B-cell Apoptosis Research Articles

Targeted Axl Inhibition Primes Chronic Lymphocytic Leukemia B Cells to Apoptosis and Shows Synergistic/Additive Effects in Combination with BTK Inhibitors.

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL. Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination. CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903-induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis. Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL.

Chronic lymphocytic leukemia: 2015 Update on diagnosis, risk stratification, and treatment.

Chronic lymphocytic leukemia (CLL) is the commonest leukemia in western countries. The disease typically occurs in elderly patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that impair apoptosis of clonal B-cells. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen as well as B-cell markers. Two prognostic staging systems exist, the Rai and Binet staging systems, which are established by physical examination and blood counts. Various biological and genetic markers also have prognostic value. Deletions of the short arm of chromosome 17 (del(17p)) predict resistance to available chemotherapies. Comprehensive prognostic scores are currently being developed. Patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. For physical fit patients, chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab remains the current standard therapy. For unfit patients, treatment with an anti-CD20 antibody (obinutuzumab or rituximab or ofatumumab) plus a milder chemotherapy (Chlorambucil) may be applied. At relapse, the initial treatment may be repeated, if the treatment-free interval exceeds two to three years. If the disease relapses earlier, therapy should be changed using alternative agents such as bendamustine (plus rituximab), alemtuzumab, lenalidomide, ofatumumab, ibrutinib, or idelalisib. Patients with a del(17p) or TP53 mutation can be treated with ibrutinib or a combination of idelalisib and rituximab. An allogeneic SCT may be considered in relapsing patients with TP53 mutations or del(17p) or patients that are refractory to repeated chemoimmunotherapies. Future challenges: Several new agents (e.g., ibrutinib, idelalisib, obinutuzumab) hold the potential to improve the outcome of patients with CLL. However, their optimal use (in terms of combination, sequence, and duration) is unknown. Therefore, CLL patients should be treated in clinical trials whenever possible.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the ‘Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim−/−Bmf−/− mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

A Two-Step Pretargeted Nanotherapy for CD20 Crosslinking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab.

The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

Axl Regulates Fibroblast Growth Factor Receptor Signaling in B-Cell Chronic Lymphocytic Leukemia: Dual Targeting in CLL Therapy

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology.We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells.Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells.To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition.In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. DisclosuresWarner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity’s Board of Directors or advisory committees; Gilead: Membership on an entity’s Board of Directors or advisory committees.

Targeted Inhibition of Axl Primes Chronic Lymphocytic Leukemia B-Cells for Apoptosis: Synergistic/Additive Effects in Combination with Bruton Tyrosine Kinase Inhibitors

