HLA-G14bp ins/del polymorphism and post-transplant weight gain in kidney transplantation: potential implications beyond tolerance.

BACKGROUND: Human leukocyte antigen (HLA)-G is a non-classical HLA molecule with immunomodulant and immunosuppressive functions, involved in transplantation tolerance. HLA-G14bp ins/del polymorphism in exon 8 has been associated with allograft rejection and kidney transplant outcome, with controversial results. We investigated associations of HLA-G14bp ins/del polymorphism on onset of some of the main post-transplant risk factors, like excess body weight, lipid abnormalities, increased fasting plasma glucose. Polymorphisms of cytokines with both immunosuppressive and metabolic effects were also assessed for comparisons and associated analysis. METHODS: The present study involved kidney transplant recipients (n = 173) in which body mass index, cholesterol, triglycerides, fasting plasma glucose were registered in the first years after transplantation and analyzed in association with genotypes. Presence of hypertension and smoking habits, demographic, transplant-related and therapeutic data of patients were also recorded. Polymerase chain reaction, sequence-specific primer amplification and Taqman allelic discrimination techniques were used for genotyping of HLA-G14bp ins/del, interleukin (IL)-10(-1082G > A,-819 T > C,-592A > C), transforming growth factor-ß(+ 869 T > C,+915C > G), IL-6(-174G > C), tumor necrosis factor-α(-308G > A) and IL-18(-137G > C,-607C > A). Effects of genotypes on clinical markers at each time point (pre-transplant and 1 to 5 years after transplant) were analyzed using a repeated-measures general linear model analysis; adjustment for potential confounders was performed. RESULTS: Results showed that HLA-G14bp ins/ins was significantly associated with obesity, in particular after transplantation (3 years, p = 0.002, OR = 4.48, 95% CI:1.76-11.41). Post-transplant body mass index was significantly increased in HLA-G14bp ins/ins carriers (3 and 4 years, p = 0.033 and p = 0.044); effects of HLA-G14bp genotypes on post-transplant BMI were confirmed by using repeated-measures analysis and after controlling for confounding variables. Cytokine genotypes did not associate with the examined factors. CONCLUSIONS: The study of transplanted patients allowed to evidence a potential relationship between post-transplant weight gain and HLA-G14bp ins/del polymorphism, previously involved in rejection for its immunosuppressive/tolerogenic activity. This novel association could widen the knowledge of the role and functions of HLA-G molecules in diseases and transplantation.

Quantitative polymerase chain reaction technology in chimerism monitoring after hematopoietic stem cell transplantation: One center experience.

Chimerism status evaluation is a routine test performed in post-hematopoietic stem cell transplantation (HSCT) period. The aim of the study was to evaluate a quantitative polymerase chain reaction (qPCR) method (GenDx, Utrecht, the Netherlands) applicability for this purpose. The study included 74 recipient/donor pairs tested for informative markers: median of four and six informative markers was found for patients (related and unrelated donor, respectively). Higher sensitivity of qPCR method was confirmed by analysis of recipient post-HSCT samples (N = 800) among which microchimerism (0.1%-1% recipient DNA) was detected in 21.8% of cases. The ability to detect less than 1% of minor population, as opposed to the short tandem repeat (STR) method for which 1% is the limit, translated into earlier identification of a disease relapse for four patients in our study sample.

Mechanisms underlying human genetic diversity: consequence for antigraft antibody responses.

This review focuses on the emerging concept of genomewide genetic variation as basis of an alloimmune response. Chronic antibody-mediated rejection is the major cause of long-term graft loss and growing evidence supports the clinical relevance of HLA but also non-HLA related alloimmune responses. Several polymorphic gene products have been identified as minor histocompatibility antigens. The formation of donor-specific alloantibodies is driven by indirect allorecognition of donor-derived peptides representing a form of conventional T-cell response. With the availability of high-throughput sequencing and genotyping technologies, the identification of genomewide genetic variation and thus mismatches between organ donors and graft recipients has become feasible. First clinical data linking genetic polymorphism and clinical outcome have been published and larger studies are currently under way. Protein arrays have successfully been used to identify a large variety of non-HLA antibodies in kidney transplant recipients and the availability of customizable peptide arrays made screening for linear epitopes on an individual patient level feasible. This review provides a summary of the recent findings in histocompatibility matching in the field of solid organ transplantation and complements it with a clear workflow for assessing the impact of genetic differences in protein-coding genes in solid organ transplantation.

Primer design for SNP genotyping based on allele-specific amplification-Application to organ transplantation pharmacogenomics.

