell anergy against the neoplastic cell clone, although the character of the immune response depends on the major histocompatibility complex (MHC) background on which the CLL or tumor antigens are presented. These findings may have important implications for the design of cellular immunotherapies for B-CLL.

B-Cell Receptor Recognition of Multiple Epitopes Correlates with An Aggressive Clinical Course of Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 56-56
Author(s):  
Mascha Binder ◽  
Antje Jackst ◽  
Barbara Léchenne ◽  
Fabian Mueller ◽  
Hendrik Veelken ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Clinical Course ◽  
Lymphocytic Leukemia ◽  
Large Set ◽  
Epitope Recognition ◽  
Fab Fragments ◽  
Recognition Pattern ◽  
Aggressive Clinical Course ◽  
Mimicking Peptides

Abstract Abstract 56 INTRODUCTION: The malignant B-cells in chronic lymphocytic leukemia (CLL) express membrane immunoglobulins (B-cell receptors; BCR) which are specific for the leukemic clone of each individual patient. Emerging evidence suggests that the development and course of CLL may be driven by antigenic stimulation through the BCR. Here we set up a model system of epitope recognition in CLL to explore how diverse epitope recognition in CLL is and whether the epitope recognition pattern has clinical relevance. METHODS: BCRs from six randomly chosen CLL patients were cloned and recombinantly expressed as IgG1 Fab fragments. Combinatorial phage-displayed peptide libraries with five different insert designs were constructed and used for the selection of epitope-mimicking peptides on the Fab fragments. We tested the binding of phage displayed epitope mimics to the respective Fab fragment by ELISA as well as to the native BCR on the cells of CLL patients. Therefore, cell-bound phage displayed epitope mimics were separated from unbound phage by differential centrifugation and bound phage were quantified by bacterial infection. The binding of six ‘index‘ epitope mimics representative for each BCR was evaluated in a set of 100 unrelated CLL cell samples. Epitope recognition patterns of CLL BCRs were correlated with the clinical course of the disease by standard biostatistical analysis including Kaplan-Meier estimator, log-rank test, cox regression analysis and Chi-square test. RESULTS: We selected epitope-mimicking peptides from phage display libraries on six CLL BCRs from randomly chosen patients. The selected peptides bound to the recombinant BCRs as well as to the native BCRs on the respective CLL cells. To model epitope recognition in a larger cohort of CLL patients we chose six representative index epitope mimics and evaluated their binding in a large set of 100 unrelated CLL cases. Surprisingly, all CLL samples recognized one or several index epitopes. Some of the CLL samples showed marked polyreactivity whereas other samples were mono- or oligoreactive. We determined whether the degree of BCR polyreactivity correlates with the clinical course of the disease using time to first treatment (TTFT) as surrogate marker of disease progression. We found that CLL patients expressing BCRs reactive with each of the epitope mimics had a significantly worse clinical course than less reactive control patients (median TTFT 27 months versus 87 months). Moreover, CLL patients whose cells express BCRs reactive with five or more epitope mimics were also characterized by an aggressive clinical course as compared to patients reacting with less than five epitopes (median TTFT 24 months versus 97 months). These outcomes were unrelated to known prognostic markers such as BCR mutational status and high risk receptor configurations. CONCLUSIONS: We introduce a system for modelling and monitoring of BCR epitope reactivity in CLL. Our findings indicate that a polyreactive epitope recognition pattern may be a determinant of an aggressive clinical course in this disease. These findings further emphasize the functional and prognostic relevance of BCR epitope recognition patterns in CLL. Disclosures: No relevant conflicts of interest to declare.

Uncoupling of BCR Internalization and Signaling in Treatment Naive and Btki-Treated Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 191-191
Author(s):  
Eve M Coulter ◽  
Andrea Buggins ◽  
Silvia Mele ◽  
Najeem Folarin ◽  
William Townsend ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Intracellular Signaling ◽  
Statistical Significance ◽  
High Surface ◽  
Lymphocytic Leukemia ◽  
Receptor Complexes ◽  
Bcr Signaling ◽  
Cell Anergy ◽  
B Cell Anergy

