HBV-related hepatocarcinogenesis: the role of signalling pathways and innovative ex vivo research models.

BACKGROUND: Hepatitis B virus (HBV) is the leading cause of liver cancer, but the mechanisms by which HBV causes liver cancer are poorly understood and chemotherapeutic strategies to cure liver cancer are not available. A better understanding of how HBV requisitions cellular components in the liver will identify novel therapeutic targets for HBV associated hepatocellular carcinoma (HCC). MAIN BODY: The development of HCC involves deregulation in several cellular signalling pathways including Wnt/FZD/ß-catenin, PI3K/Akt/mTOR, IRS1/IGF, and Ras/Raf/MAPK. HBV is known to dysregulate several hepatocyte pathways and cell cycle regulation resulting in HCC development. A number of these HBV induced changes are also mediated through the Wnt/FZD/ß-catenin pathway. The lack of a suitable human liver model for the study of HBV has hampered research into understanding pathogenesis of HBV. Primary human hepatocytes provide one option; however, these cells are prone to losing their hepatic functionality and their ability to support HBV replication. Another approach involves induced-pluripotent stem (iPS) cell-derived hepatocytes. However, iPS technology relies on retroviruses or lentiviruses for effective gene delivery and pose the risk of activating a range of oncogenes. Liver organoids developed from patient-derived liver tissues provide a significant advance in HCC research. Liver organoids retain the characteristics of their original tissue, undergo unlimited expansion, can be differentiated into mature hepatocytes and are susceptible to natural infection with HBV. CONCLUSION: By utilizing new ex vivo techniques like liver organoids it will become possible to develop improved and personalized therapeutic approaches that will improve HCC outcomes and potentially lead to a cure for HBV.

Antigen-specific CD4+ CD25+ T cells induced by locally expressed ICOS-Ig: the role of Foxp3, Perforin, Granzyme B and IL-10 - an experimental study.

We have previously reported that ICOS-Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4+ CD25+ Foxp3+ T cells and specific xenograft prolongation. In the present study we isolated and purified CD4+ CD25+ T cells from ICOS-Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4+ CD25+ T cells isolated from xenografts secreting ICOS-Ig were analysed by flow cytometry and gene expression by real-time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre-existing Foxp3+ Treg, IL-10, perforin and granzyme B. CD4+ CD25+ Foxp3+ T cells isolated from xenografts secreting ICOS-Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.

Adenovirus vector produced Zika virus-like particles induce a long-lived neutralising antibody response in mice.

Countermeasures against Zika virus (ZIKV) epidemics are urgently needed. In this study we generated a ZIKV virus-like particle (VLP) based vaccine candidate and assessed the immunogenicity of these particles in mice. The ZIKV-VLPs were morphologically similar to ZIKV by electron microscopy and were recognized by anti-Flavivirus neutralising antibodies. We observed that a single dose of unadjuvanted ZIKV-VLPs, or inactivated ZIKV, generated an immune response that lasted over 6 months, but did not neutralize ZIKV infection of cells in vitro. However, when we co-administered the ZIKV VLPs with either Aluminium hydroxide (Alhydrogel®; Alum), AddaVax or Pam2Cys we observed that Alum was the most effective in a single dose regime, since it not only produced antibodies that neutralized the virus, but also generated a greater number of antigen-specific memory B cells. We additionally observed that the generation of the neutralising antibodies persisted for up to 6 months. Our results suggest that a single dose ZIKV VLPs could be a suitable single dose vaccine candidate for use in outbreak settings.

Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates.

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.

Foxp3+ T cells in peripheral blood of renal transplant recipients and clinical correlations.