Introduction: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite that still requires options for treatment despite recent advances with BTK therapy. We previously found that Axl receptor tyrosine kinase (RTK), a member of the TAM (Tyro3, Axl, MER) family of RTKs, plays a critical role in CLL B-cell survival (Blood 2011, 117:1928). Here, we explored the levels of apoptosis induction of CLL B-cells using a novel high-affinity Axl inhibitor as a single agent or in combination with BTK inhibitors.Methods: Expression/activation status of the Tyro3, Axl and MER in CLL B-cells was evaluated by Immunoprecipitation/Western blots. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 (Tolero Pharmaceuticals) or FGFR inhibitor TKI-258 (Novartis) with or without the presence of CLL bone marrow stromal cells (BMSCs). Impact of Axl or FGFR signal inhibition on CLL B-cell survival was also evaluated by flow cytometry after staining with annexin V/propidium iodide. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination in induction of apoptosis. Combination index (CI) of two agents was calculated using the method of Chou and Talalay, 1984.Results: Although we defined pro-survival role of Axl in CLL B-cells, contribution of other members of the TAM family of RTKs including Tyro3 and MER to prolonged survival of the leukemic B-cells remains largely undefined. In this study we found that CLL B-cells overexpress Tyro3, but not MER, and that Tyro3 is constitutively phosphorylated. Interestingly, immunoprecipitation of Tyro3 from CLL B-cell lysates shows retention of highly active Axl in the immune complex suggesting that these two RTKs either heterodimerize or form a molecular complex in CLL B-cells.Our earlier work found that Axl regulates multiple cell survival signaling pathways including PI3K/AKT and Src and that, Axl inhibition using a first generation Axl inhibitor induced apoptosis in CLL B-cells (Blood 2011, 117:1928). Thus, we hypothesized that targeting Axl using a very high affinity Axl inhibitor would induce even higher levels of apoptosis in CLL B-cells compared to the first generation Axl inhibitor. To address this, we utilized a high-affinity orally bioavailable Axl inhibitor TP-0903 to generate preliminary information on its efficiency to specifically target Axl and the level of apoptosis induction in CLL B-cells. Indeed TP-0903 as a single agent induced a robust apoptotic cell death in CLL B-cells within 24 hours of exposure at nanomolar doses, which are easily achievable in vivo (information provided by Tolero Pharmaceuticals). Interestingly, while TP-0903 effectively reduces phosphorylation on Axl in CLL B-cells, it was unable to target P-Tyro3 suggesting that Axl is likely the predominant RTK of the TAM family in CLL B-cells. A marked reduction of the anti-apoptotic proteins Mcl-1, Bcl-2, XIAP and upregulation of the pro-apoptotic protein BIM in CLL B-cells via activation of its upstream regulator FOXO3a were noted as a result of inhibition of P-Axl and its downstream signaling pathways, P-AKT/P-Src. Importantly, TP-0903 was able to overcome CLL BMSC mediated protection of the leukemic B-cells from apoptosis induction. Given the observed robust induction of apoptosis with TP-0903 and the known impact of BTK inhibitors on CLL patient clinical outcome, we then exposed CLL B-cells to TP-0903 in combination with two BTK inhibitors that included Ibrutinib and a reversible BTK inhibitor TP-4216 (a kind gift from Tolero Pharmaceuticals). Based on the CI values, we found that the in vitro combination of TP-0903 and Ibrutinib showed additive effects in CLL B-cells from 6 of 11 CLL patients, CLL B-cells from the remaining 5 CLL patients showed antagonistic effects (Fig. 1A). However, we observed synergistic effects of TP-0903 in combination with TP-4216 in the induction of apoptosis in CLL B-cells from 2 of 10 patients and additive effects in CLL B-cells from 6 patients (Fig. 1B). Cells from the remaining 2 patients exhibited a slightly antagonisticeffect.Conclusion: In total, these results suggest that TP-0903 is quite effective in inducing robust CLL B-cell apoptosis as a single agent even at a nanomolar concentration and its combination with the reversible BTK inhibitor TP-4216 could be an effective and unique strategy for the treatment of CLL patients. [Display omitted] DisclosuresWarner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int’l: Research Funding. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity’s Board of Directors or advisory committees; Gilead: Membership on an entity’s Board of Directors or advisory committees.

Anti-CD20 and B-Cell Receptor-Mediated Growth Inhibition and Apoptosis: A Preclinical Study of Ibrutinib and Rituximab Combination Therapy in Mantle Cell Lymphoma in Vitro and in Vivo

