IRF4 Has a Unique Role in Early B Cell Development and Acts Prior to CD21 Expression to Control Marginal Zone B Cell Numbers.

Strict control of B lymphocyte development is required for the ability to mount humoral immune responses to diverse foreign antigens while remaining self-tolerant. In the bone marrow, B lineage cells transit through several developmental stages in which they assemble a functional B cell receptor in a stepwise manner. The immunoglobulin heavy chain gene is rearranged at the pro-B stage. At the large pre-B stage, cells with a functional heavy chain expand in response to signals from IL-7 and the pre-BCR. Cells then cease proliferation at the small pre-B stage and rearrange the immunoglobulin light chain gene. The fully formed BCR is subsequently expressed on the surface of immature B cells and autoreactive cells are culled by central tolerance mechanisms. Once in the periphery, transitional B cells develop into mature B cell subsets such as marginal zone and follicular B cells. These developmental processes are controlled by transcription factor networks, central to which are IRF4 and IRF8. These were thought to act redundantly during B cell development in the bone marrow, with their functions diverging in the periphery where IRF4 limits the number of marginal zone B cells and is required for germinal center responses and plasma cell differentiation. Because of IRF4's unique role in mature B cells, we hypothesized that it may also have functions earlier in B cell development that cannot be compensated for by IRF8. Indeed, we find that IRF4 has a unique role in upregulating the pre-B cell marker CD25, limiting IL-7 responsiveness, and promoting migration to CXCR4 such that IRF4-deficient mice have a partial block at the pre-B cell stage. We also find that IRF4 acts in early transitional B cells to restrict marginal zone B cell development, as deletion of IRF4 in mature B cells with CD21-cre impairs plasma cell differentiation but has no effect on marginal zone B cell numbers. These studies highlight IRF4 as the dominant IRF family member in early B lymphopoiesis.

Sensitization to Drug Treatment in Precursor B-Cell Acute Lymphoblastic Leukemia Is Not Achieved by Stromal NF-κB Inhibition of Cell Adhesion but by Stromal PKC-Dependent Inhibition of ABC Transporters Activity.

Cell adhesion to stromal support and the associated intracellular signaling are central to drug resistance, therefore blocking both has been effective in increasing drug sensitization in leukemia. The stromal Ser/Thr protein kinase C (PKC) has been found to be important for conferring protection to leukemic cells. We aimed at elucidating the intracellular signals connected to cell adhesion and to stromal PKC. We found that NF-κB and Akt were up-regulated in mesenchymal stem cells (MSC) after binding of B-cell acute lymphoblastic leukemia (B-ALL) cells. Nevertheless, Akt inhibition did not induce B-ALL cell detachment. In spite of a clear activation of the NF-κB signaling pathway after B-ALL cell binding (up-regulation NF-κB1/2, and down-regulation of the IKBε and IKBα inhibitors) and an important reduction in cell adhesion after NF-κB inhibition, sensitization to the drug treatment was not observed. This was opposite to the PKC inhibitors Enzastaurin and HKPS, a novel chimeric peptide inhibitor, that were able to increase sensitization to dexamethasone, methotrexate, and vincristine. PLCγ1, Erk1/2, and CREB appear to be related to PKC signaling and PKC effect on drug sensitization since they were contra-regulated by HKPS when compared to dexamethasone-treated cells. Additionally, PKC inhibition by HKPS, but not by Enzastaurin, in MSC reduced the activity of three ABC transporters in leukemic cells treated with dexamethasone, a new indirect mechanism to increase sensitization to drug treatment in B-ALL cells. Our results show the validity of targeting the functional characteristic acquired and modulated during cell-to-cell interactions occurring in the leukemic niche.

Treatment-Induced BAFF Expression and B Cell Biology in Multiple Sclerosis.