AIM: Immunophenotype peripheral blood T cells from renal transplant recipients (RTR) using cellular markers of regulatory T cells (Tregs) and flow cytometry, including Foxp3, and correlate these findings with clinical parameters. METHODS: Expression of phenotypic markers of Tregs was assessed by flow cytometric analysis of peripheral blood lymphocytes (PBL) from (i) RTR (n = 95); (ii) patients with end-stage renal failure (ESRF) awaiting transplantation (n = 17); and (iii) normal healthy controls (n = 18). RESULTS: The percentage of CD4(+) CD25(+) Foxp3(+) cells within the CD4(+) cell population did not significantly alter at different time points post-transplant. However, the percentage of CD4(+) CD25(+) Foxp3(+) cells within the CD4(+) population was significantly lower in RTR compared with patients with ESRF. In contrast, RTR and ESRF had a similar percentage of CD4(+) CD25(+) cells expressing Foxp3. Multivariate analysis of PBL and clinical parameters demonstrated (i) a positive linear relationship between the percentage CD4(+) CD25(+) cells expressing Foxp3 and estimated glomerular filtration rate and (ii) a higher percentage of CD4(+) CD25(+) cells in the CD4(+) cell population in patients with malignancy (the majority were skin cancers). Malignancy also correlated strongly with time post-transplant and age of the RTR. CONCLUSION: Immune monitoring of the PBL phenotype in RTR using CD4, CD25 and Foxp3 may stratify RTR and predict graft outcome and function, and risk of complications from immunosuppression. Longitudinal and functional studies of Tregs are essential to extend the findings of the present study.

Inducible Co-Stimulator (ICOS) in transplantation: A review.

Prevention of T cell activation is one of the goals of successful organ and tissue transplantation. Blockade of T cell co-stimulation, particularly of the CD28:B7 interaction, has been shown to prolong graft survival. Inducible Co-Stimulator (ICOS) is the third member of the B7 family and here we review the literature on ICOS, its receptor (B7RP-1), and blockade of this pathway in transplant models. ICOS:B7RP-1 are a single receptor:ligand pair with a loss of function of either being implicated in some autoimmune diseases. ICOS has multiple functions, related to its constitutive expression on B cells and activated T cells. In in vitro transplant models, ICOS:B7RP-1 blockade has produced mixed results as to its ability to modulate lymphocyte proliferation. Several in vivo transplant models demonstrate varying degrees of success in prolonging graft survival. Timing and dose of treatment appear important, and combination with other immunosuppressive treatments may also be of benefit. As ICOS has multiple functions, it may be that the observed variable results are due to inadvertent inactivation of graft protective functions. If these barriers can be overcome, ICOS:B7RP-1 blockade could provide an important target for future immunosuppression regimens.

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells.

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.

Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.

Investigating virus-host cell interactions: Comparative binding forces between hepatitis C virus-like particles and host cell receptors in 2D and 3D cell culture models.

Cell cultures have been successfully used to study hepatitis C virus (HCV) for many years. However, most work has been done using traditional, 2-dimensional (2D) cell cultures (cells grown as a monolayer in growth flasks or dishes). Studies have shown that when cells are grown suspended in an extra-cellular-matrix-like material, they develop into spherical, 'organoid' arrangements of cells (3D growth) that display distinct differences in morphological and functional characteristics compared to 2D cell cultures. In liver organoids, one key difference is the development of clearly differentiated apical and basolateral surfaces separated and maintained by cellular tight junctions. This phenomenon, termed polarity, is vital to normal barrier function of hepatocytes in vivo. It has also been shown that viruses, and virus-like particles, interact very differently with cells derived from 2D as compared to 3D cell cultures, bringing into question the usefulness of 2D cell cultures to study virus-host cell interactions. Here, we investigate differences in cellular architecture as a function of cell culture system, using confocal scanning laser microscopy, and determine differences in binding interactions between HCV virus-like particles (VLPs) and their cognate receptors in the different cell culture systems using atomic force microscopy (AFM). We generated organoid cultures that were polarized, as determined by localization of key apical and basolateral markers. We found that, while uptake of HCV VLPs by both 2D and 3D Huh7 cells was observed by flow cytometry, binding interactions between HCV VLPs and cells were measurable by AFM only on polarized cells. The work presented here adds to the growing body of research suggesting that polarized cell systems are more suitable for the study of HCV infection and dynamics than non-polarized systems.

Dendritic cells expressing soluble CTLA4Ig prolong antigen-specific skin graft survival.

