Statins impair antitumor effects of rituximab by inducing conformational changes of CD20

Background

Rituximab is used in the treatment of CD20⁺ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.

Methods and Findings

Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a ⁵¹Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.

Conclusions

Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.     

Inhibition of Increased Circulating Tfh Cell by Anti-CD20 Monoclonal Antibody in Patients with Type 1 Diabetes

Objectives

Follicular helper T (Tfh) cells exert an important role in autoimmune diseases. Whether it might be involved in type 1 diabetes (T1D) is unknown. Our aim was to investigate the role of Tfh cells in patients with T1D and the effect of anti-CD20 monoclonal antibody (rituximab) on Tfh cells from T1D patients.

Patients and Methods

Fifty-four patients with T1D and 37 healthy controls were enrolled in the current study. 20 of those patients were treated with rituximab. The frequencies of circulating CD4⁺CXCR5⁺ICOS⁺T cells were analyzed by flow cytometry. The serum autoantibodies were detected by radioligand assay. The levels of IL-21, IL-6 and BCL-6 were assessed using ELISA and/or real-time PCR.

Results

Increased frequencies of circulating Tfh cells together with enhanced expression of IL-21 were detected in patients. The correlation between the frequencies of circulating Tfh cells and the serum autoantibodies or C-peptide level was comfirmed. After rituximab therapy, follow-up analysis demonstrated that the frequencies of circulating Tfh cell and serum IA2A were decreased. The levels of IL-21, IL-6 and Bcl-6 mRNA were decreased after treatment. Furthermore, beta cell function in 10 of 20 patients was improved.

Conclusions

These data indicate Tfh cells may participate in the T1D-relatede immune responses and B cells might play a role in the development of Tfh responses in the disease progression.     

Fibroblast Growth Factor Receptor 3 (FGFR3) Associated with the CD20 Antigen Regulates the Rituximab-induced Proliferation Inhibition in B-cell Lymphoma Cells

Rituximab is reported to inhibit the proliferation of lymphoma cells through an unknown CD20-mediated signal transduction pathway. Herein, we investigated cell surface molecules involved in the CD20-mediated signal transduction pathway by using a recently developed technique named enzyme-mediated activation of radical sources. Using this method, we found that under stimulation with rituximab and another anti-CD20 antibody B-Ly1, CD20 was physically associated with fibroblast growth factor receptor 3 (FGFR3) as well as some other receptor tyrosine kinases in Raji cells. However, under stimulation with a noncytotoxic anti-CD20 antibody 2H7, CD20 was not associated with FGFR3 but with the PDGF receptor β. When the tyrosine kinase activity of FGFR3 was inhibited by the chemical inhibitor PD173074 or an siRNA knockdown strategy, the proliferation inhibition by rituximab was attenuated, indicating that FGFR3 participates in the rituximab-dependent signal transduction pathway leading to proliferation inhibition. These observations raise the possibility that concomitant targeted therapy toward FGFR3 might improve the efficacy and safety of the rituximab therapy.     

Antibodies against CD20 or B-cell receptor induce similar transcription patterns in human lymphoma cell lines

Background

CD20 is a cell surface protein exclusively expressed on B cells. It is a clinically validated target for Non-Hodgkin''s lymphomas (NHL) and autoimmune diseases. The B cell receptor (BCR) plays an important role for development and proliferation of pre-B and B cells. Physical interaction of CD20 with BCR and components of the BCR signaling cascade has been reported but the consequences are not fully understood.

Methodology

In this study we employed antibodies against CD20 and against the BCR to trigger the respective signaling. These antibodies induced very similar expression patterns of up- and down-regulated genes in NHL cell lines indicating that CD20 may play a role in BCR signaling and vice versa. Two of the genes that were rapidly and transiently induced by both stimuli are CCL3 and CCL4. 4 hours after stimulation the concentration of these chemokines in culture medium reaches a maximum. Spleen tyrosine kinase Syk is a cytoplasmic tyrosine kinase and a key component of BCR signaling. Both siRNA mediated silencing of Syk and inhibition by selective small molecule inhibitors impaired CCL3/CCL4 protein induction after treatment with either anti-CD20 or anti-BCR antibodies.