Abstract Intruduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor outcome and therapeutic challenge. Oral single-agent ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, elicited a response rate of 68% in phase II clinical trial and has been approved by FDA for the treatment of MCL patients who received at least one prior therapy. To increase the response rate, the combination of ibrutinib with rituximab would be an ideal regimen because: (1) Both BTK and CD20 are effective therapeutic targets for MCL; (2) CD20-dependent apoptosis occurs associated with B-cell receptor (BCR) activity. Regardless rituximab-mediated immune response (ADCC and CDC) and ibrutinib-mediated ITK inhibition (Th1 polarization), an important potential effect of ibrutinib and rituximab may enhance the direct anti-MCL cytotoxicity of the combination. Methods and Results We investigated the effects of ibrutinib and/or rituximab in 6 established MCL cell lines and 3 freshly isolated primary MCL cells in vitro. First, ibrutinib, as well as BTK-siRNA, inhibited the growth of MCL cells. Then we found that ibrutinib induced the growth inhibition and apoptosis of most MCL cell lines and primary MCL cells in a dose-dependent manner during 5-7 days of culture (IC50 range from 1.5 to 15 µM). On the other hand, rituximab alone induced 10-30% growth inhibition and apoptosis of both established and primary MCL cells in vitro. Importantly, our results showed that ibrutinib and rituximab combination synergistically induced apoptosis of MCL cells during 5-7 days of culture in vitro. Next, our animal study confirmed the compartmental shift phenomenon mediated by ibrutinib which was similar with the data from clinical study. Briefly, SCID mice were subcutaneously implanted with human fetal bone chips (SCID-hu). After 6 weeks of bone implantation, the freshly isolated MCL cells from patients were directly engrafted into the human fetal bone chips. The engrafted MCL cells produced measurable levels of human β2-microglobulin (β2M) in mouse serum. Once human β2M had been detected in mouse serum, the primary MCL-bearing SCID-hu mice were treatment with 25 mg/kg ibrutinib oral gavage daily. A transient increase of human CD5+CD20+ cells in mouse peripheral blood was detected by flow cytometry from day 8 of treatment, representing a shift of human MCL cells from human fetal bone chip to mouse peripheral blood. At this time, 10 mg/kg rituximab was intravenously administrated every 3 days for total 7 doses. Our results demonstrated that rituximab and ibrutinib combination eradicated MCL cells in vivo and kept mice survival longer than either ibrutinib or rituximab alone. Conclusions Based on our preclinical data of rituximab and ibrutinib combination in vitro and in primary MCL-bearing SCID-hu mice, ibrutinib plus rituximab could be an effective regimen in a clinical trial of relapsed or refractory MCL. Disclosures Off Label Use: Ibrutinib plus rituximab may enhance the direct anti-MCL cytotoxicity. Wang:Pharmacyclics: Honoraria, Research Funding.

Contractile activity of human follicular dendritic cells.

Follicular dendritic cells (FDCs) present antigens to B cells in the lymphoid follicle and inhibit B-cell apoptosis. In previous work, we obtained human FDC lines that allowed us to study the antigen phenotype and functions of these cells, finding that they expressed α-smooth muscle (SM) actin (a protein involved in cell contraction) and were able to contract collagen gel matrixes in gel contraction assays. Actin polymerization associated with cell contractility is essential for many cellular functions. We report here that interleukin (IL)-2 and interferon (IFN)-γ increased FDC contractility, and IL-10 reduced contractility, whereas IL-4 had no effect. Tumor necrosis factor (TNF) and lymphotoxin (LT)-α1β2, cytokines involved in FDC differentiation, also increased FDC contractility. In different cell systems, cell contraction is related with the incorporation of α-SM actin into stress fibers. By confocal microscopy, we showed that cytochalasin D, an inhibitor of actin polymerization, inhibited α-SM actin incorporation and relaxed FDCs. Likewise, IL-10 significantly decreased the proportion of FDCs with α-SM actin-positive stress fibers, whereas cytokines that increased FDC contractility also increased this proportion. However, none of the cytokines tested significantly affected α-SM actin expression as determined by flow cytometry. IL-10, in addition to decreasing FDC contractility, increased the inhibitory activity of FDC in spontaneous B-cell apoptosis (P<0.05), but the other cytokines did not affect this activity. We conclude that cytokines related with FDC physiology regulate the contractility of these cells, and IL-10 also regulates the effect of FDC on B-cell apoptosis.

A new hope: novel therapeutic approaches to treatment of chronic lymphocytic leukaemia with defects in TP53.

Chronic lymphocytic leukaemia (CLL) is an indolent B-cell malignancy with heterogeneous outcomes. Chromosomal abnormalities in CLL are predictive of the natural disease course; del(11q) and del(17p) are recognized as high risk genetic lesions. Del(17p) is associated with an impaired function of TP53, a key tumour suppressor, and is particularly problematic. Such patients respond poorly to chemo-immunotherapy and have significantly shorter survival compared to patients with standard and low-risk cytogenetics. While TP53 pathway defects are rare at initial diagnosis, their frequency increases in relapsed CLL. Until very recently, this group of patients represented an unmet clinical need with few therapeutic options. However, the advent of targeted therapies has expanded the drug armamentarium and introduced new hope for these highly refractory patients. Agents that target B-cell receptor signalling, BH3-mimetics and others induce apoptosis of the neoplastic B-cells in a TP53-independent manner. Their use in the clinic is associated with remarkable activity in patients with del(17p). In this review we discuss the frequency and clinical significance of del(17p) and genetic mutations leading to disrupted TP53, the putative role of other TP53 homologues, and the results of key clinical trials involving both conventional chemotherapy and novel agents.