Dendritic cells (DCs) and CTLA4Ig are important in regulating T-cell responses and therefore represent potential therapeutic tools in transplantation. In this study, CTLA4Ig was expressed in a C57BL/6 murine DC line (JAWS II) by lentiviral transduction and these cells were used to examine T-cell immunomodulatory effects in vitro and in vivo. A lower stimulation index to C57BL/6 was observed with splenocytes from BALB/c mice primed with JAWS II-CTLA4Ig compared with control JAWS II-green fluorescent protein (JAWS II-GFP). Mice primed with JAWS II-CTLA4Ig cells had significantly prolonged antigen-specific C57BL/6 skin graft survival compared with either JAWS II-GFP-primed or naïve mice (median 13, 11 and 11 days, respectively, P=0.0001). Furthermore, JAWS II-CTLA4Ig-primed mice that had been previously transplanted with skin grafts were re-transplanted with skin grafts 6 months later without immune manipulation. These mice demonstrated specific prolongation of second-set rejection responses, indicating systemic immune modulation induced by genetically modified DC. The mechanism was not due to expression of indoleamine 2,3-dioxygenase or induction of circulating regulatory T cells as assessed by flow cytometry of the peripheral blood lymphocytes. This potent effect demonstrated with skin grafts and second-set responses highlights the potential use of this strategy for transplantation more generally.

The cytoplasmic and transmembrane domains of secretor type alpha1,2fucosyltransferase confer atypical cellular localisation.

Carbohydrate structures influence many aspects of cell biology. Manipulating the glycosyltransferase enzymes, that sequentially add carbohydrate moieties to proteins and lipids as they pass through the Golgi and secretory pathway, can alter these carbohydrate epitopes. We previously demonstrated that the eight amino acid cytoplasmic tail of alpha1,2fucosyltransferase (FT) contained a sequence for Golgi localisation. In this study, we examined the localisation of the closely related secretor type alpha1,2fucosyltransferase (Sec) which has a smaller, yet apparently unrelated, five amino acid cytoplasmic tail. In contrast to the Golgi localisation of FT, Sec displayed atypical cytoplasmic vesicular-like staining. However, replacing just the five amino acid tail of Sec with FT was sufficient to relocalise the enzyme to a perinuclear region with Golgi-like staining. The biological significance of this relocalisation was this chimaeric enzyme was more effective than FT at competing for N-Acetyl-lactosamine and thus was superior in reducing expression of the Galalpha(1,3)Gal xenoepitope.

Probing and pressing surfaces of hepatitis C virus-like particles.

Hepatitis C virus-like particles (VLPs) are being developed as a quadrivalent vaccine candidate, eliciting both humoral and cellular immune responses in animal trials. Biophysical, biomechanical and biochemical properties are important for virus and VLP interactions with host cells and recognition by the immune system. Atomic force microscopy (AFM) is a powerful tool for visualizing surface topographies of cells, bionanoparticles and biomolecules, and for determining biophysical and biomechanical attributes such as size and elasticity. In this work, AFM was used to define morphological and nanomechanical properties of VLPs representing four common genotypes of hepatitis C virus. Significant differences in size of the VLPs were observed, and particles demonstrated a wide range of elasticity. Ordered packing of the core and potentially envelope glycoproteins was observed on the surfaces of the VLPs, but detailed structural characterization was hindered due to intrinsic dynamic fluctuations or AFM probe-induced damage of the VLPs. All VLPs were shown to be glycosylated in a manner similar to native viral particles. Together, the results presented in this study further our understanding of the nanostructure of hepatitis C VLPs, and should influence their uptake as viable vaccine candidates.

A Hepatitis C Virus DNA Vaccine Encoding a Secreted, Oligomerized Form of Envelope Proteins Is Highly Immunogenic and Elicits Neutralizing Antibodies in Vaccinated Mice.

Hepatitis C virus (HCV) persistently infects approximately 71 million people globally. To prevent infection a vaccine which elicits neutralizing antibodies against the virus envelope proteins (E1/E2) which are required for entry into host cells is desirable. DNA vaccines are cost-effective to manufacture globally and despite recent landmark studies highlighting the therapeutic efficacy of DNA vaccines in humans against cervical cancer, DNA vaccines encoding E1/E2 developed thus far are poorly immunogenic. We now report a novel and highly immunogenic DNA vaccination strategy that incorporates secreted E1 and E2 (sE1 and sE2) into oligomers by fusion with the oligomerization domain of the C4b-binding protein, IMX313P. The FDA approved plasmid, pVax, was used to encode sE1, sE2, or sE1E2 with or without IMX313P, and intradermal prime-boost vaccination studies in BALB/c mice showed that vaccines encoding IMX313P were the most effective in eliciting humoral and cell-mediated immunity against the envelope proteins. Further boosting with recombinant E1E2 proteins but not DNA nor virus-like particles (VLPs) expressing E1E2 increased the immunogenicity of the DNA prime-boost regimen. Nevertheless, the antibodies generated by the homologous DNA prime-boost vaccinations more effectively inhibited the binding of VLPs to target cells and neutralized transduction with HCV pseudoparticles (HCVpp) derived from different genotypes including genotypes 1, 2, 3, 4, 5, and 6. This report provides the first evidence that IMX313P can be used as an adjuvant for E1/E2-based DNA vaccines and represents a translatable approach for the development of a HCV DNA vaccine.