Conclusion

Our results suggest that treatment with anti-CD20 antibodies triggers at least partially a BCR activation-like response in NHL cell lines.     

Anti-CD22/CD20 Bispecific Antibody with Enhanced Trogocytosis for Treatment of Lupus

The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma, leukemia and autoimmune diseases, treating currently over 1500 cases of non-Hodgkin lymphoma, acute lymphoblastic leukemias, Waldenström’s macroglobulinemia, Sjögren’s syndrome, and systemic lupus erythematosus. Because epratuzumab reduces on average only 35% of circulating B cells in patients, and has minimal antibody-dependent cellular cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, its therapeutic activity may not result completely from B-cell depletion. We reported recently that epratuzumab mediates Fc/FcR-dependent membrane transfer from B cells to effector cells via trogocytosis, resulting in a substantial reduction of multiple BCR modulators, including CD22, CD19, CD21, and CD79b, as well as key cell adhesion molecules, including CD44, CD62L, and β7 integrin, on the surface of B cells in peripheral blood mononuclear cells obtained from normal donors or SLE patients. Rituximab has clinical activity in lupus, but failed to achieve primary endpoints in a Phase III trial. This is the first study of trogocytosis mediated by bispecific antibodies targeting neighboring cell-surface proteins, CD22, CD20, and CD19, as demonstrated by flow cytometry and immunofluorescence microscopy. We show that, compared to epratuzumab, a bispecific hexavalent antibody comprising epratuzumab and veltuzumab (humanized anti-CD20 mAb) exhibits enhanced trogocytosis resulting in major reductions in B-cell surface levels of CD19, CD20, CD21, CD22, CD79b, CD44, CD62L and β7-integrin, and with considerably less immunocompromising B-cell depletion that would result with anti-CD20 mAbs such as veltuzumab or rituximab, given either alone or in combination with epratuzumab. A CD22/CD19 bispecific hexavalent antibody, which exhibited enhanced trogocytosis of some antigens and minimal B-cell depletion, may also be therapeutically useful. The bispecific antibody is a candidate for improved treatment of lupus and other autoimmune diseases, offering advantages over administration of the two parental antibodies in combination.     

Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

Background

Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.

Methodology/Principal Findings

After transplantation with CD34⁺CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2^−/−IL-2Rγc^−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19⁺CD27⁺ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively.

Conclusion/Significance

This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.     

CD100 Up-Regulation Induced by Interferon-α on B Cells Is Related to Hepatitis C Virus Infection

Objectives

CD100, also known as Sema4D, is a member of the semaphorin family and has important regulatory functions that promote immune cell activation and responses. The role of CD100 expression on B cells in immune regulation during chronic hepatitis C virus (HCV) infection remains unclear.

Materials and Methods

We longitudinally investigated the altered expression of CD100, its receptor CD72, and other activation markers CD69 and CD86 on B cells in 20 chronic HCV-infected patients before and after treatment with pegylated interferon-alpha (Peg-IFN-α) and ribavirin (RBV) by flow cytometry.

Results

The frequency of CD5⁺ B cells as well as the expression levels of CD100, CD69 and CD86 was significantly increased in chronic HCV patients and returned to normal in patients with sustained virological response after discontinuation of IFN-α/RBV therapy. Upon IFN-α treatment, CD100 expression on B cells and the two subsets was further up-regulated in patients who achieved early virological response, and this was confirmed by in vitro experiments. Moreover, the increased CD100 expression via IFN-α was inversely correlated with the decline of the HCV-RNA titer during early-phase treatment.

Conclusions

Peripheral B cells show an activated phenotype during chronic HCV infection. Moreover, IFN-α therapy facilitates the reversion of disrupted B cell homeostasis, and up-regulated expression of CD100 may be indirectly related to HCV clearance.     