Although fingolimod and interferon-ß are two mechanistically different multiple sclerosis (MS) treatments, they both induce B cell activating factor (BAFF) and shift the B cell pool towards a regulatory phenotype. However, whether there is a shared mechanism between both treatments in how they influence the B cell compartment remains elusive. In this study, we collected a cross-sectional study population of 112 MS patients (41 untreated, 42 interferon-ß, 29 fingolimod) and determined B cell subsets, cell-surface and RNA expression of BAFF-receptor (BAFF-R) and transmembrane activator and cyclophilin ligand interactor (TACI) as well as plasma and/or RNA levels of BAFF, BAFF splice forms and interleukin-10 (IL-10) and -35 (IL-35). We added an in vitro B cell culture with four stimulus conditions (Medium, CpG, BAFF and CpG+BAFF) for untreated and interferon-ß treated patients including measurement of intracellular IL-10 levels. Our flow experiments showed that interferon-ß and fingolimod induced BAFF protein and mRNA expression (P ≤ 3.15 x 10-4) without disproportional change in the antagonizing splice form. Protein BAFF correlated with an increase in transitional B cells (P = 5.70 x 10-6), decrease in switched B cells (P = 3.29 x 10-4), and reduction in B cell-surface BAFF-R expression (P = 2.70 x 10-10), both on TACI-positive and -negative cells. TACI and BAFF-R RNA levels remained unaltered. RNA, plasma and in vitro experiments demonstrated that BAFF was not associated with increased IL-10 and IL-35 levels. In conclusion, treatment-induced BAFF correlates with a shift towards transitional B cells which are enriched for cells with an immunoregulatory function. However, BAFF does not directly influence the expression of the immunoregulatory cytokines IL-10 and IL-35. Furthermore, the post-translational mechanism of BAFF-induced BAFF-R cell surface loss was TACI-independent. These observations put the failure of pharmaceutical anti-BAFF strategies in perspective and provide insights for targeted B cell therapies.

Dermatan Sulfate Is a Potential Regulator of IgH via Interactions With Pre-BCR, GTF2I, and BiP ER Complex in Pre-B Lymphoblasts.

Dermatan sulfate (DS) and autoantigen (autoAg) complexes are capable of stimulating autoreactive CD5+ B1 cells. We examined the activity of DS on CD5+ pre-B lymphoblast NFS-25 cells. CD19, CD5, CD72, PI3K, and Fas possess varying degrees of DS affinity. The three pre-BCR components, Ig heavy chain mu (IgH), VpreB, and lambda 5, display differential DS affinities, with IgH having the strongest affinity. DS attaches to NFS-25 cells, gradually accumulates in the ER, and eventually localizes to the nucleus. DS and IgH co-localize on the cell surface and in the ER. DS associates strongly with 17 ER proteins (e.g., BiP/Grp78, Grp94, Hsp90ab1, Ganab, Vcp, Canx, Kpnb1, Prkcsh, Pdia3), which points to an IgH-associated multiprotein complex in the ER. In addition, DS interacts with nuclear proteins (Ncl, Xrcc6, Prmt5, Eftud2, Supt16h) and Lck. We also discovered that DS binds GTF2I, a required gene transcription factor at the IgH locus. These findings support DS as a potential regulator of IgH in pre-B cells at protein and gene levels. We propose a (DS•autoAg)-autoBCR dual signal model in which an autoBCR is engaged by both autoAg and DS, and, once internalized, DS recruits a cascade of molecules that may help avert apoptosis and steer autoreactive B cell fate. Through its affinity with autoAgs and its control of IgH, DS emerges as a potential key player in the development of autoreactive B cells and autoimmunity.

Precursor B-ALL Cell Lines Differentially Respond to SYK Inhibition by Entospletinib.

