(1/163) Induction of complement attack on human cells by Gal(alpha1,3)Gal xenoantigen expression as a gene therapy approach to cancer.

Galactose(alpha1,3)galactose on the surface of cells of non-primate organs is the major xenoantigen responsible for hyperacute rejection in xenotransplantation. The antigen is synthesised by (alpha1, 3)galactosyl transferase. Humans lack this enzyme and their serum contains high levels of pre-existing natural antibody which recognises the structure and activates complement. We have evaluated in vitro the potential for delivery of this enzyme to sensitise human cells to complement attack as a gene therapy approach to cancer. Retrovirus-mediated delivery of (alpha1,3)galactosyl transferase resulted in high level expression which led to serum-mediated lysis of five human cell targets, including endothelial and primary melanoma cells. Lysis was specific for those cells expressing the antigen in a mixed cell population. The mechanism of cell lysis mimicked that involved in hyperacute rejection: activation of the classical complement pathway by natural antibody specific for galactose(alpha1,3)galactose. The degree of lysis was determined by both the level of specific antibody and the expression of glycophosphatidylinositol-linked complement regulatory proteins. We conclude that expression of (alpha1,3)galactosyl transferase is a promising new therapeutic approach for cancer gene therapy, avoiding toxicity problems associated with application of prodrugs and with the potential to elicit further immunological responses.  (+info)

(2/163) The human antibody response to porcine xenoantigens is encoded by IGHV3-11 and IGHV3-74 IgVH germline progenitors.

Preformed and induced Ab responses present a major immunological barrier to the use of pig organs for human xenotransplantation. We generated IgM and IgG gene libraries established from lymphocytes of patients treated with a bioartificial liver (BAL) containing pig hepatocytes and used these libraries to identify IgVH genes that encode human Ab responses to pig xenoantigens. Genes encoded by the VH3 family are increased in expression in patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated, and the relative frequency of expression of genes used to encode the Ab response was determined at days 0, 10, and 21. Ig genes derived from the IGHV3-11 and IGHV3-74 germline progenitors increase in frequency post-BAL. The IGHV3-11 gene encodes 12% of VH3 cDNA clones expressed as IgM Abs at day 0 and 32.4-39.0% of cDNA clones encoding IgM Abs in two patients at day 10. IGHV3-11 and IGHV3-74 genes encoding IgM Abs in these patients are expressed without evidence of somatic mutation. By day 21, an isotype switch occurs and IGHV3-11 IgVH progenitors encode IgG Abs that demonstrate somatic mutation. We cloned these genes into a phagemid vector, expressed these clones as single-chain Abs, and demonstrated that the IGHV3-11 gene encodes Abs with the ability to bind to the gal alpha (1,3) gal epitope. Our results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline progenitors. IgM Abs are expressed in germline configuration and IgG Abs demonstrate somatic mutations by day 21.  (+info)

(3/163) The Th1/Th2 nature of concurrent immune responses to unrelated antigens can be independent.

We tested the independence hypothesis, namely that the Th1/Th2 nature of concurrent immune responses, generated in the same secondary lymphoid organ to non-cross-reacting Ags, can be independently determined. Some infectious agents and some adjuvants contain modulatory molecules that affect the Th1/Th2 nature of immune responses in a non-Ag-specific manner. We therefore excluded infectious agents as Ags and the use of adjuvants to generate immune responses. We first show that the dose of xenogeneic RBC administered i.v. determines the Th1/Th2 nature of the splenic immune response. Low doses generate a virtually exclusive Th1 response, whereas a higher dose induces either a mixed Th1/Th2 or a predominantly Th2 response, and stimulates the production of specific Abs. We immunized individual mice simultaneously with a low dose of one kind of xenogeneic RBC and with a higher dose of another non-cross-reacting xenogeneic RBC and assessed the Th1/Th2 nature of the immune responses generated in the spleen to each kind of RBC. The Th1/Th2 nature of the response to each RBC in doubly immunized mice was indistinguishable from that of the corresponding immune response in singly immunized mice. We discuss the significance of our findings for understanding immune class regulation, and the possible reasons why such independence is not always seen.  (+info)

(4/163) Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.  (+info)

(5/163) HLA-E and HLA-G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2-dependent and -independent pathways.