Binding to CD20 by Anti-B1 Antibody or F(ab')2 is sufficient for induction of apoptosis in B-cell lines

CD20 is a B-cell-specific cell surface protein expressed on mature B lymphocytes and is a target for monoclonal antibody therapy for non-Hodgkin's lymphoma (NHL). Though clear clinical efficacy has been demonstrated with several anti-CD20 antibodies, the mechanisms by which the antibodies activate CD20 and kill cells remain unclear. Proposed mechanisms of action include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and induction of apoptosis. In this report we compared the activity of two anti-CD20 antibodies, Anti-B1 Antibody (tositumomab) and rituximab (C2B8), in a variety of cellular assays using a panel of B-cell lines. Anti-B1 Antibody showed a low level of activity in a CDC assay against complement-sensitive B-cell lines, Ramos and Daudi. We found that there is an inverse correlation between the expression of CD55 and CD59 and CDC mediated by either Anti-B1 Antibody or rituximab. Rituximab was more potent at inducing CDC when compared to Anti-B1 Antibody. Using Raji cells as target cells and human peripheral blood leukocytes as effector cells, Anti-B1 Antibody was a potent inducer of ADCC. The activities of Anti-B1 Antibody and rituximab were nearly identical in the ADCC assay. In addition, Anti-B1 Antibody showed direct induction of apoptosis in all B-cell lines tested. In general, crosslinking Anti-B1 Antibody with a goat anti-mouse Ig did not further enhance the percentage of cells undergoing apoptosis. Importantly, a F(ab')(2) fragment of Anti-B1 Antibody induced apoptosis, while the Fab fragment did not, indicating that the Fc region was not required and dimerization of CD20 may be sufficient for induction of apoptosis. In contrast, rituximab, which binds to an overlapping epitope on CD20 with a three-fold lower affinity than Anti-B1 Antibody, did not efficiently induce apoptosis in the cell lines tested in the absence of crosslinking. In conclusion, these two anti-CD20 antibodies have overlapping, but distinct mechanisms of action on B-cell lines.     

CD1d Expression in Paneth Cells and Rat Exocrine Pancreas Revealed by Novel Monoclonal Antibodies Which Differentially Affect NKT Cell Activation

Background

CD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated.

Methodology/Principal Findings

Two mAbs, WTH-1 and WTH-2, were generated which bound equally well to cell surface-expressed rat and mouse CD1d. Their non-overlapping epitopes were mapped to the CD1d heavy chain. Flow cytometry and immunohistological analyses revealed a nearly identical degree and pattern of CD1d expression for hematopoieitic cells of both species. Notable is also the detection of CD1d protein in mouse and rat Paneth cells as well as the extremely high CD1d expression in acinar exocrine cells of the rat pancreas and the expression of CD4 on rat marginal zone B cells. Both mAbs blocked α-galactosylceramide recognition by primary rat and mouse NKT cells. Interestingly, the two mAbs differed in their impact on the activation of various autoreactive T cell hybridomas, including the XV19.2 hybridoma whose activation was enhanced by the WTH-1 mAb.

Conclusions/Significance

The two novel monoclonal antibodies described in this study, allowed the analysis of CD1d expression and CD1d-restricted T cell responses in the rat for the first time. Moreover, they provided new insights into mechanisms of CD1d-restricted antigen recognition. While CD1d expression by hematopoietic cells of mice and rats was extremely similar, CD1d protein was detected at not yet described sites of non-lymphatic tissues such as the rat exocrine pancreas and Paneth cells. The latter is of special relevance given the recently reported defects of Paneth cells in CD1d^−/− mice, which resulted in an altered composition of the gut flora.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

Lymphoma and Myeloma Cell Resistance to Cytotoxic Agents and Ionizing Radiations Is Not Affected by Exposure to Anti–IL-6 Antibody

Background

Production of high levels of IL-6 is often correlated with resistance to cytotoxics or ionizing radiations, in cancer cell lines as in various cancer patients. We investigated whether monoclonal antibodies directed against IL-6 may enable to reverse resistance of cancer cell lines.