BACKGROUND: Impaired B-cell receptor (BCR) function has been associated with the progress of several B-cell malignancies. The spleen tyrosine kinase (SYK) represents a potential therapeutic target in a subset of B-cell neoplasias. In precursor B-acute lymphoblastic leukemia (B-ALL), the pathogenic role and therapeutic potential of SYK is still controversially discussed. We evaluate the application of the SYK inhibitor entospletinib (Ento) in pre- and pro-B-ALL cell lines, characterizing the biologic and molecular effects. METHODS: SYK expression was characterized in pre-B-ALL (NALM-6) and pro-B-ALL cell lines (SEM and RS4;11). The cell lines were exposed to different Ento concentrations and the cell biological response analyzed by proliferation, metabolic activity, apoptosis induction, cell-cycle distribution and morphology. BCR pathway gene expression and protein modulations were further characterized. RESULTS: Ento significantly induced anti-proliferative and pro-apoptotic effects in NALM-6 and SEM, while barely affecting RS4;11. Targeted RNAseq revealed pronounced gene expression modulation only in NALM-6, while Western Blot analyses demonstrated that vital downstream effector proteins, such as pAKT, pERK, pGSK3ß, p53 and BCL-6, were affected by Ento exposure in the inhibitor-sensitive cell lines. CONCLUSION: Different acting modes of Ento, independent of pre-BCR dependency, were characterized, unexpected in SEM. Accordingly, SYK classifies as a potential target structure in a subset of pro-B-ALLs.

Value of flow cytometry for MRD-based relapse prediction in B-cell precursor ALL in a multicenter setting.

PCR of TCR/Ig gene rearrangements is considered the method of choice for minimal residual disease (MRD) quantification in BCP-ALL, but flow cytometry analysis of leukemia-associated immunophenotypes (FCM-MRD) is faster and biologically more informative. FCM-MRD performed in 18 laboratories across seven countries was used for risk stratification of 1487 patients with BCP-ALL enrolled in the NOPHO ALL2008 protocol. When no informative FCM-marker was available, risk stratification was based on real-time quantitative PCR. An informative FCM-marker was found in 96.2% and only two patients (0.14%) had non-informative FCM and non-informative PCR-markers. The overall 5-year event-free survival was 86.1% with a cumulative incidence of relapse (CIR5y) of 9.5%. FCM-MRD levels on days 15 (HzR 4.0, p < 0.0001), 29 (HzR 2.7, p < 0.0001), and 79 (HzR 3.5, p < 0.0001) associated with hazard of relapse adjusted for age, cytogenetics, and WBC. The early (day 15) response associated with CIR5y adjusted for day 29 FCM-MRD, with higher levels in adults (median 2.4 × 10-2 versus 5.2 × 10-3, p < 0.0001). Undetectable FCM- and/or PCR-MRD on day 29 identified patients with a very good outcome (CIR5y = 3.2%). For patients who did not undergo transplantation, day 79 FCM-MRD > 10-4 associated with a CIR5y = 22.1%. In conclusion, FCM-MRD performed in a multicenter setting is a clinically useful method for MRD-based treatment stratification in BCP-ALL.

Reversal of autoimmunity by mixed chimerism enables reactivation of ß cells and transdifferentiation of α cells in diabetic NOD mice.

Type 1 diabetes (T1D) results from the autoimmune destruction of ß cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of ß cells. It is known that ß cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of ß cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments ß cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing ß cells, Sox9+ ductal progenitors, Nestin+ mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulinlo) ß cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of ß cells and transdifferentiation of α cells can provide sufficient new functional ß cells to reach euglycemia in firmly established T1D.

A Functional Assay to Assess Toxicity During Murine B Cell Development In Vitro.

B lymphocytes, or B cells, are important players in immunity that produce antigen-specific immunoglobulins. As a result, they are involved in various immune-linked pathologies. To better understand, prevent, or treat B cell-associated disease and immunotoxicity, we developed an in vitro assay to model early murine B cell differentiation within the bone marrow. This model uses sorted B cell precursors cultured on a supporting stromal cell layer, which over time acquire markers of further differentiated B cells, such as surface antigens and rearranged immunoglobulin light chain. Importantly, we utilized our in vitro model to validate our previous observations that xenobiotics, such as tungsten and organotins, alter B cell development in vivo. Furthermore, gene expression can be modulated in this model using retroviral transduction, making it amenable to investigating signaling pathways involved in disruption of B cell differentiation. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Assessment of early B lymphocyte differentiation in vitro Support Protocol: Isolation of murine bone marrow Alternate Protocol 1: Addition of recombinant interleukin-7 Alternate Protocol 2: Genetic manipulation via retroviral transduction.