Diagnostic methods based on single nucleotide polymorphism (SNP) biomarkers are essential for the real adoption of personalized medicine. Allele specific amplification in a homogeneous format or combined to microarray hybridization are powerful approaches for SNP genotyping. However, primers must be properly selected to minimize cross-reactivity, dimer formation and nonspecific hybridization. This study presents a design workflow diagram for the selection of required oligonucleotides for multiplex assays. Based on thermodynamic restrictions, the oligonucleotide sets are chosen for a specific amplification of wild- and mutant-type templates. Design constraints include the structural stability of primer-template duplexes, template-probe duplexes and self-annealing complexes or hairpins for each targeted gene. The performance of the design algorithm was evaluated for the simultaneous genotyping of three SNPs related to immunosuppressive drugs administered after solid organ transplantation. The assayed polymorphisms were rs1045642 (ABCB1 gene), rs1801133 (MTHFR gene) and rs776746 (CYP3A5 gene). Candidates were confirmed by discriminating homozygote and heterozygote populations using a fluorescence solution method and two colorimetric microarray methods on polycarbonate chips. The analysis of patient samples provided excellent genotyping results compared to those obtained by a reference method. The study demonstrates that the development of the allele-specific methods as pharmacogenetic tools can be simplified.

Knockout of microRNA-155 ameliorates the Th1/Th17 immune response and tissue injury in chronic rejection.

BACKGROUND: MicroRNAs (miRNAs) are integral for maintaining immune homeostasis and self-tolerance. The influence of miRNAs on T-cell differentiation and plasticity are critical in the development of chronic rejection of transplanted hearts. In this study, we sought to determine whether the knockout of miR-155 affects the development of cardiac allograft vasculopathy (CAV) in a murine model. METHODS: miRNA microarray and quantitative polymerase chain reaction (qPCR) analyses were performed for allograft neointimal lesion samples in chronic rejection. A model of heterotopic murine heart transplantation (bm12 to miR-155+/+ or miR-155-/- mice) was then used to analyze allograft survival, histology, mRNA expression and T-cell sub-populations in spleens. The accelerated experiments were performed by intraperitoneal injection of either recombinant interleukin-17A or phosphate-buffered saline (PBS) after heart transplantation. For the competitive transfer experiments, CD4+ splenocytes from wild-type (WT) or miR-155-/- mice were mixed and injected into Rag1-/- mice, and cardiac transplantation was performed after 24 hours. The differentiation of T-helper subsets (Th1/Th17/iTreg) was investigated in vitro. RESULTS: miR-155-/- mice showed resistance to cardiac rejection along with weakened T-cell-mediated inflammation, especially for Th17 cells. Recombinant IL-17A could restore this relieved injury. The competitive experiments implied that miR-155 plays a vital role in the stability of the Th17 phenotype. In vitro, we also demonstrated that miR-155-/- mice exhibit a defect in Th17 differentiation. CONCLUSIONS: miR-155 regulates Th1/Th17-related inflammation in chronic cardiac rejection and may be a potential therapeutic target to attenuate cardiac allograft rejection. Despite advancements in immunosuppressive therapy, the immunologic mechanisms responsible for allograft rejection remain an important issue for both clinicians and researchers. Allograft rejection is a T-cell-dependent phenomenon and is critically dependent on inflammation mediated by CD4+ Th subsets, including Th1, Th2, Th17, Th9 and regulatory T (Treg) cells.

[Genetic mechanisms of adaptive immunity emergence in vertebrates].

The adaptive immune system in vertebrates emerged in a multistep process that can be reconstructed on the basis of the data concerning the structure of immune systems of modern cartilaginous and bony fishes, as well as of cyclostomes. The most probable evolutionary scenario is likely to be as follows: the T cell receptor loci emerged on the basis of NK cell-like receptor genes; the antibody loci evolved on the basis of T cell receptor loci; the MHC locus arose on the basis of the locus responsible for innate immunity of early chordates. The ancestral MHC molecules likely participated in the transplantation immunity before they acquired the ability of antigen peptide presentation.

Longitudinal studies of a B cell-derived signature of tolerance in renal transplant recipients.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Human Leukocyte Antigen Diversity: A Southern African Perspective.

Despite the increasingly well-documented evidence of high genetic, ethnic, and linguistic diversity amongst African populations, there is limited data on human leukocyte antigen (HLA) diversity in these populations. HLA is part of the host defense mechanism mediated through antigen presentation to effector cells of the immune system. With the high disease burden in southern Africa, HLA diversity data is increasingly important in the design of population-specific vaccines and the improvement of transplantation therapeutic interventions. This review highlights the paucity of HLA diversity data amongst southern African populations and defines a need for information of this kind. This information will support disease association studies, provide guidance in vaccine design, and improve transplantation outcomes.