Durable Remission of Pemphigus With a Fixed-Dose Rituximab Protocol

Rituximab induces B-lymphocyte apoptosis by targeting CD20 antigen and has shown efficacy in antibody-mediated autoimmune disease. Rituximab is increasingly being acknowledged as an effective and safe treatment option for pemphigus. To assess the clinical response of patients with pemphigus to rituximab using a modified fixed-dose rheumatoid arthritis protocol (1 g intravenously on days 1 and 15, followed by 500 mg intravenously if clinically warranted at 6-month intervals or repeated full dosing). A retrospective cohort study was conducted using records from a tertiary referral center for autoimmune bullous disorders. Participants included 92 patients (pemphigus vulgaris, 84 [91%], and pemphigus foliaceus, 8 [9%]) who received rituximab treatment between May 1, 2006, and August 30, 2012. The primary outcomes were time to relapse and achievement of a complete response with or without treatment at the end of the study. Median time to relapse after the first treatment cycle was 15 months (95% CI, 10.3-19.7). All patients experienced improvement. Complete remission rates with or without adjuvant treatment at final follow-up were 89% (56 patients [61%] were in complete remission without treatment and 26 patients [28%] were in complete remission during adjuvant treatment). No serious infectious adverse events occurred. The fixed-dose, modified rheumatoid arthritis protocol for rituximab was efficacious and well tolerated in patients with pemphigus. Patients who do not achieve remission after 1 cycle or patients who experience relapse benefit from further cycles of rituximab. Our results need to be confirmed in larger and controlled trials.

Effect of Mycophenolic Acid and Bortezomib on Purified Human B Cells: An In Vitro Study of Long-Term Functionally Stable MICA-Sensitized Renal Recipients

We sought to develop a B-cell in vitro culture system and test B cells isolated from sensitized kidney recipients and healthy controls, and assess the effectiveness of proteasome inhibitors and mycophenolic acid on antibody secretion and cell apoptosis. CD19(+) B cells and CD19(+)CD27(+) memory B-cell subsets were detected from peripheral blood mononuclear cells obtained from 6 MICA-sensitized kidney recipients and 6 healthy controls. Peripheral blood B cells were isolated and cultured with CpG2006, PMA, MICA antigen, B-cell activating factor, CD40 ligand (CD40L), human recombinant IL-2 (rhuIL-2), rhuIL-10, rhuIL-4, and rhuIL-21. After culturing for 7 days, we tested several variables of B-cell activity including differentiation, apoptosis, and IgM production. We also assessed the effects of 2 immunosuppressive drugs (mycophenolic acid and bortezomib) on antibody secretion and cellular apoptosis. Kidney recipients had a lower ratio of CD19+ B cells in peripheral blood mononuclear cells than did healthy controls. However, the percentage of CD19(+)CD27(+) B cells was higher in kidney recipients than in healthy controls. In the cell stimulation culture system, the ratio of CD19(+) B cells, CD19(+)CD27(+) B cells, and CD19(+)CD138(+) B cells increased after culturing for 7 days compared with unstimulated controls. In addition, the percentage of apoptotic B cells decreased, and antibody production increased in sensitized transplant patients and healthy controls. Treatment with bortezomib or mycophenolic acid induced B-cell apoptosis and inhibited secretion of antibodies. This study describes establishment of a B-cell in vitro culture system, showing that B cells may be stimulated to secrete antibodies. The study also provides an assay for studying B cells in vitro. This study provides information suggesting that bortezomib and mycophenolic acid can inhibit B-cell antibody secretion.