The Roles of Bursal Nonapeptide (BP9) on AIV Vaccine Immune Response in Chick Immunization and on Avian Immature B Cell.

BACKGROUND: Bursa of Fabricius plays the vital functions on B cell development and antibody production in poultry. The bursal-derived peptide plays the essential roles on avian immature B cell development. OBJECTIVES: Here we explored the functions of the recently reported bursal nonapeptide (BP9) on the antibody production and the molecular basis of BP9 on avian immature B cell. METHODS: Chicken were twice immunized with Avian Influenza Virus (AIV) inactivated vaccine plus with BP9 at three dosages, respectively. On two weeks after the second immunization, sera samples were collected from all experimental groups to measure AIV-specific Agglutination Inhibition (HI) antibody titers. Also, on 7th day after the second immunization, spleen lymphocytes were isolated from the immunized chicken to detect the lymphocyte viabilities. DT40 cells were treated with BP9 from 0.02 to 2 µg/mL for 4 and 20h to detect sIgM mRNA levels, and total RNAs from BP9-treated DT40 cells were collected to investigate the gene expression profiles of DT40 cells, and to analyze the enriched pathways and functional biological processes. Finally, nine gene expressions were validated with quantitative PCR (qPCR). RESULTS: Our investigation proved the strong regulatory roles of BP9 on AIV-specific HI antibody titers and lymphocyte viabilities. BP9 promoted sIgM mRNA levels in DT40 cells, and upregulated 598 gene expressions and downregulated 395 gene expressions in DT40 cells with 0.2µg/mL BP9 treatment. Moreover, our findings verified the significantly enriched six pathways and various the biological functional processes of BP9 on avian immature B cell. Also, we found eight signaling pathways in the enriched biological processes of BP9-treated DT40 cells, and the expressions of nine selected genes with qPCR were identical to that of microarray data. CONCLUSION: BP9 promoted the antibody production in the 21-old-day chicken immunization, and stimulated the sIgM expression in DT40 cells. Furthermore, we analyzed the gene expression profile and immune-related biological processes of DT40 cells treated with BP9, which provided some new insights into the mechanism on immature B cell development, and provided important references for adjuvant development on vaccine improvement and clinical application.

EGFR-Mutated Lung Cancers Resistant to Osimertinib through EGFR C797S Respond to First-Generation Reversible EGFR Inhibitors but Eventually Acquire EGFR T790M/C797S in Preclinical Models and Clinical Samples.

INTRODUCTION: Osimertinib is approved for advanced EGFR-mutated NSCLC, and identification of on-target mechanisms of resistance (i.e., EGFR C797S) to this third-generation EGFR inhibitor are evolving. Whether durable control of subsequently osimertinib-resistant NSCLC with the EGFR-sensitizing mutation (SM)/C797S is possible with first-generation EGFR inhibitors (such as gefitinib or erlotinib) remains underreported, as does the resultant acquired resistance profile. METHODS: We used N-ethyl-N-nitrosourea mutagenesis to determine the profile of EGFR SM/C797S preclinical models exposed to reversible EGFR inhibitors. In addition, we retrospectively probed a case of EGFR SM lung adenocarcinoma treated with first-line osimertinib, followed by second-line erlotinib in the setting of EGFR SM/C797S. RESULTS: Use of N-ethyl-N-nitrosourea mutagenesis against the background of EGFR L858R/C797S in conjunction with administration of gefitinib revealed preferential outgrowth of cells with EGFR L858R/T790M/C797S. A patient with EGFR delE746_T751insV NSCLC was treated with osimertinib with sustained response for 10 months before acquiring EGFR C797S. The patient was subsequently treated with erlotinib, with response for a period of 4 months, but disease progression ensued. Liquid biopsy disclosed EGFR delE746_T751insV with T790M and C797S present in cis. CONCLUSION: EGFR SM NSCLC can acquire resistance to osimertinib through development of the EGFR C797S mutation. In this clinical scenario, the tumor may respond transiently to reversible first-generation EGFR inhibitors (gefitinib or erlotinib), but evolving mechanisms of on-target resistance-in clinical specimens and preclinical systems-indicate that EGFR C797S along with EGFR T790M can evolve. This report adds to the growing understanding of tumor evolution or adaptability to sequential EGFR inhibition and augments support for exploring combination therapies to delay or prevent on-target resistance.

