Suchitra Sumitran-Holgersson

The polymorphic major histocompatibility complex class I–related chain A (MICA) antigen is being increasingly recognized as a potential target molecule for immune cells during allograft rejection. Here we studied whether MICA is a target antigen for antibodies in liver transplant patients. Eighty-four patients were investigated for the presence of MICA antibodies before and after liver transplantation with MICA-transfected cells and flow cytometry. MICA typing was performed by polymerase chain reaction. Expression of MICA in liver cells was determined by reverse-transcription polymerase chain reaction, Western blotting, and flow cytometry. Liver biopsy specimens from liver transplant patients were examined for MICA expression. A total of 22 of 84 (26%) patients had MICA antibodies either pre-transplant (8/84, 9.5%) or post-transplant (14/84, 17%). No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibody versus those without MICA antibody (29/62, 47% P = not significant). We found weak messenger RNA expression for MICA in liver cells but no protein or cell surface expression. In addition, no MICA expression in liver biopsy sections from liver transplant patients was observed at any time point, including rejections. Thus, our preliminary results demonstrate no causal relationship between the presence of MICA antibodies and liver allograft rejections. Therefore, it is likely that MICA may not be an important target antigen during liver allograft rejections.

Background. Reconstitution of hematopoiesis after hematopoietic stem cell transplantation (HSCT) occurs through a reservoir of donor CD34⁺ HSC. CD34⁺/VEGFR-2⁺ is a primitive, quiescent subpopulation of HSC known to generate hematopoietic or endothelial progeny in vitro and in vivo. We hypothesized that donor-specific antibodies to the CD34⁺/VEGFR-2⁺ stem cells may be associated with rejection after HSCT. Methods. We studied 30 patients without and 11 with rejections after HSCT and 20 nontransplanted healthy individuals. Ninety-three sera taken pre and posttransplantation from patients receiving HSCT were studied for the presence of donor CD34⁺/VEGFR-2⁺ cell-specific antibodies. Results. We provide evidence that significantly higher numbers of patients with rejections 9/11 (81%), whereas 1/30 (3%) (P=0.001) without rejections had antibodies against donor CD34⁺/VEGFR-2⁺ cells, but not CD34⁻/VEGFR-2⁻ cells. In eight transplantations, antibodies against donor CD34⁺/VEGFR-2⁺ cells were detected already before transplantation. Purified IgG fractions from patients with rejections but not controls significantly decreased the ability of these cells to form hematopoietic and endothelial colonies. In multivariate analysis, antibodies against CD34⁺/VEGFR-2⁺ cells proved to be the most significant risk factor for rejection. Conclusions. These novel findings document a high rate of rejections in patients with donor-specific antibodies to CD34⁺/VEGFR-2⁺ HSC. These antibodies may significantly and commonly contribute to HSCT rejections.

Purpose of review The clinical importance of HLA-specific antibodies for organ allograft outcome is well established. Antibody-mediated rejections of HLA-identical kidney transplants and rejections in the absence of detectable HLA antibodies, however, indicate that non-HLA antibodies also play a significant role. Recent findings Non-HLA antigens, such as the polymorphic major histocompatibility complex class I-related chain A, expressed on endothelial cells, have been implicated in the pathogenesis of hyperacute, acute, and chronic organ allograft rejections. The use of donor endothelial cells as targets in pretransplant and posttransplant cross-matches will facilitate detection and specificity determination of other clinically relevant non-HLA antibodies. Non-swine leukocyte antigen–antibody responses are also receiving increasing interest as important mediators of acute vascular rejection of porcine organ xenografts; acute vascular rejection is the next big hurdle preventing clinical xenotransplantation. Summary Non-HLA antibody responses are receiving increasing interest in acute and chronic rejection, and specificity, affinity, and pathogenicity need to be investigated to estimate their contribution. This review summarizes past and current knowledge of the clinical importance and specificities of non-HLA antibodies and mechanisms by which these antibodies may contribute to graft destruction in organ allotransplant and xenotransplant recipients.

Background: The liver may have a role in peripheral tolerance, by serving as a site for trapping, apoptosis and phagocytosis of activated T cells. It is not known which hepatic cells are involved in these processes. It was hypothesised that liver sinusoidal endothelial cells (LSEC) which are strategically placed for participation in the regulation of sinusoidal blood flow, and express markers involved in recognition, sequestration and apoptosis, may contribute to peripheral tolerance by inducing apoptosis of activated T cells. Methods: By using immunoassays and western blot analysis, the fate of activated T cells when incubated with human LSEC isolated from normal healthy livers was investigated. Results: Evidence that activated (approximately 30%) but not non-activated T cells undergo apoptosis on incubation with human LSEC in mixed cell cultures is provided. No difference in the results was observed when unstimulated and cytokine-stimulated LSEC were used. T cell–LSEC contact is required for induction of apoptosis. Apoptosis induced by LSEC was associated with caspase 8 and 3 activity and strong expression of the proapoptotic molecule Bak. Transforming growth factor β (TGFβ) produced constitutively by LSEC is partly responsible for the caspase-induced apoptosis, as neutralising antibodies to TGFβ markedly attenuated apoptosis, up regulated the antiapoptotic molecule Bcl-2 and partially blocked caspase-3 activity. Conclusion: These findings broaden the potential role of LSEC in immune tolerance and homeostasis of the immune system. This study may provide insight for exploring the mechanisms of immune tolerance by liver allografts, immune escape by some liver pathogens including hepatitis C and pathogenesis of liver diseases.