Porcine cells express more than one functional ligand for the human lymphocyte activating receptor NKG2D.

BACKGROUND: Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically-modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti-pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8(+) and gammadelta T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co-stimulation of T cells. METHODS: Chinese hamster ovary P (CHOP) cells were transfected with human NKG2D and used in cell-cell binding studies with porcine epithelial, and endothelial cell lines. Soluble recombinant NKG2D-Fc was used to stain various porcine cells and tissues to indicate ligand expression. Porcine cells were used as targets in cytotoxicity assays with the HuNK cell lines NKL and YT, with and without enzymatic removal of pULBP1 and antibody blockade of NKG2D signaling. RESULTS AND CONCLUSIONS: In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D-dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.

Local expression of IDO, either alone or in combination with CD40Ig, IL10 or CTLA4Ig, inhibits indirect xenorejection responses.

BACKGROUND: To overcome cell-mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3-dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo. METHODS: The PIEC stable lines expressing IDO, CD40Ig, and IL10 as single molecules were generated. In addition, PIEC lines expressing IDO with either CD40Ig, IL10 or CTLA4Ig were generated to produce cell lines expressing two molecules. BALB/c mice were primed with wild type PIEC, followed by harvesting splenocytes used as responder cells and PIEC expressing immunomodulatory molecules as stimulators, in proliferation and cytokine assays. In vivo effects of modified PIEC were examined by transplantation of PIEC lines expressing the immunomodulatory molecules under the renal capsule of naïve mice. PIEC grafts were harvested for histological evaluation at days 7 and 14. RESULTS: Proliferation of primed BALB/c splenocytes was inhibited most significantly by IDO compared with control cells (49%, P = 0.02). In addition both Th1 (interferon-gamma) and Th2 (IL4 and IL10) cytokines were markedly inhibited in vitro by IDO expression. IL10 expressing cells did not inhibit proliferation as potently (37%, P = 0.03) whilst CD40Ig lead to an increase in proliferative responses (59%, P = 0.02). Co-expression of CD40Ig, IL10, and CTLA4Ig with IDO resulted in further modest reductions in proliferation compared with IDO expression alone. When transplanted under the renal capsule of BALB/c mice, those grafts expressing IDO demonstrated significantly lower levels of lymphocyte infiltration at days 7 and 14 than control grafts and those expressing CD40Ig, CTLA4Ig or IL10 alone. Grafts co-expressing IDO and a second molecule were no better protected than those expressing IDO alone. Graft cell viability (PIECs) was reduced in some IDO expressing grafts suggesting high levels of IDO expression may inhibit PIEC viability, however, grafts co-expressing IDO-CTLA4Ig and IDO-IL10 were not affected in this way. CONCLUSION: Indoleamine 2,3-dioxygenase appears to be a potent molecule for protecting xenografts from cell-mediated rejection responses activated via the indirect pathway. Co-expression of IDO with both CTLA4Ig and IL10 warrants further investigation. Overall these findings support pursuing further studies, in larger animal models, to determine whether increased IDO activity within the graft itself can attenuate xenorejection responses.

Gamma-secretase-dependent proteolysis of CD44 promotes neoplastic transformation of rat fibroblastic cells.

The metalloprotease-dependent extracellular domain cleavage of the adhesion molecule CD44 is frequently observed in human tumors and is thought to promote metastasis. This cleavage is followed by gamma-secretase-dependent release of CD44 intracellular domain (CD44-ICD), which exhibits nuclear signaling activity. Using a reversible Ret-dependent oncogenic conversion model and a restricted proteomic approach, we identified a positive correlation between the neoplastic transformation of Rat-1 cells and the expression of standard CD44. In these transformed cells, CD44 was found to undergo a sequential metalloprotease and gamma-secretase cleavage, resulting in an increase in expression of CD44-ICD. We showed that this proteolytic fragment possesses a transforming activity. In support of this role, a significant and specific reduction in Ret-induced transformation of Rat-1 cells was observed following drug-mediated inhibition of gamma-secretase. Taken together, these findings suggest that the shedding of CD44 may not only modulate metastasis but also affects earlier events in tumorigenesis through the release of CD44-ICD.