Sensitivity and Resistance of MET Exon 14 Mutations in Lung Cancer to Eight MET Tyrosine Kinase Inhibitors In Vitro.

BACKGROUND: MNNG HOS transforming gene (MET) exon 14 mutations in lung cancer, including exon 14 skipping and point mutations, have been attracting the attention of thoracic oncologists as new therapeutic targets. Tumors with these mutations almost always acquire resistance, which also occurs in other oncogene-addicted lung cancers. However, the resistance mechanisms and treatment strategies are not fully understood. METHODS: We generated Ba/F3 cells expressing MET exon 14 mutations by retroviral gene transfer. The sensitivities of these cells to eight MET-tyrosine kinase inhibitors (TKIs) were determined using a colorimetric assay. In addition, using N-ethyl-N-nitrosourea mutagenesis, we generated resistant clones, searched for secondary MET mutations, and then examined the sensitivities of these resistant cells to different TKIs. RESULTS: Ba/F3 cells transfected with MET mutations grew in the absence of interleukin-3, indicating their oncogenic activity. These cells were sensitive to all MET-TKIs except tivantinib. We identified a variety of secondary mutations. D1228 and Y1230 were common sites for resistance mutations for type I TKIs, which bind the active form of MET, whereas L1195 and F1200 were common sites for type II TKIs, which bind the inactive form. In general, resistance mutations against type I were sensitive to type II, and vice versa. CONCLUSIONS: MET-TKIs inhibited the growth of cells with MET exon 14 mutations. We also identified mutation sites specific for TKI types as resistance mechanisms and complementary activities between type I and type II inhibitors against those mutations. These finding should provide relevant clinical implication for treating patients with lung cancer harboring MET exon 14 mutations.

The Inducing Role and Molecular Basis of Bursal Hexapeptide (BHP) on Avian Immature B Cell.

BACKGROUND: The Bursa of Fabricius is an acknowledged central humoral immune organ unique to birds, which provides an ideal research model on the immature B cell development. OBJECTIVE: In this article, our motivation is to study the role on sIgM and establish the molecular basis and functional processes of Bursal Hexapeptide (BHP) in avian immature B cells DT40 cell lines. METHODS: In this article, we detected the expressions of sIgM mRNA with qPCR in DT40 cells with BHP treatment, and investigated the gene expression profiles of BHP-treated DT40 cells, employing microarray analyses. Also, to validate the differentially expressed genes, we performed KEGG pathway and Gene Ontology analysis in the BHP-treated DT40 cells. Finally, we comparatively analyzed the similar regulated genes and their involved immune functional processes between DT40 cell and mouse immature B cell line WEHI231 cell with BHP treatment. RESULTS: Following the proposed framework, we proved that the BHP enhanced the mRNA expression levels of IgM in DT40 cells, and induced 460 upregulated genes and 460 downregulated genes in BHP-treated DT40 cells. The pathway analysis showed that the differentially regulated genes in DT40 cell line with BHP treatment were involved in 12 enrichment pathways, in which Toll-like receptor signaling pathway was the vital pathways, and cytokine-cytokine receptor interaction and Jak-STAT signaling pathway were another two important pathways in BHP-treated DT40 cells. Moreover, BHP induced the immune related biological processes in BHP-treated DT40 cells, including T cell related, cytokine related, lymphocyte related, and innate immune response GO terms. Finally, the comparatively analysis showed that there were two downregulated genes GATA3 and IFNG to be found co-existed among the differentially expressed genes in BHP-treated DT40 cell and WEHI231 cells, which shared some same immune related functional processes in both cell lines. CONCLUSION: After the applying the framework, we proved the inducing roles and the gene expression profiles of BHP on avian immature B cells, and verified some molecular basis from the KEGG and GO analysis. These results provided the insight for mechanism on immature B cell differentiation, and offer the essential direction for the vaccine improvement.