Differential Effects Of Triggering Via Toll Like Receptors In Stable Or Aggressive Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood, secondary lymphoid tissues and bone marrow of functionally defective clonal B lymphocytes with prolonged survival in vivo. Despite therapeutic achievements have been accomplished in the management of this disease, however CLL remains incurable partially because of the resistance to apoptosis of CLL B-cells and of the altered immune system of CLL patients. CLL is hetereogenous, but its evolution is mostly slow, probably linked to some level of immune control of the leukemic cells. Indeed, clinical observations have been reported of spontaneous remissions associated with the intense immunological activity following a viral infection. Clinical responses have also been observed after treatment by immunomodulating cytokines and long-term survival is described, without disease, after allogeneic stem cells transplantation. All these data suggest that immunotherapy could be useful in the treatment of CLL, possibly as an adjuvant therapy after classical immunochemotherapy schedules.Toll like receptors (TLR) are proteins of the innate immune system belonging to the family of Pattern Recognition receptors (PRRs). Recognition of their ligands by the TLR present on neutrophils, macrophages, dendritic cells and B-cells are important in the initiation of adaptive immune responses. TLR-7 and 9 are expressed by CLL B-cells (Grandjenette, Hematologica, 2007). Previous studies have shown that stimulation of TLR-9 by CpG ODN (oligodinucleotides) induces an activation of CLL B-cells while triggering TLR-7 increases in vitro apoptosis of these cells. Here we show that these effects of TLR-9 and TLR-7 stimulation differ depending on the clinical form (stable or aggressive) of the disease and on the mutational status of CLL B-cells.In vitro stimulation with three doses of Imiquimod R837 or ODN CpG M362 was carried out for three days on cells from 40 patients (22 stable, 18 aggressive, mutational status known for 35, 18 IgVH mutated, 17 unmutated). Flow cytometry was used to measure apoptosis, proliferation after carboxyfluorescein succinimidyl ester (CFSE) labeling and modulation of surface differentiation antigens. Signaling pathways after incubation were further studied by antibody arrays and western blot.Spontaneous apoptosis occurring in vitro was demonstrated to involve the mitochondrial pathway. CLL B-cells were also confirmed to proliferate strongly and produce large amounts of IL-6 and IL-8 upon triggering by phorbol myrsistate (positive control), this compound almost completely aborting in vitro apoptosis.Cells from patients with a stable disease were significantly more prone to rapid apoptosis after TLR-7 triggering with Imiquimod, compared to cells from patients with an aggressive disease which displayed only spontaneous apoptosis. This rapid apoptosis in stable patients involved the p38 MAP-Kinase pathway. It was concomitant to an important production of IL-8 and IL-6.Conversely, CpG ODN conferred a protection against apoptosis to CLL B-cells from patients with an aggressive disease. This was accompanied by the activation of numerous anti-apoptotic proteins in the cells. CpG ODN also significantly increased CLL B-cells proliferation, concomitantly to the phosphorylation of Erk and Akt proteins. ODN finally increased the expression of CD20 and CD19 on the cells'surface.The same differences in reactivity were observed comparing mutated (∼stable) and unmutated (∼aggressive) cases.These data indicate that CLL B-cells from patients with a stable or aggressive (mutated/unmutated) disease answer differently when triggered through their surface TLRs. This might have an incidence on the behavior of these cells in vivo, in answer to stimulations by microbial compounds naturally binding these structures. These properties could also be used to develop adjuvant immunotherapies by loading CpG ODN-activated CLL B-cells with autologous apoptotic fragments issued from stimulation of part of the same cells with Imiquimod. Disclosures:No relevant conflicts of interest to declare.

Deletion Of The Von Hippel-Lindau Gene Interferes With The Development Of Peripheral B-Cell Subsets

