Fatimah Bte Mustafa

Honeybee venom secretory phospholipase A2 induces leukotriene production but not histamine release from human basophils.

The role of basophils in an anaphylactic response is well recognized but is usually masked by mast cells, which contain similar mediators for the induction of generalized vasodilatation and laryngeal constriction. The rapid onset of systemic anaphylactic symptoms, particularly in insect stings and ingested food, suggest that basophils, a circulating pool of cells containing histamine and other potent mediators such as leukotrienes, may be more involved in systemic anaphylaxis than originally thought. We wished to examine if secretory phospholipase A2, a systemic allergen found in honey bee venom (HBV‐sPLA2) may activate basophils directly leading to rapid systemic mediator release. Basophils were isolated from human blood and stimulated with increasing concentrations of HBV‐sPLA2. We found that physiological concentrations of HBV‐sPLA2 induce rapid leukotriene C4 production from purified human basophils within 5 min, while interleukin (IL)‐4 expression and production was induced at later time‐points. Histamine release was not induced, signifying that HBV‐sPLA2 did not induce generalized degranulation. Surface expression of CD63, CD69 and CD11b were up‐regulated following HBV‐sPLA2 treatment. Stimulation of basophils with anti‐immunoglobulin E (IgE) following treatment with HBV‐sPLA2 did not induce more leukotriene release. To investigate the mechanism of leukotriene production, 9–12 octadecadiynioc acid, a cyclooxygenase‐1 (COX‐1) and 15‐lipoxygenase inhibitor, was used and this abrogated leukotriene production. These results indicate that HBV‐sPLA2 can directly activate human basophils in vitro to induce leukotriene production.

Annexin‐1‐deficient mice exhibit spontaneous airway hyperresponsiveness and exacerbated allergen‐specific antibody responses in a mouse model of asthma

Glucocorticoids are the mainstream drugs used in the treatment and control of inflammatory diseases such as asthma. Annexin‐1 (ANXA1) is an anti‐inflammatory protein which has been described as an endogenous protein responsible for some anti‐inflammatory glucocorticoid effects. Previous studies have identified its importance in other immune diseases such as rheumatoid arthritis and cystic fibrosis. ANXA1‐deficient (−/−) mice are Th2 biased, and ANXA1 N‐terminus peptide exhibits anti‐inflammatory activity in a rat model of pulmonary inflammation.

Stimulation of Human Endothelium with IL-3 Induces Selective Basophil Accumulation In Vitro

Basophils have been shown to accumulate in allergic airways and other extravascular sites. Mechanisms responsible for the selective recruitment of basophils from the blood into tissue sites remain poorly characterized. In this study, we characterized human basophil rolling and adhesion on HUVECs under physiological shear flow conditions. Interestingly, treatment of endothelial cells with the basophil-specific cytokine IL-3 (0.01–10 ng/ml) promoted basophil and eosinophil, but not neutrophil, rolling and exclusively promoted basophil adhesion. Preincubation of HUVECs with an IL-3R-blocking Ab (CD123) before the addition of IL-3 inhibited basophil rolling and adhesion, implicating IL-3R activation on endothelial cells. Incubation of basophils with neuraminidase completely abolished both rolling and adhesion, indicating the involvement of sialylated structures in the process. Abs to the β1 integrins, CD49d and CD49e, as well as to P-selectin and P-selectin glycoprotein ligand 1, inhibited basophil rolling and adhesion. Furthermore, blocking chemokine receptors expressed by basophils, such as CCR2, CCR3, and CCR7, demonstrated that CCR7 was involved in the observed recruitment of basophils. These data provide novel insights into how IL-3, acting directly on endothelium, can cause basophils to preferentially interact with blood vessels under physiological flow conditions and be selectively recruited to sites of inflammation.

Identification of two proteins, S14 and UIP1, that interact with UCH37

By the use of the yeast two‐hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co‐immunoprecipitation analysis. The C‐terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two‐hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.

Characterization of Human Umbilical Cord Lining-Derived Epithelial Cells and Transplantation Potential

In this study we describe the derivation and immunological characterization of a primary epithelial cell type from the human umbilical cord membrane. These cord lining epithelial cells (CLECs) expressed and/or secreted isoforms of the nonclassical human leukocyte antigen class I (HLA-1b) glycoproteins, HLA-G and E. Conditioned media from CLECs inhibited mitogen-stimulated T-lymphocyte responses, and in a mixed leukocyte reaction (MLR) assay, cocultured CLECs inhibited allogeneic responses with a concomitant reduction in proinflammatory cytokines. Using a transwell coculture system, it was demonstrated that these immunoregulatory effects were mediated by soluble factors secreted by CLECs, in a dose-dependent manner. Functional studies using HLA-G blocking antibody showed that the effects of CLEC-secreted products could be inhibited, thus demonstrating a significant and important role for soluble HLA-G. In vivo, we show that transplanted CLECs could be maintained for extended periods in immunocompetent mice where xenorejection rapidly destroyed primary keratinocytes, a control human epithelial cell type. Additionally, CLECs delayed the rejection of keratinocytes and extended their survival when cotransplanted, indicating an ability to protect adjacent human cell types that would otherwise be rejected if transplanted alone. We also show that CLECs transduced with a modified human proinsulin gene were transplanted intraperitoneally into streptozotocin (STZ)-induced diabetic mice, resulting in significantly lower levels of serum glucose compared to control mice. This study has characterized the immunological properties of CLECs and tested a potential therapeutic application in the treatment of a type 1 diabetes mouse model.

Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities

Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases.

Microfabrication of A Si Mesh Structure Depth Filter

The uTAS proposed the full incorporation of analytical procedures, which include sampling, sample preparation or pretreatment and actual analysis, into flowing systems. To incorporate a full uTAS, it is important to develop the individual microfluidic components. Microfilter is one of the microfluidic devices for sample preparation. This paper presents a fabrication method for a vertical silicon mesh structure depth filter as well as the application of white blood cell filtration. The results are also compared with another fabricated horizontal flow silicon filter.

TCR–like antibodies mediate complement and antibody-dependent cellular cytotoxicity against Epstein-Barr virus–transformed B lymphoblastoid cells expressing different HLA-A*02 microvariants

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV–transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.

Abstract B095: Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities

Introduction: Epstein-Barr virus (EBV) is a gamma herpesvirus that is found in the majority of the human population. Though commonly established as a life-long, asymptomatic infection, EBV has also been implicated in a number of human malignancies including Hodgkin9s and Burkitt9s lymphomas, nasopharyngeal carcinoma as well as post-transplant lymphoproliferative disease in immunosuppressed patients. While EBV displays restricted gene expression during different latency programs, the viral gene products that are frequently detected in EBV-associated tumors include Epstein-Barr virus Nuclear Antigen 1 (EBNA1), Latent Membrane Protein 1 (LMP1) and Latent Membrane Protein 2A (LMP2A). Our lab has previously described the generation and characterization of monoclonal antibodies with T cell receptor-like specificities (TCR-like mAbs) targeting EBV latent epitopes in association with HLA-A0201. These antibodies have been shown to bind to their targets with high specificities and affinities, and were capable of recognizing antigens displayed on human nasopharyngeal carcinoma biopsies, highlighting their immunotherapeutic potential for targeting EBV-associated tumors. Methods: EBV is particularly capable of transforming resting B cells into latently infected, actively proliferating B lymphoblastoid cell lines (BLCL) in vitro. As a proof-of-concept, we first generated EBV BLCLs from HLA-A0201+ human peripheral blood mononuclear cells (PBMCs) and compared the ability of our TCR-like mAbs to detect their endogenous targets before and after establishment of the cell lines. The xenograft model involving the inoculation of EBV BLCL into immunodeficient mice resembles B cell lymphoma that develops in post-transplant, immunosuppressed patients. Hence to test the ability of our TCR-like mAbs to target the BLCLs in vivo, we next inoculated PBMC-derived BLCLs into immunodeficient NSG mice and administered weekly dosage of TCR-like mAbs. Mice were assessed for changes in weight, survival as well as end-point organs analysis. Results: Here, we showed that endogenous EBV latent epitopes could be detected by our TCR-like mAbs on PBMC-derived BLCLs after EBV establishment of the cell line. Despite the inherent heterogeneities between different donors PBMC-derived BLCLs, several of the HLA-A0201+ BLCLs tested displayed similar binding activities with these TCR-like mAbs. In addition, BLCL-injected NSG mice that received weekly treatment of TCR-like mAbs displayed reduced tumor burden, splenomegaly and liver tumor spots in comparison to mice that received isotype antibody or PBS control. Importantly, BLCL-injected NSG mice that received the TCR-like mAb targeting the EBNA1 latent epitope exhibited delayed weight loss and survival advantage. Conclusion: We have utilized TCR-like mAbs with specificities against EBV latent epitopes expressed on HLA-A0201 and showed that these antibodies could recognize endogenous targets on EBV transformed, PBMC-derived BLCLs. Furthermore, weekly administration of TCR-like mAbs into BLCL xenograft NSG mice resulted in an overall reduction of tumor load and improved survival in one of the three described antibodies. Taken together, our data provide preliminary evidence for the therapeutic usage of antibodies with TCR-like specificities for the targeting EBV transformed BLCL in vivo. Citation Format: Junyun Lai, Wei Jian Tan, Chien Tei Too, Nalini Srinivasan, Lan Hiong Wong, Fatimah Mustafa, Soh Ha Chan, Jianzhu Chen, Paul Anthony MacAry. Targeting Epstein-Barr virus transformed B lymphoblastoid cells using antibodies with T cell receptor-like specificities. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B095.