Deletion of WASp and N-WASp in B cells cripples the germinal center response and results in production of IgM autoantibodies.

Humoral immunodeficiency caused by mutations in the Wiskott-Aldrich syndrome protein (WASp) is associated with failure to respond to common pathogens and high frequency of autoimmunity. Here we addressed the question how deficiency in WASp and the homologous protein N-WASp skews the immune response towards autoreactivity. Mice devoid of WASp or both WASp and N-WASp in B cells formed germinal center to increased load of apoptotic cells as a source of autoantigens. However, the germinal centers showed abolished polarity and B cells retained longer and proliferated less in the germinal centers. While WASp-deficient mice had high titers of autoreactive IgG, B cells devoid of both WASp and N-WASp produced mainly IgM autoantibodies with broad reactivity to autoantigens. Moreover, B cells lacking both WASp and N-WASp induced somatic hypermutation at reduced frequency. Despite this, IgG1-expressing B cells devoid of WASp and N-WASp acquired a specific high affinity mutation, implying an increased BCR signaling threshold for selection in germinal centers. Our data provides evidence for that N-WASp expression alone drives WASp-deficient B cells towards autoimmunity.

A novel GPI-anchored dominant-negative TGF-ß receptor II renders T cells unresponsive to TGF-ß signaling.

Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine expressed by a wide range of cell types and is known for hampering the effectiveness of cancer immune cell therapeutic approaches. We have designed a novel construct containing the extracellular domain of the TGF-ß receptor II linked to a glycosylphosphatidylinositol (GPI) anchor (GPI-ecto-TßRII) lacking the transmembrane and cytoplasmic signaling domain of TGF-ß receptor II (TßRII). T cells transduced with lentivirus expressing the GPI-ecto-TßRII construct show 5 to 15 times higher membrane expression compared with a previously established dominant-negative receptor carrying a truncated signaling domain. GPI-ecto-TßRII expression renders T cells unresponsive to TGF-ß-induced signaling seen by a lack of SMAD phosphorylation upon exogeneous TGF-ß treatment. Transduced T cells continue to express high levels of IFNγ and granulocyte-macrophage colony-stimulating factor (GM-CSF), among other cytokines, in the presence of TGF-ß while cytokine expression in untransduced T cells is being markedly suppressed. Furthermore, T cells expressing GPI-ecto-TßRII constructs have been shown to efficiently capture and inactivate TGF-ß from their environment. These results indicate the potential benefits of GPI-ecto-TßRII expressing cytotoxic T cells (CTLs) in future cell therapies.

The role of tetraspanin CD63 in antigen presentation via MHC class II.

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.

BZD9L1 sirtuin inhibitor as a potential adjuvant for sensitization of colorectal cancer cells to 5-fluorouracil.

BACKGROUND: This study aims to investigate the combination effect of a novel sirtuin inhibitor (BZD9L1) with 5-fluorouracil (5-FU) and to determine its molecular mechanism of action in colorectal cancer (CRC). METHODS: BZD9L1 and 5-FU either as single treatment or in combination were tested against CRC cells to evaluate synergism in cytotoxicity, senescence and formation of micronucleus, cell cycle and apoptosis, as well as the regulation of related molecular players. The effects of combined treatments at different doses on stress and apoptosis, migration, invasion and cell death mechanism were evaluated through two-dimensional and three-dimensional cultures. In vivo studies include investigation on the combination effects of BZD9L1 and 5-FU on colorectal tumour xenograft growth and an evaluation of tumour proliferation and apoptosis using immunohistochemistry. RESULTS: Combination treatments exerted synergistic reduction on cell viability on HCT 116 cells but not on HT-29 cells. Combined treatments reduced survival, induced cell cycle arrest, apoptosis, senescence and micronucleation in HCT 116 cells through modulation of multiple responsible molecular players and apoptosis pathways, with no effect in epithelial mesenchymal transition (EMT). Combination treatments regulated SIRT1 and SIRT2 protein expression levels differently and changed SIRT2 protein localization. Combined treatment reduced growth, migration, invasion and viability of HCT 116 spheroids through apoptosis, when compared with the single treatment. In addition, combined treatment was found to reduce tumour growth in vivo through reduction of tumour proliferation and necrosis compared with the vehicle control group. This highlights the potential therapeutic effects of BZD9L1 and 5-FU towards CRC. CONCLUSION: This study may pave the way for use of BZD9L1 as an adjuvant to 5-FU in improving the therapeutic efficacy for the treatment of colorectal cancer.

Anticancer activities of a benzimidazole compound through sirtuin inhibition in colorectal cancer.

AIM: This study aims to investigate the mode of action of a novel sirtuin inhibitor (BZD9L1) and its associated molecular pathways in colorectal cancer (CRC) cells. MATERIALS & METHODS: BZD9L1 was tested against metastatic CRC cell lines to evaluate cytotoxicity, cell cycle and apoptosis, senescence, apoptosis related genes and protein expressions, as well as effect against major cancer signaling pathways. RESULTS & CONCLUSION: BZD9L1 reduced the viability, cell migration and colony forming ability of both HCT 116 and HT-29 metastatic CRC cell lines through apoptosis. BZD9L1 regulated major cancer pathways differently in CRC with different mutation profiles. BZD9L1 exhibited anticancer activities as a cytotoxic drug in CRC and as a promising therapeutic strategy in CRC treatment.

Isoledene from Mesua ferrea oleo-gum resin induces apoptosis in HCT 116 cells through ROS-mediated modulation of multiple proteins in the apoptotic pathways: A mechanistic study.

Colorectal cancer (CRC) is one of the most common human malignant tumors worldwide. Arising from the transformation of epithelial cells in the colon and/or rectum into malignant cells, the foundation of CRC pathogenesis lies in the progressive accumulation of mutations in oncogenes and tumor-suppressor genes, such as KRAS and APC. Resistance to apoptosis is one of the key mechanisms in the development of CRC as it is for any other kind of cancer. Natural products have been shown to induce the expression of apoptosis regulators that are blocked in cancer cells. In the present study, a series of in vitro assays were employed to study the apoptosis-inducing attributes of Isoledene rich sub-fraction (IR-SF) collected from the oleo-gum resin of M. ferrea. Data obtained, showed that IR-SF inhibited cell proliferation and induced typical apoptotic changes in the overall morphology of all the CRC cell lines tested. Fluorescent staining assays revealed characteristic nuclear condensation, and marked decrease in mitochondrial outer membrane potential in the treated cells. In addition, an increment in the levels of ROS, caspase-8, -9 and -3 was observed. Proteomic analysis revealed that IR-SF up-regulated the expression of pro-apoptotic proteins, i.e., Bid, Bim and cytochrome c. Cytochrome c in turn activated caspases cascade resulting in the induction of apoptosis. Moreover, IR-SF significantly down-regulated Bcl-2, Bcl-w, survivin, xIAP and HSPs pro-survival proteins and induced DNA fragmentation and G0/G1-phase arrest in HCT 116 cells. Chemical characterization of IR-SF by GC-MS and HPLC methods identified Isoledene as one of the major compounds. Altogether, results of the present study demonstrate that IR-SF may induce apoptosis in human colorectal carcinoma cells through activation of ROS-mediated apoptotic pathways.