The development of hematopoietic cells occurs in highly specialized microenvironments within bone marrow, thymus and spleen. Hematopoietic stem and progenitor cells are positioned at the lowest end of a bone marrow oxygen gradient, which implies a role for the hypoxic-response pathway in regulating hematopoiesis. In this pathway the von Hippel-Lindau protein (pVhl) is the central negative regulator and continuously mediates the proteasomal destruction of the hypoxia-inducible factor-1α (Hif-1α). Under hypoxic conditions Hif-1α destruction is inhibited and results in the expression of hypoxia-inducible genes.In order to study the role of pVhl in hematopoiesis we crossed Vhl conditional mice with vav-iCre mice to induce a constitutive hematopoiesis-specific Vhl deletion (VhlKOvav). As expected, we observed a dramatic expansion of spleen erythropoiesis, however bone marrow lin-Sca-1+c-kit+CD48-CD150+ hematopoietic stem cell numbers of VhlKOvav mice did not significantly differ from control mice. The most striking observation in VhlKOvav mice was that B-cell numbers in bone marrow and spleen were decreased by 53% and 78%, respectively. In order to exclude that the loss of B-cells in VhlKOvav mice was due to a B-cell extrinsic effect we crossed Vhl conditional mice to the B-cell specific deleter lines CD19-Cre and Mb1-Cre (VhlKOCD19 and VhlKOMb1 mice). Flow cytometric analysis also revealed decreased peripheral B-cell numbers in VhlKOCD19 mice and an even more pronounced B-cell loss in VhlKOMb1 mice (B-cells spleen, mean±SEM; control (n=9), 77±2.4x106; VhlKOMb1 (n=12), 1.3±0.2x106; p<0.001). This demonstrated that pVhl is cell-autonomously required for the normal development of the B-cell compartment. To more accurately define the Vhl-null B-cell developmental defect we analyzed bone marrow B-cell subsets of VhlKOMb1 and control mice. Early B-cell progenitor numbers defined by the surface markers B220, CD43, CD24, BP-1 (Hardy's classification, fractions A-D) of VhlKOMb1 mice were not altered. In contrast, we observed a significant 41% reduction of the immature B220+CD43-IgM+IgD- and a 46% reduction of the mature B220+CD43-IgM+IgD+ bone marrow VhlKOMb1 B-cell numbers compared to controls. In peripheral blood VhlKOMb1 immature and mature B-cell numbers were even more decreased (by 81% and 86%, respectively). We hypothesized that increased CXCR4 expression, which is negatively regulated by pVhl, could be responsible for the decreased bone marrow egress of immature bone marrow B-cells. Indeed, we observed a more than twofold increase of CXCR4 expression of VhlKOMb1 compared to control bone marrow immature B-cells (MFI, mean±SEM; control (n=3), 72±15; VhlKOMb1 (n=3), 162±23; p<0.05). Strikingly, VhlKOMb1 spleens were almost devoid of follicular B220+CD21/35intmCD23+IgD+IgM+ and marginal zone B220+CD21/35highCD23-IgMhigh B-cells and accordingly follicular structures could not be observed in histological sections. In VhlKOMb1 lymph nodes the follicular B-cell numbers were also dramatically decreased. Next, we flow sorted residual splenic VhlKOMb1 B-cells and were able to confirm deletion of the Vhl-gene by PCR. Target genes of the hypoxic-response pathway such as Pgk1, Vegf and Bnip3 were 10- to more than 100-fold higher expressed in sorted VhlKOMb1 compared to control B-cells. As a possible reason for the low VhlKOMb1 peripheral B-cell numbers we identified a more than twofold reduction in CD62L expression by immature blood B-cells (MFI, mean±SEM; control (n=3), 3127±250; VhlKOMb1 (n=3), 1528±66; p<0.05) which presumably impaired their homing ability to peripheral lymphoid organs. Additionally, we detected an increased B-cell apoptosis rate of VhlKOMb1 B-cells in the spleen. Finally, we were able to show that decreased follicular splenic B-cell numbers of VhlKOCD19 mice could be completely rescued by additionally breeding Hif-1α conditional alleles into the system (Hif-1αVhlKOCD19mice).In summary, we identified pVhl as a key regulator of peripheral B-cell maturation. We show that pVhl-mediated negative regulation of the hypoxic-response pathway is required for normal peripheral B-cell differentiation. Our data suggest that B-cell pVHL loss-of-function leads to decreased bone marrow egress and decreased lymphoid organ homing of immature B-cells mediated by the dysregulation of CXCR4 and CD62L. Disclosures:No relevant conflicts of interest to declare.