Background: Liver diseases include a wide spectrum of both acute and chronic conditions which are associated with significant morbidity and mortality worldwide. Hepatocyte transplantation has therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue. Generation of hepatocytes in vitro from adult or fetal liver cell progenitors or, alternatively, identification of a progenitor population which in vivo can generate mature liver cells could solve this problem. Methods: CD117+/CD34+/Lin− human fetal liver cells were isolated by magnetic cell sorting and expanded in culture. Both freshly isolated and in vitro expanded cells in various passages were studied for their ability to be functional in hepatic parenchyma following d -galactosamine (GalN) induced injury in nude C57 black mice. Results: Freshly isolated and in vitro expanded CD117+/CD34+/Lin− cells, when transplanted intrasplenically into GalN treated mice, morphologically and functionally differentiated into hepatocytes and cholangiocytes. Human specific albumin, α fetoprotein, cytokeratin 19, and antitrypsin mRNA were expressed in mouse liver. In addition, the human progenitor cells expressed glucose-6-phosphatase, glycogen, albumin, gamma glutamyl transpeptidase, and dipeptidyl peptidase IV after transplantation. Expanded cells in various passages maintained their capacity to differentiate into functional liver cells. Conclusions: Fetal liver CD117+/CD34+/Lin− progenitors and their progeny proliferated in vitro and also functionally differentiated into mature hepatic cells in an acute liver injury model. Successful in vitro expansion of liver progenitor cells provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.

Background— Receptor tyrosine kinases that include vascular endothelial growth factor (VEGFR)-1, VEGFR-2, and Tie-2 regulate cardiovascular development and physiological and pathological angiogenesis. We were interested in the phenotypic and functional characterization of peripheral blood cells expressing these receptors and their therapeutic potential in vascular injury. Methods and Results— VEGFR-1 ⁺ , VEGFR-2 ⁺ , and Tie-2 ⁺ cells constituted ≈3.0±0.2%, 0.8±0.5%, and 2.0±0.3%, respectively, of the total population of mononuclear cells in blood. Phenotypic analysis demonstrated that all 3 cell populations mainly expressed markers of monocytic/macrophage lineage. Only VEGFR-2 ⁺ and Tie-2 ⁺ cells phenotypically, morphologically, and functionally differentiated to endothelial cells after culture, whereas VEGFR-1 ⁺ cells did not. None of the cell types proliferated in vitro. Only freshly isolated VEGFR-2 ⁺ or Tie-2 ⁺ cells but not VEGFR-2 ⁻ or Tie-2 ⁻ cell populations significantly contributed to efficient endothelialization of balloon-injured femoral arteries of nude mice. Furthermore, these cells also differentiated into α-actin–positive smooth muscle cells. Administration of bromodeoxyuridine to animals transplanted with human endothelial progenitor cells showed that VEGFR-2 ⁺ and Tie-2 ⁺ cells proliferated in vivo. Conclusions— These data demonstrate that expression of VEGFR-2 and/or Tie-2 on peripheral blood cells defines functionally competent cell populations that proliferate in vivo and that contribute to reendothelialization. These findings may have implications for a cell-based approach in vascular diseases.

Liver sinusoidal endothelial cells (LSECs) may be implicated in the induction of liver allograft rejections. We studied the clinical consequences of LSEC-reactive antibodies and their functional capacity in modulating T-cell responses during acute rejections. Pre- and posttransplant sera and T lymphocytes from 95 liver transplant patients were used in this study. LSECs were isolated from normal healthy liver. Binding of antibodies to LSECs was detected using flow cytometry. To study whether LSEC antibodies facilitated cell-mediated immunity, a mixed cell culture (MCC) assay was used. Cytokines in the supernatants of MCC were measured by enzyme-linked immunosorbent assay. Liver biopsy sections were stained to detect the deposition of immunoglobulins in LSECs during rejections. The 2-year patient survival was 86.3%. A significantly higher number of patients with rejections had LSEC antibodies (35/50; 70%) than those without rejections (8/45; 18%) (P < .0001). Purified fractions of LSEC antibodies induced the expression of the costimulatory molecule CD86 on LSECs. A significantly higher number of patients with LSEC antibodies and rejections had an increased proliferation of T cells and markedly decreased levels of transforming growth factor β (TGF-β) in the MCC than those without antibodies and rejections (P < .0001, P < .0001, respectively). Deposition of antibodies in LSECs during rejection episodes was observed in the biopsies of patients with LSEC antibodies but not in those without LSEC antibodies. In conclusion, antibodies to LSECs may facilitate acute liver allograft rejections by down-regulating the immune modulating cytokine TGF-β and thus up-regulating alloreactive T-cell proliferation. (Hepatology 2004;40:1211-1221.)