Abstract Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR. In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi). BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p<0.0001, n=40) although there was considerable inter-patient variation. We observed a significant correlation between pHrodo-αIgM uptake and high surface IgM (sIgM) expression (r2=0.529, p=0.0001, n=40), and an association with markers of adverse prognosis including, CD38 positivity (p=0.001, n=40), and the capacity to mobilize calcium ions following BCR ligation (p=0.001, n=26). Cases of CLL with unmutated IGHV genes showed higher uptake of pHrodo-αIgM than mutated cases however this did not reach statistical significance (p=0.053, n=40). Similarly, when BCR function within individual CLL patients was examined, we also found that pHrodo-αIgM uptake varied substantially and was maximal in lymph node (LN) derived CLL cells (p=0.03, n=6) and those in the peripheral blood (PB) that express the highest levels of CD5 (p=0.0001, n=26), a marker that is upregulated following BCR activation. In addition, we found that LN CLL cells expressed higher levels of sIgM than those derived from the PB (p=0.03, n=6). This was a surprising finding, as BCR stimulation is thought to occur within LNs, which might be expected to result in down regulated BCR expression. When the level of BCR internalization and accumulation in the endosomes was adjusted for the number of sIgM molecules, we found that BCR internalization and retention was actually more efficient in anergic cases of CLL (defined by lack of ability to mobilize Ca2+ following stimulation with anti-IgM; p=0.0002, n=26). This observation was supported by direct measurement of the rate of BCR endocytosis, which showed a more rapid internalization in anergic B-cells compared to signaling competent and normal B-cells. Similar findings have previously been reported in a murine model of B-cell anergy. Finally, data from BTKi treated CLL patients showed that, 12 months after commencing treatment, CLL B-cells exhibit lower levels of sIgM expression (p=0.02, n=7) and have more efficient BCR internalization than at the outset (p=0.05, n=7). Since low sIgM expression and more efficient BCR internalization is a feature of B-cell anergy, these results suggest that therapy with BTK inhibitors selectively depletes B-cells that are capable of signaling through the BCR and enriches for those displaying features of anergy. Overall our data show that BCR internalization is uncoupled from intracellular signaling in CLL and is most efficient in cells that demonstrate poor downstream BCR signaling or show features of recent BCR stimulation. These observations are similar to those previously reported in anergic B-cells and provide further evidence for ongoing BCR activation and anergy in CLL. Disclosures Patten: Gilead: Research Funding. Devereux:Gilead: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Other: Travel, Accommodations, Expenses ; GSK: Consultancy.

scholarly journals B-cell receptor epitope recognition correlates with the clinical course of chronic lymphocytic leukemia

Cancer ◽  
2010 ◽  
Vol 117 (9) ◽  
pp. 1891-1900 ◽  
Author(s):  
Mascha Binder ◽  
Fabian Müller ◽  
Antje Jackst ◽  
Barbara Léchenne ◽  
Milena Pantic ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Clinical Course ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Epitope Recognition

A Molecular Signature of Anergy Detected in a Subset of CLL Patients.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 742-742
Author(s):  
Paolo Ghia ◽  
Marta Muzio ◽  
Benedetta Apollonio ◽  
Cristina Scielzo ◽  
Michela Frenquelli ◽  
...  
Keyword(s):  
B Cell ◽  
Clinical Course ◽  
Molecular Signature ◽  
Leukemic Cells ◽  
Quiescent State ◽  
Akt Activation ◽  
Cell Anergy ◽  
B Cell Anergy ◽  
Functional Unresponsiveness

Abstract It has been long assumed that BCR signaling plays a relevant role in the natural history of Chronic Lymphocytic Leukemia (CLL), by regulating various and possibly opposing mechanisms. A proportion of CLL cases carry a surface Immunoglobulin (sIg) which is responsive to crosslinking, resulting in effective activation of leukemic cells in vitro. In clear contrast, the remaining cases show a typical functional unresponsiveness through the Immunoglobulin, as indicated by a lack of tyrosine phosphorylation following BCR cross-linking. This feature together with the characteristic decreased expression of the BCR, have led to the time-honored hypothesis that these cells might have been anergized in vivo upon their encounter with a (self-) antigen. Of note, this functional feature tends to correlate with an indolent clinical course of the disease, which may derive from the quiescent state of the functionally silenced leukemic lymphocytes. Despite extensive studies, the concept of B cell anergy in the human immune system and in particular of the functional unresponsiveness in CLL lacks a molecular and biochemical definition. These observations prompted us to examine the expression and activation of key molecules involved in the signaling pathways originating from the BCR in 37 CLL patients and to investigate potential association of the distinct signaling patterns with clinical prognostic features. Our studies showed that in those CLL patients characterized by cellular unresponsiveness to sIg ligation, the MAP kinases ERK1/2 were constitutively phosphorylated and remained unchanged after stimulation with anti-IgM. These cases also displayed constitutive phosphorylation of MEK1/2 and Raf-1 and increased NF-AT transactivation; interestingly, these biochemical features were associated with a lack of AKT activation. In contrast, PMA that bypasses BCR proximal events was able to induce phosphorylation of ERK of these CLL cases indicating that the MAPK signaling machinery is intact but is selectively silenced and unresponsive to BCR-mediated signals. Notably, CLL cases with a constitutive ERK1/2 activation showed a more favorable clinical presentation as compared to the ERK1/2 negative cases. Constitutive activation of MAPK, along with NF-AT transactivation and lack of AKT activation has been previously described as a hallmark of anergic B lymphocytes in the murine system. Taking together all the above, we propose that the “molecularly anergic” subset of CLL described in our work may represent a human cellular model of anergic human B cells aberrantly expanded as part of the malignant process. These molecular features of constitutive BCR-mediated activation may provide evidence for BCR engagement by a putative anergizing antigen occurring in vivo. In addition, these data raise the intriguing hypothesis that this molecular signature of in vivo anergization may also be responsible for the quiescent state of the leukemic cells resulting in the indolent clinical course. In conclusion, our results may provide a molecular basis for the concept of human B-cell anergy and for the distinct functional and clinical behavior observed in a subset of CLL patients.