Chronic treatment with valproate protects INS1 cell from palmitate-induced ER stress and apoptosis by inhibiting GSK3β

Objective Reduction of b-cell mass is increasingly recognized as one of the main contributing factors to the pathogenesis of type 2 diabetes. Chronic free fatty acid (FFA) exposure has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic b-cell apoptosis. In the present study, we first investigated whether anticonvulsant sodium valproate (VPA), at clinically relevant doses, protects pancreatic b-cell from palmitate-induced apoptosis and the mechanism underlying anti-apoptosis.

Akt inhibitor MK2206 selectively targets CLL B‐cell receptor induced cytokines, mobilizes lymphocytes and synergizes with bendamustine to induce CLL apoptosis

<scp>Akt</scp> inhibitor <scp>MK</scp>2206 selectively targets <scp>CLL B</scp>‐cell receptor induced cytokines, mobilizes lymphocytes and synergizes with bendamustine to induce <scp>CLL</scp> apoptosis

EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, including Burkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDS-associated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinase-dead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis.

Chronic lymphocytic leukemia: 2013 update on diagnosis, risk stratification and treatment

Chronic lymphocytic leukemia (CLL) is the commonest leukemia in western countries. The disease typically occurs in elderly patients and has a highly variable clinical course. Leukemic transformation is initiated by specific genomic alterations that impair apoptosis of clonal B-cells. The diagnosis is established by blood counts, blood smears, and immunophenotyping of circulating B-lymphocytes, which identify a clonal B-cell population carrying the CD5 antigen as well as B-cell markers. Two prognostic staging systems exist, the Rai and Binet staging systems, which are established by physical examination and blood counts. Various biological and genetic markers also have prognostic value. Deletions of the short arm of chromosome 17 (del(17p)) predict resistance to most available therapies. Patients with active or symptomatic disease or with advanced Binet or Rai stages require therapy. For physical fit patients, chemoimmunotherapy with fludarabine, cyclophosphamide and rituximab represents the current standard therapy. For unfit patients, treatment with an anti-CD20 antibody plus a milder chemotherapy (chlorambucil) is currently established as standard treatment. At relapse, the initial treatment may be repeated, if the treatment-free interval exceeds two years. If the disease relapses earlier, alternative therapies such as bendamustine alone or with rituximab, alemtuzumab, lenalidomide, or ofatumumab should be used. Patients with a del(17p) or TP53 should be considered for an allogeneic SCT. Several new agents (e.g., ibrutinib, obinutuzumab) hold the potential to change standard of CLL treatment in the next 6-12 months. Therefore, CLL patients should be included into current clinical trials whenever possible.

Toll-like receptor agonists induce apoptosis in mouse B-cell lymphoma cells by altering NF-κB activation

Toll-like receptor 9 (TLR9) recognizes microbial DNA containing unmethylated cytosyl guanosyl (CpG) sequences, induces innate immune responses, and facilitates antigen-specific adaptive immunity. Recent studies report that in addition to stimulating innate immunity, TLR9 ligands induce apoptosis of TLR9 expressing cancer cells. To understand the mechanism of TLR9-induced apoptosis, we compared the effects of CpG containing oligodeoxynucleotides (CpG ODN) on a mouse B-cell lymphoma line, CH27, with those on mouse splenic B cells. CpG ODN inhibited constitutive proliferation and induced apoptosis in the CH27 B-cell lymphoma line. In contrast, CpG ODN-treated primary B cells were stimulated to proliferate and were rescued from spontaneous apoptosis. The induction of apoptosis required the ODNs to contain the CpG motif and the expression of TLR9 in lymphoma B cells. A decrease in Bcl-xl expression and an increase in Fas and Fas ligand expression accompanied lymphoma B-cell apoptosis. Treatment with the Fas ligand-neutralizing antibody inhibited CpG ODN-induced apoptosis. CpG ODN triggered a transient NF-κB activation in the B-cell lymphoma cell line, which constitutively expresses a high level of c-Myc, while CpG ODN induced sustained increases in NF-κB activation and c-Myc expression in primary B cells. Furthermore, an NF-κB inhibitor inhibited the proliferation of the CH27 B-cell lymphoma line. Our data suggest that the differential responses of lymphoma and primary B cells to CpG ODN are the result of differences in NF-κB activation. The impaired NF-κB activation in the CpG ODN-treated B-cell lymphoma cell line alters the balance between NF-κB and c-Myc, which induces Fas/Fas ligand-dependent apoptosis.