Methodology/Principal Findings

We exposed ten haematological cancer cells from lymphoma, myeloma, or leukemia origins to cytotoxics or ionizing radiations and assessed the effects of anti–IL-6 antibody addition on cell proliferation, apoptosis, or IL-6 signaling. A strong correlation between IL-6 secretion, measured by ELISA, and resistance to doxorubicin as ionizing radiations was observed in the multiple myeloma U266 and the Burkitt''s lymphoma Daudi and Namalwa cells. Although an anti–IL-6 antibody combined to both treatments efficiently blocked IL-6 signaling in U266 cells, expressing the IL-6 receptor gp80, it did not increase treatment-induced anti-proliferative and pro-apoptotic effects on these cells, as well as on Daudi and Namalwa cells. This lack of effect could be related to diverse factors: 1) a higher release of the soluble form of IL-6 receptor gp80 in response to doxorubicin and irradiation from all cell lines, 2) an impaired level of the IL-6 pathway inhibitor SOCS3 in Daudi cells, and 3) an increased release of IL-10 and TNFα, two cytokines involved in cell radio- and chemoresistance.

Conclusions/Significance

These data support the fact that IL-6 is not the preponderant actor of cell resistance to cytotoxics and ionizing radiations, which seems to be regulated by a complex network of proteins.     

Lymphoid Hyperplasia and Lymphoma in Transgenic Mice Expressing the Small Non-Coding RNA,EBER1 of Epstein-Barr Virus

Background

Non-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure.

Methodology/Principal Findings

To investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer Eμ. Mice of several of these EμEBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitt''s lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with EμEBER1 and EμMyc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some EμEBER1 single transgenic tumours, indicative of a functional cooperation.

Conclusions/Significance

This study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease.     

Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes.     

Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement-dependent Cytotoxicity

Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.     

Proteomic-based identification of CD4-interacting proteins in human primary macrophages

Background

Human macrophages (Mφ) express low levels of CD4 glycoprotein, which is constitutively recycled, and 40–50% of its localization is intracellular at steady-state. Although CD4-interacting proteins in lymphoid cells are well characterised, little is known about the CD4 protein interaction-network in human Mφ, which notably lack LCK, a Src family protein tyrosine kinase believed to stabilise CD4 at the surface of T cells. As CD4 is the main cellular receptor used by HIV-1, knowledge of its molecular interactions is important for the understanding of viral infection strategies.

Methodology/Principal Findings

We performed large-scale anti-CD4 immunoprecipitations in human primary Mφ followed by high-resolution mass spectrometry analysis to elucidate the protein interaction-network involved in induced CD4 internalization and degradation. Proteomic analysis of CD4 co-immunoisolates in resting Mφ showed CD4 association with a range of proteins found in the cellular cortex, membrane rafts and components of clathrin-adaptor proteins, whereas in induced internalization and degradation CD4 is associated with components of specific signal transduction, transport and the proteasome.

Conclusions/Significance

This is the first time that the anti-CD4 co-immunoprecipitation sub-proteome has been analysed in human primary Mφ. Our data have identified important Mφ cell surface CD4-interacting proteins, as well as regulatory proteins involved in internalization and degradation. The data give valuable insights into the molecular pathways involved in the regulation of CD4 expression in Mφ and provide candidates/targets for further biochemical studies.     

High Glucose Induces CCL20 in Proximal Tubular Cells via Activation of the KCa3.1 Channel

Background

Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-β1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-β1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN.

Methods

Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34.

Results

In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB.

Conclusions

Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.     

Engagement of SIRPα Inhibits Growth and Induces Programmed Cell Death in Acute Myeloid Leukemia Cells

Background

Recent studies show the importance of interactions between CD47 expressed on acute myeloid leukemia (AML) cells and the inhibitory immunoreceptor, signal regulatory protein-alpha (SIRPα) on macrophages. Although AML cells express SIRPα, its function has not been investigated in these cells. In this study we aimed to determine the role of the SIRPα in acute myeloid leukemia.

Design and Methods

We analyzed the expression of SIRPα, both on mRNA and protein level in AML patients and we further investigated whether the expression of SIRPα on two low SIRPα expressing AML cell lines could be upregulated upon differentiation of the cells. We determined the effect of chimeric SIRPα expression on tumor cell growth and programmed cell death by its triggering with an agonistic antibody in these cells. Moreover, we examined the efficacy of agonistic antibody in combination with established antileukemic drugs.

