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.

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.

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.

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 beta(1) 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.