Targeting CD22 in B-cell Malignancies: Current Status and Clinical Outlook

CD22 is a B-cell-specific transmembrane glycoprotein found on the surface of most B cells; it modulates B-cell function, survival and apoptosis. CD22 has emerged as an ideal target for monoclonal antibody (mAb)-based therapy of B-cell malignancies including most lymphomas and many leukemias. Epratuzumab, an anti-CD22 mAb, has been developed in various forms, including as an unlabeled (naked) mAb, as a radioimmunotherapeutic, as an antibody drug conjugate (ADC), and as a vehicle for CD22-targeted nanoparticles. While clinical trials with unlabeled epratuzumab have demonstrated modest results, its combination with rituximab in phase II studies has been more encouraging. Based on the potential for CD22 to become internalized, CD22-targeted constructs carrying radioisotopes or toxins have generated promising results. Radioimmunotherapy, utilizing ⁹⁰Y-labeled epratuzumab, was shown to be highly effective in patients with follicular lymphoma, generating a complete response (CR) rate of 92 % and progression-free survival of more than 2 years. ADC therapy is a promising therapeutic approach to B-cell malignancies which includes the direct conjugation of mAbs with cytotoxic agents. Phase II studies of inotuzumab ozogamicin, an ADC which combines anti-CD22 mAb with calicheamicin, an enediyne antibiotic which mediates apoptosis, in patients with acute lymphoblastic leukemia have produced an overall response rate (ORR) of greater than 50 % in treatment-refractory patients. Phase I trials of moxetumomab pasudotox, an ADC which combines anti-CD22 with PE38, a fragment of Pseudomonas exotoxin A, have been completed in hairy cell leukemia with a ORR of 86 %. Finally, a review of CD22-targeted nanoparticles, that include a doxorubicin-containing lipid complex that uses synthetic high-affinity CD22 ligand mimetics as well as anti-CD22 mAb-coated pegylated liposomas doxorubin (PLD), has demonstrated promising results in pre-clinical models of human lymphoma. Moreover, novel anti-CD22 mAb that block CD22 ligand binding as well as second generation ADC that utilize biodegradable linkers and more potent toxins hold great hope for the future of CD22-targeted therapeutics that may translate into better outcomes for patients with CD22-positive malignancies.

Mycobacterium tuberculosis culture filtrate protein of 10 kDa inhibits B cell function (P3120)

Abstract Culture filtrate protein 10 kD (CFP10) and early secreted antigenic target of 6 kD (ESAT-6), are required in pathogenesis of tuberculosis in part by manipulation of host immunity. We reported the immune modulatory effects of ESAT-6, and the effect of CFP10 in this regard remains unexplored. We now demonstrate that recombinant CFP10 binds to peripheral blood B cells and monocytes, but not to T-cells. Stimulation of B cells through the B cell receptor BCR plus TLR9 induced IL-6, IL-8 and IL-10 production, and CFP10 but not rAg85 markedly reduced IL-10 secretion and mRNA expression, without affecting IL-6 and IL-8. CFP10 did not affect LPS-stimulated cytokine production by monocytes. CFP10 reduced BCR plusTLR9 stimulated B cell proliferation and IgG secretion. Using B cells as antigen-presenting cells, we found that CFP10 inhibited expression of CD80 and CD86, but not that of CD69 and MHC-II expression, resulting in reduced M. tuberculosis-specific T cell IFN-γ production. The effects of CFP10 on B cells were not due to changes in B-cell TLR9 expression, viability or apoptosis. However, CFP10 inhibited BCR plus TLR9 stimulated nuclear translocation of NF-κB, activation of AP-1 transcription factors and expression of BLIMP1, the master regulator of B cell maturation and activation. We conclude that CFP10 inhibits B functions by blocking TLR9 and BCR signaling, suggesting potential cell specific immune regulatory functions for CFP10 during tuberculosis infection.