Results

By microarray analysis of an extensive cohort of primary AML samples, we demonstrated that SIRPα is differentially expressed in AML subgroups and its expression level is dependent on differentiation stage, with high levels in FAB M4/M5 AML and low levels in FAB M0–M3. Interestingly, AML patients with high SIRPα expression had a poor prognosis. Our results also showed that SIRPα is upregulated upon differentiation of NB4 and Kasumi cells. In addition, triggering of SIRPα with an agonistic antibody in the cells stably expressing chimeric SIRPα, led to inhibition of growth and induction of programmed cell death. Finally, the SIRPα-derived signaling synergized with the activity of established antileukemic drugs.

Conclusions

Our data indicate that triggering of SIRPα has antileukemic effect and may function as a potential therapeutic target in AML.     

Curcumin Inhibits CD4+ T Cell Activation,but Augments CD69 Expression and TGF-β1-Mediated Generation of Regulatory T Cells at Late Phase

Background

Curcumin is a promising candidate for a natural medicinal agent to treat chronic inflammatory diseases. Although CD4⁺ T cells have been implicated in the pathogenesis of chronic inflammation, whether curcumin directly regulates CD4⁺ T cells has not been definitively established. Here, we showed curcumin-mediated regulation of CD2/CD3/CD28-initiated CD4⁺ T cell activation in vitro.

Methodology/Principal Findings

Primary human CD4⁺ T cells were stimulated with anti-CD2/CD3/CD28 antibody-coated beads as an in vitro surrogate system for antigen presenting cell-T cell interaction and treated with curcumin. We found that curcumin suppresses CD2/CD3/CD28-initiated CD4⁺ T cell activation by inhibiting cell proliferation, differentiation and cytokine production. On the other hand, curcumin attenuated the spontaneous decline of CD69 expression and indirectly increased expression of CCR7, L-selectin and Transforming growth factor-β1 (TGF-β1) at the late phase of CD2/CD3/CD28-initiated T cell activation. Curcumin-mediated up-regulation of CD69 at late phase was associated with ERK_1/2 signaling. Furthermore, TGF-β1 was involved in curcumin-mediated regulation of T cell activation and late-phase generation of regulatory T cells.

Conclusions/Significance

Curcumin not merely blocks, but regulates CD2/CD3/CD28-initiated CD4⁺ T cell activation by augmenting CD69, CCR7, L-selectin and TGF-β1 expression followed by regulatory T cell generation. These results suggest that curcumin could directly reduce T cell-dependent inflammatory stress by modulating CD4⁺ T cell activation at multiple levels.     

Near-Term Anti-CD25 Monoclonal Antibody Administration Protects Murine Liver from Ischemia-Reperfusion Injury Due to Reduced Numbers of CD4+ T Cells

Background

CD4⁺ T cell is acknowledged as a key factor in the initiation phase of liver ischemia reperfusion injury. The purpose of current study is to demonstrate the effect of antecedent near-term anti-CD25 monoclonal antibody treatment on IR-induced liver injury by modulation of CD4⁺ T cells.

Methods

70% liver warm IR was induced in male C57BL/6 mice after anti-CD25 mAb or non-specific IgG administration. Liver function, histological damage, in vitro Proliferation, FACS, cytokine production, and immunofluorescence were assessed to evaluate the impact of antecedent near-term PC61 treatment on IR-induced liver injury.

Results

After 70% liver ischemia, mice preconditioned with PC61 displayed significantly preserved liver function as characterized by less histological damage and reduced serum enzymes level. Mechanistic studies revealed that the protection effect of anti-CD25 mAb was associated with ameliorated intrahepatic inflammatory milieu and reduced CD4⁺ T lymphocytes as manifested by the decrease of proinflammatory cytokine production (less expression of TNF-α, IFN-γ, IL-2, and IL-6) and the lower CD4/CD8 proportion.

Conclusions

Our results provide first line of evidence indicating that near-term treatment with anti-CD25 monoclonal antibody might provide protection for livers against IR-induced injury by reducing CD4⁺ T cells, but not influencing functional Treg population. Therefore, our results demonstrate a potential function of anti-CD25 monoclonal antibody which was neglected in the past, and may be helpful in various clinical conditions, particularly in liver and kidney transplantations.