Interferon-beta treated-multiple sclerosis patients exhibit a decreased ratio between immature/transitional B cell subset and plasmablasts.

Our aim was to quantify circulating B cell subsets; immature/transitional, naïve, CD27- and CD27+ memory cells and plasmablasts, in relapsing-remitting multiple sclerosis patients treated with IFN-ß. The most relevant findings were a significant increase of plasmablasts and a decrease of immature/transitional B cells, resulting in a decreased ratio between those cells in relapse RRMS, together with an increase of CD27- and CD27+IgM+ memory B cell subsets in both phases of the disease. These alterations point to an active B cell response, particularly in relapse, and the above referred ratio could constitute a good biomarker of relapse in patients that underwent IFN-ß treatment.

Mechanisms of cell death induced by arginase and asparaginase in precursor B-cell lymphoblasts.

Arginase has therapeutic potential as a cytotoxic agent in some cancers, but this is unclear for precursor B acute lymphoblastic leukaemia (pre-B ALL), the commonest form of childhood leukaemia. We compared arginase cytotoxicity with asparaginase, currently used in pre-B ALL treatment, and characterised the forms of cell death induced in a pre-B ALL cell line 697. Arginase and asparaginase both efficiently killed 697 cells and mature B lymphoma cell line Ramos, but neither enzyme killed normal lymphocytes. Arginase depleted cellular arginine, and arginase-treated media induced cell death, blocked by addition of arginine or arginine-precursor citrulline. Asparaginase depleted both asparagine and glutamine, and asparaginase-treated media induced cell death, blocked by asparagine, but not glutamine. Both enzymes induced caspase cleavage and activation, chromatin condensation and phosphatidylserine exposure, indicating apoptosis. Both arginase- and asparaginase-induced death were blocked by caspase inhibitors, but with different sensitivities. BCL-2 overexpression inhibited arginase- and asparaginase-induced cell death, but did not prevent arginase-induced cytostasis, indicating a different mechanism of growth arrest. An autophagy inhibitor, chloroquine, had no effect on the cell death induced by arginase, but doubled the cell death induced by asparaginase. In conclusion, arginase causes death of lymphoblasts by arginine-depletion induced apoptosis, via mechanism distinct from asparaginase. Therapeutic implications for childhood ALL include: arginase might be used as treatment (but antagonised by dietary arginine and citrulline), chloroquine may enhance efficacy of asparaginase treatment, and partial resistance to arginase and asparaginase may develop by BCL-2 expression. Arginase or asparaginase might potentially be used to treat Burkitt lymphoma.

Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.

Proteasome inhibition with bortezomib induces a therapeutically relevant depletion of plasma cells in SLE but does not target their precursors.

