Activated CD4+ T Cells and Highly Differentiated Alloreactive CD4+ T Cells Distinguish Operationally Tolerant Liver Transplantation Recipients.

Spontaneous operational tolerance to the allograft develops in a proportion of liver transplantation (LT) recipients weaned off immunosuppressive (IS) drugs. Several studies have investigated whether peripheral blood circulating T cells could play a role in the development or identify operational tolerance, but never characterized alloreactive T cells in detail due to the lack of a marker for these T cells. In this study, we comprehensively investigated phenotypic and functional characteristics of alloreactive circulating T cell subsets in tolerant LT recipients (n = 15) using multiparameter flow cytometry and compared these with LT recipients on IS drugs (n = 23) and healthy individuals (n = 16). Activation-induced CD137 was used as a marker for alloreactive T cells upon allogenic stimulation. We found that central and effector memory CD4+ T cells were hyporesponsive against donor and third-party splenocyte stimulation in tolerant LT recipients, whereas an overall hyperresponsiveness was observed in alloreactive terminally differentiated effector memory CD4+ T cells. In addition, elevated percentages of circulating activated T helper cells were observed in these recipients. Lastly, tolerant and control LT recipients did not differ in donor-specific antibody formation. In conclusion, a combination of circulating hyperresponsive highly differentiated alloreactive CD4+ T cells and circulating activated T helper cells could discriminate tolerant recipients from a larger group of LT recipients.

Donor-specific anti-HLA antibodies are not associated with nonanastomotic biliary strictures but both are independent risk factors for graft loss after liver transplantation.

Donor-specific alloantibodies (DSA) have been associated with rejection and shorter graft survival after orthotopic liver transplantation (OLT). We examined the role of DSA in nonanastomotic biliary strictures (NAS) after OLT. Patients receiving first OLT who developed NAS (n = 68) and a control group without NAS (n = 83), with pre-OLT and 12 months post-OLT serum samples, were included. DSA were specified using the Luminex single antigen test. Risk factors for NAS and graft survival were analyzed. The presence of preformed DSA was not significantly different between patients with NAS and controls (P = .89). After 12 months, 26.5% of NAS patients and 16.9% of controls had generated de novo DSA (P = .15). Neither de novo class I DSA nor de novo class II DSA were associated with NAS. De novo DSA generally developed after the diagnosis of NAS. Time-dependent regression analysis identified both NAS (aHR 8.05, CI 3.28 - 19.77, P < .01) and de novo class II DSA (aHR 2.84, CI 1.38 - 5.82, P < .01) as independent risk factors for graft loss. Preformed or de novo DSA were not associated with the development of NAS. However, NAS as well as de novo class II DSA were independent risk factors for graft loss after OLT.

Immunogenicity of human embryonic stem cell-derived beta cells.

AIMS/HYPOTHESIS: To overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells. METHODS: We comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice. RESULTS: We found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells. CONCLUSIONS/INTERPRETATION: hESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.

Innate and adaptive immunity to human beta cell lines: implications for beta cell therapy.

AIMS/HYPOTHESIS: Genetically engineered human beta cell lines provide a novel source of human beta cells to study metabolism, pharmacology and beta cell replacement therapy. Since the immune system is essentially involved in beta cell destruction in type 1 diabetes and after beta cell transplantation, we investigated the interaction of human beta cell lineswith the immune system to resolve their potential for immune intervention protocol studies. METHODS: Human pancreatic beta cell lines (EndoC-ßH1 and ECi50) generated by targeted oncogenesis in fetal pancreas were assessed for viability after innate and adaptive immune challenges. Beta cell lines were pre-conditioned with T helper type 1 (Th1) cytokines or high glucose to mimic inflammatory and hyperglycaemia-stressed conditions. Beta cells were then co-cultured with auto- and alloreactive cytotoxic T cells (CTL), natural killer (NK) cells, supernatant fraction from activated autoreactive Th1 cells, or alloantibodies in the presence of complement or effector cells. RESULTS: Low HLA expression protected human beta cell lines from adaptive immune destruction, but it was associated with direct killing by activated NK cells. Autoreactive Th1 cell inflammation, rather than glucose stress, induced increased beta cell apoptosis and upregulation of HLA, increasing beta cell vulnerability to killing by auto- and alloreactive CTL and alloreactive antibodies. CONCLUSIONS/INTERPRETATION: We demonstrate that genetically engineered human beta cell lines can be used in vitro to assess diverse immune responses that may be involved in the pathogenesis of type 1 diabetes in humans and beta cell transplantation, enabling preclinical evaluation of novel immune intervention strategies protecting beta cells from immune destruction.

Low incidence of IgA isotype of HLA antibodies in alloantigen exposed individuals.

Human leukocyte antigen (HLA) antibodies are induced by pregnancy, transfusion, or transplantation. Serum from transplant recipients is regularly screened for IgG HLA antibodies because of their clinical relevance for transplant outcome. While other isotypes of HLA antibodies, such as IgA may also contribute to the alloimmune response, validated detection assays for IgA HLA antibody detection are lacking. Therefore, we modified the commonly used luminex screening assay for IgG HLA antibody detection (IgG-LMX) into an IgA HLA antibody screening assay (IgA-LMX). Optimization and validation was performed with IgG, IgA1, and IgA2 isotype variants of HLA-specific human recombinant monoclonal antibodies (mAbs). Reactivity patterns of IgA1 and IgA2 isotype HLA-specific mAbs in IgA-LMX were identical to those of the IgG isotype. Cross-reactivity with IgG and IgM antibodies and nonspecific binding to the beads were excluded. Further assay validation showed the absence of IgA HLA antibodies in serum from individuals without alloantigen exposure (n = 18). When the IgA-LMX assay was applied to sera from 289 individuals with known alloantigen exposure through pregnancy (n = 91) or kidney transplantation (n = 198), IgA HLA antibodies were detected in 3.5% of individuals; eight patients on the kidney retransplant waitlist and two women immunized through pregnancy. The majority (90%) of IgA HLA antibodies were directed against HLA class II and were always present in conjunction with IgG HLA antibodies. Results of this study show that this validated IgA-LMX method can serve as a screening assay for IgA HLA antibodies and that the incidence of IgA HLA antibodies in alloantigen exposed individuals is low.