B-Cell Anergy Underlies Indolent Clinical Behavior Of CLL Stereotyped Subset #4

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4115-4115
Author(s):  
Stavroula Ntoufa ◽  
Nikos Papakonstantinou ◽  
Benedetta Apollonio ◽  
Maria Gounari ◽  
Achilles Anagnostopoulos ◽  
...  
Keyword(s):  
B Cell ◽  
Biochemical Characterization ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Immune Signaling ◽  
Erk Phosphorylation ◽  
Cell Anergy ◽  
B Cell Anergy

Abstract Mounting evidence suggests that specific modalities of B-cell receptor (BcR) and Toll-like receptor (TLR) collaboration and/or regulation may exist in chronic lymphocytic leukemia (CLL), eventually impacting on the behavior of the malignant clones. CLL patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BcRs, SS4), the largest subset within mutated CLL, are characterized clinically by early age at diagnosis and indolent disease course and genetically by few lesions, with deletion of chromosome 13q representing the exclusive recurrent aberration. Examination of the IG sequences of SS4 clonotypic BcRs allows for parallels to be drawn with pathogenic anti-DNA antibodies since both carry long positively charged VH CDR3s. However, SS4 IGs also display distinctive somatic hypermutation (SHM) patterns, the most notable concerning the obligatory introduction of negatively charged residues in either the heavy or the light chains or both, alluding to editing of an autoreactive progenitor. Moreover, SS4 BcRs continue to acquire SHMs within their IG genes overtime, implying antigen selection in the clonal evolution of CLL SS4, albeit without a negative clinical impact. Altogether, these findings suggest that CLL SS4 cells may exist in an anergic state showing attenuated responses to selecting (auto)antigenic elements. To test this hypothesis, after BcR engagement in vitro we analysed basal phosphorylated ERK (pERK) levels and calcium mobilization, both known to be features of B-cell anergic state. Nine of 10 SS4 cases expressed high pERK levels and were unable to increase intracellular Ca2+ upon BcR crosslinking; moreover, stimulation through the BcR had no effect on pERK levels. Primary cell culture for 50min caused a statistically significant reduction of pERK, while culture for 6-24 hours restored the ability of the cells to respond to BcR activation by inducing ERK phosphorylation. Furthermore, analysis of serial samples of 4 SS4 cases over a period spanning 5-7 years revealed consistently positive pERK expression. On these grounds, we conclude that SS4 CLL clones are anergic through the BcR possibly because of chronic (auto)antigen activation. To determine if this profile is SS4-biased or linked to the usage of the IGHV4-34 gene, we also performed similar experiments in 6 cases using this gene in mutated non-stereotyped rearrangements of the MD isotype and found a more heterogeneous pattern: 3/4 cases were pERK negative, while 3/6 cases showed Ca2+ fluxes after BcR crosslinking. Given that SS4 cells are responsive to TLR1/2 ligands, we next investigated if TLR signals can modulate BcR anergy. To this end, SS4 CLL cells pre-stimulated through TLR1/2 with Pam3CSK4 for 50min were then subjected to BcR crosslinking for 10min. TLR1/2 stimulation up-regulated pERK levels, confirming our previous finding that CLL SS4 cells are responsive to TLR1/2 signalling. Notably, BcR crosslinking in TLR1/2-pretreated cells induced even higher pERK levels, indicating that TLR1/2 triggering restores BcR functionality, likely breaching the anergic state. We have previously demonstrated that TLR1/2 stimulation induces up-regulation of the miR-17∼92 cluster eventually leading to down-regulation of critical BcR and/or TLR signaling molecules (i.e. MAP3K1, MAP2K3, MAPK8, CASP8, IKBKB) which are potential targets of these miRNAs. In order to experimentally validate the predicted mRNA-miRNA interactions, we performed 3’ UTR luciferase reporter assay experiments in HeLa cells and documented 5 novel interactions between miR-17∼92 cluster members and MAP3K1, MAP2K3 and MAPK8. To ensure that these interactions are valid also in CLL, we performed transfection experiments with miRNA mimics in negatively selected CLL B cells and obtained identical results. In conclusion, we demonstrate that SS4 CLL cells are anergic through the BcR and that stimulation through TLR1/2 may break B-cell anergy. Moreover, we propose a regulatory mechanism whereby TLR1/2 signals induce miR-17∼92 cluster up-regulation, leading to down-regulation of MAP3K1, MAP2K3 and MAPK8 and eventual modulation of ERK phosphorylation, a key to the anergic state. On a broader scale, the detailed molecular and biochemical characterization of immune signaling in SS4, a prototype for clinically indolent, good prognosis CLL, is also relevant for the advancement of therapeutic strategies incorporating immune signaling inhibitors. Disclosures: Stamatopoulos: Roche: Research Funding.

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