Long-lived plasma cells (PCs) not only provide protective humoral immunity, they are also an essential component of the autoreactive immunologic memory that may drive chronic immune responses in systemic autoimmunity, such as systemic lupus erythematosus (SLE). The therapeutic relevance of their targeting has been demonstrated in preclinical models and severe, treatment-refractory cases of autoimmune diseases using the proteasome inhibitor bortezomib. Herein, we describe in detail the dynamic serologic changes and effects on immune effector cells in eight SLE patients receiving a median two cycles of 1.3 mg/m2 intravenous bortezomib. Upon proteasome inhibition, immunoglobulin levels gradually declined by ∼30%, associated with a significant reduction of autoantibodies, and serum complement whereas B-cell activation factor levels increased. While proteasome inhibition was associated with a significant depletion of short- and long-lived PCs in peripheral blood and bone marrow by ∼50%, including those with a distinctly mature CD19- phenotype, their precursor B cells and T cells largely remained unaffected, resulting in a rapid repopulation of short-lived PCs after bortezomib withdrawal, accompanied by increasing autoantibody levels. Collectively, these findings identify proteasome inhibitors as a promising treatment option for refractory SLE, but also indicate that PC depletion needs to be combined with targeted B-cell therapies for sustained responses in systemic autoimmunity.

Hesperidin inhibits insulin-induced phosphoinositide 3-kinase/Akt activation in human pre-B cell line NALM-6.

CONTEXT: It has been shown that hesperidin induces apoptosis in NALM-6 cells through inhibition of nuclear factor-kappa B (NF-κB) activation. AIMS: To investigate the effect of hesperidin on inhibition of NF-κB activation through blocking phosphoinositide 3-kinase (PI3K)/Akt pathway as a main target in cancer treatment, in NALM-6 cells. MATERIALS AND METHODS: NALM-6 cells were incubated with two concentrations of hesperidin (25, 50 µM) in the presence or absence of insulin (100 nM), as a potent activator of Akt. The cytotoxic activity of hesperidin was determined by 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptotic death was measured by ELISA test using cell death detection ELISA Plus kit. To assay the effect of hesperidin on Akt pathway, the phosphorylation levels of Akt, inhibitor of kappa B alpha (IκBα), and glycogen synthase kinase-3 beta (GSK-3ß) and expression level of IκB kinase alpha (IKKα) were determined by Western blot analysis. RESULTS: Hesperidin (both concentrations) significantly reduced cells survival in the presence and absence of insulin compared to untreated cells in a time-dependent manner (P < 0.05). Hesperidin also significantly increased apoptosis in NALM-6 cells even in hyperinsulinemia condition (P < 0.0001). Hesperidin inhibited insulin-induced phosphorylation and activation of Akt, IκBα, and GSK-3ß and decreased expression of IKKα. CONCLUSION: The results of this study demonstrated that cytotoxic and proapoptotic actions of hesperidin are partly mediated through the suppression of PI3K3/Akt/IKK signaling pathway. So, hesperidin might act as a chemotherapeutic agent by targeting cell survival pathways.

Transitional B Cells and TLR9 Responses Are Defective in Selective IgA Deficiency.

Selective IgA deficiency (IgAD) is the most common primary antibody deficiency in the western world with affected individuals suffering from an increased burden of autoimmunity, atopic diseases and infections. It has been shown that IgAD B cells can be induced with germinal center mimicking reactions to produce IgA. However, IgA is the most prevalent antibody in mucosal sites, where antigen-independent responses are important. Much interest has recently focused on the role of TLR9 in both naïve and mature B cell differentiation into IgA secreting plasma cells. Here, we analyze the phenotype and function of T and B cells in individuals with IgAD following IgA-inducing CpG-TLR9 stimulations. The IgAD individuals had significantly lower numbers of transitional B cells (CD19+CD24hiCD38hi) and class-switched memory B cells (CD20+CD27+IgD-) ex vivo. However, proportions of T cell populations ex vivo as well as in vitro induced T effector cells and T regulatory cells were comparable to healthy controls. After CpG stimulation, the transitional B cell defect was further enhanced, especially within its B regulatory subset expressing IL-10. Finally, CpG stimulation failed to induce IgA production in IgAD individuals. Collectively, our results demonstrate a defect of the TLR9 responses in IgAD that leads to B cell dysregulation and decreased IgA production.