Antibody Responses to a Quadrivalent Hepatitis C Viral-Like Particle Vaccine Adjuvanted with Toll-Like Receptor 2 Agonists.

The development of an effective preventative hepatitis C virus (HCV) vaccine will reside, in part, in its ability to elicit neutralizing antibodies (NAbs). We previously reported a genotype 1a HCV virus like particle (VLP) vaccine that produced HCV specific NAb and T cell responses that were substantially enhanced by Toll-like receptor 2 (TLR2) agonists. We have now produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine and tested the ability of two TLR2 agonists, R4Pam2Cys and E8Pam2Cys, to stimulate the production of NAb. We now show that our vaccine with R4Pam2Cys or E8Pam2Cys produces strong antibody and NAb responses in vaccinated mice after just two doses. Total antibody titers were higher in mice inoculated with vaccine plus E8Pam2Cys compared to HCV VLPs alone. However, the TLR2 agonists did not result in stronger NAb responses compared to vaccine without adjuvant. Such a vaccine could provide a substantial addition to the overall goal to eliminate HCV.

The incidence of HBV and HCV infection in Australian travelers to Asia.

We analyzed paired pre- and post-travel sera in a cohort of Australian travelers to Asia and demonstrated the acquisition of hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. The incidence density in nonimmune travelers for HCV infection was calculated as 1.8 infections per 10,000 traveler-days and for HBV infection 2.19 per 10,000 traveler-days.

Studies on carbohydrate xenoantigens.

Naturally occurring and elicited anti-carbohydrate antibodies play a major role in immune responses to xenografts. The original obstacles associated with the Gal antigen have been largely resolved by the generation of knockout pigs. In contrast, much less is known about the nature and role of non-Gal carbohydrate antigens and the antibodies recognizing these. These antibodies can be identified and characterized by enzyme-linked immunosorbent assay. Furthermore, the biological significance of the non-Gal antigen(s) can be determined by expression of the relevant glycosyltransferase(s) by transfection and analyzed by antibody and/or lectin binding.

Prolonged xenograft survival induced by inducible costimulator-Ig is associated with increased forkhead box P3(+) cells.

BACKGROUND: Blockade of the inducible costimulator (ICOS) pathway has been shown to prolong allograft survival; however, its utility in xenotransplantation is unknown. We hypothesize that local expression of ICOS-Ig by the xenograft will suppress the T-cell response resulting in significant prolonged graft survival. METHODS: Pig iliac artery endothelial cells (PIEC) secreting ICOS-Ig were generated and examined for the following: (1) inhibition of allogeneic and xenogeneic proliferation of primed T cells in vitro and (2) prolongation of xenograft survival in vivo. Grafts were examined for Tregs by flow cytometry and cytokine levels determined by quantitative reverse-transcriptase polymerase chain reaction. RESULTS: Soluble ICOS-Ig markedly decreased allogeneic and xenogeneic primed T-cell proliferation in a dose-dependent manner. PIEC-ICOS-Ig grafts were significantly prolonged compared with wild-type grafts (median survival, 34 and 12 days, respectively) with 20% of PIEC-ICOS-Ig grafts surviving more than 170 days. Histological examination showed a perigraft cellular accumulation of Forkhead box P3 (Foxp3(+)) cells in the PIEC-ICOS-Ig grafts, these were also shown to be CD3(+)CD4(+)CD25(+). Survival of wild-type PIEC grafts in a recipient simultaneously transplanted with PIEC-ICOS-Ig were also prolonged, with a similar accumulation of Foxp3(+) cells at the periphery of the graft demonstrating ICOS-Ig induces systemic graft prolongation. However, this prolongation was specific for the priming xenograft. Intragraft cytokine analysis showed an increase in interleukin-10 levels, suggesting a potential role in induction/function of CD4(+)CD25(+)Foxp3(+) cells. CONCLUSIONS: This study demonstrates prolonged xenograft survival by local expression of ICOS-Ig, we propose that the accumulation of CD4(+)CD25(+)Foxp3(+) cells at the periphery of the graft and secretion of interleukin-10 is responsible for this novel observation.