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab')2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.  (+info)

(6/163) Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection.

The transplantation of organs from other species into humans is considered to be a potential solution to the shortage of human donor organs. Organ transplantation from pig to human, however, results in hyperacute rejection, initiated by the binding of human natural antidonor antibody and complement. The major target antigen of this natural antibody is the terminal disaccharide Galalphal,3Gal, which is synthesized by Galbeta1,4GlcNAc alpha1,3-galactosyltransferase. Here we review our current knowledge of this key enzyme. A better understanding of structure, enzyme properties, and expression pattern of alpha1,3-galactosyltransferase has opened up several novel therapeutic approaches to prevent hyperacute vascular rejection. Cloning, and expression in vitro of the corresponding cDNA, has allowed to develop strategies to induce immune tolerance, and deplete or neutralize the natural xenoreactive antibody. Elucidation of the genomic structure has led to the production of transgenic animals that are lacking alpha1,3-galactosyltransferase activity. A detailed knowledge of the enzyme properties has formed the basis of approaches to modify donor organ glycosylation by intracellular competition. Study of the expression pattern of alpha1,3-galactosyltransferase has helped to understand the mechanism of hyperacute rejection in discordant xenotransplantation, and that of complement-mediated, natural immunity against interspecies transmission of retroviruses.  (+info)

(7/163) Taking lessons from dendritic cells: multiple xenogeneic ligands for leukocyte integrins have the potential to stimulate anti-tumor immunity.

Expression of large numbers of different costimulatory integrin ligands (CILs) attributes dendritic cells with an ability to induce primary anti-tumor immune responses. Here, we show that optimized gene transfer of the xenogeneic (human) CILs VCAM-1, MAdCAM-1 and ICAM-1 causes rapid and complete rejection of established mouse EL-4 tumors, and generates prolonged systemic anti-tumor immunity; whereas human E-cadherin weakly slows tumor growth. In each case the immune response was mediated by CD8+ T cells and NK cells, accompanied by augmented tumor-specific cytolytic T cell (CTL) activity involving both the perforin and Fas-ligand pathways. Adoptive transfer of splenocytes from cured mice rapidly cleared established tumors in recipients. The mechanism for CIL-mediated immunity is unknown, but may involve CTL-facilitated tumor lysis, since CTLs were generally twice as efficient at killing CIL-transfected tumor cells than parental tumor cells. Optimized CIL-based gene therapy may provide an approach to complement or replace conventional DC adoptive cell therapy for suppressing tumor growth.  (+info)

(8/163) Naturally developing memory T cell xenoreactivity to swine antigens in human peripheral blood lymphocytes.

Naturally developing xenospecific Abs are well-documented barriers to xenograft transplantation in humans, but whether analogous xenoreactive T cell immunity develops is not known. We used an enzyme-linked immunospot assay to determine the frequency and cytokine profiles of xenoreactive PBLs from a panel of human volunteers. Because naive T cells produce only IL-2 in short term culture, IFN-gamma production by this approach is a measure of a memory immune response. Stimulation of human PBLs or purified T lymphocytes with stimulator cells from inbred swine revealed a high frequency of IFN-gamma producers with 5-fold fewer IL-2 producers. In contrast, lymphocytes obtained from neonatal umbilical cord blood contained swine-specific IL-2 producers but few IFN-gamma producers, which is what one would expect to find with a naive phenotype. Moreover, PBLs from adults with a history of abstention from pork consumption responded to swine cells with a significantly lower frequency of IFN-gamma producers than PBLs from adults with unrestricted diets did, suggesting that pork consumption may result in priming of swine-specific T cell immunity. Our findings provide the first evidence for naturally occurring xenospecific T cell immunity in humans. The detected strength of this memory response suggests that it will present a formidable barrier to transplantation of swine organs.  (+info)