Clinical Role of Human Leukocyte Antigen in Health and Disease.

Most of the genes in the major histocompatibility complex (MHC) region express high polymorphism that is fundamental for their function. The most important function of human leukocyte antigen (HLA) molecule is in the induction, regulation of immune responses and the selection of the T cell repertoire. A clinician's attention is normally drawn to a system only when it malfunctions. The HLA system is no exception in this regard, but in contrast to other systems, it also arouses interest when it functions well - too well, in fact. Population studies carried out over the last several decades have identified a long list of human diseases that are significantly more common among individuals that carry particular HLA alleles including inflammatory, autoimmune and malignant disorders. HLA-disease association is the name of this phenomenon, and the mechanism underlying is still a subject of hot debate. Social behaviours are affected by HLA genes and preference for HLA disparate mates may provide 'good genes' for an individual's offspring. Also, certain HLA genes may be associated with shorter life and others with longer lifespan, but the effects depend both on the genetic background and on the environmental conditions. The following is a general overview of the important functional aspects of HLA in health and diseases.

[Pharmacogenetics: a promising tool to personalize immunosuppressive therapy in renal transplantation]. / Farmacogenetica: un possibile strumento per la personalizzazione della terapia immunosoppressiva nel trapianto renale.

Renal transplantation is the gold standard therapy for patients affected by end stage renal disease. It is a clinical condition characterized by severe biological/biochemical alterations that requires renal replacement therapy to ensure patients survival. In most cases, it is followed by a significant improvement of patients quality of life, reduction in medical expenses and prolongation of life. However, to reach these positive clinical effects, patients need to take several immunosuppressive medications (calcineurin inhibitors, mTOR inhibitors and antimetabolites) characterized by a narrow therapeutic index, that, in some cases, could cause important adverse effects. To avoid toxicities and adverse drug reactions, immunosuppressors should be correctly administered, according to the blood trough levels. Nevertheless, in most of the times, this methodology to adjust drug doses gives inadequate and non-reproducible results. Additionally, as largely described, inherited differences in drug metabolism and disposition and genetic variability in therapeutic targets (e.g. receptors) need to be taken into account because of their role in modulating drug effects and toxicities. Therefore, worldwide researchers are working together to identify biomarkers, useful to personalize therapy based on genetic characteristics of patients. In this context, we believe that the omics techniques could represent a future powerful instruments that, whether employed routinely, could help to reach this objective.

A Three-Gene Assay for Monitoring Immune Quiescence in Kidney Transplantation.

Organ transplant recipients face life-long immunosuppression and consequently are at high risk of comorbidities. Occasionally, kidney transplant recipients develop a state of targeted immune quiescence (operational tolerance) against an HLA-mismatched graft, allowing them to withdraw all immunosuppression and retain stable graft function while resuming immune responses to third-party antigens. Methods to better understand and monitor this state of alloimmune quiescence by transcriptional profiling may reveal a gene signature that identifies patients for whom immunosuppression could be titrated to reduce patient and graft morbidities. Therefore, we investigated 571 unique peripheral blood samples from 348 HLA-mismatched renal transplant recipients and 101 nontransplant controls in a four-stage study including microarray, quantitative PCR, and flow cytometry analyses. We report a refined and highly validated (area under the curve, 0.95; 95% confidence interval, 0.92 to 0.97) peripheral blood three-gene assay (KLF6, BNC2, CYP1B1) to detect the state of operational tolerance by quantitative PCR. The frequency of predicted alloimmune quiescence in stable renal transplant patients receiving long-term immunosuppression (n=150) was 7.3% by the three-gene assay. Targeted cell sorting of peripheral blood from operationally tolerant patients showed a significant shift in the ratio of circulating monocyte-derived dendritic cells with significantly different expression of the genes constituting the three-gene assay. Our results suggest that incorporation of patient screening by specific cellular and gene expression assays may support the safety of drug minimization trials and protocols.

Defining the alloreactive T cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture.

The cellular immune response is the most important mediator of allograft rejection and is a major barrier to transplant tolerance. Delineation of the depth and breadth of the alloreactive T cell repertoire and subsequent application of the technology to the clinic may improve patient outcomes. As a first step toward this, we have used MLR and high-throughput sequencing to characterize the alloreactive T cell repertoire in healthy adults at baseline and 3 months later. Our results demonstrate that thousands of T cell clones proliferate in MLR, and that the alloreactive repertoire is dominated by relatively high-abundance T cell clones. This clonal make up is consistently reproducible across replicates and across a span of three months. These results indicate that our technology is sensitive and that the alloreactive TCR repertoire is broad and stable over time. We anticipate that application of this approach to track donor-reactive clones may positively impact clinical management of transplant patients.

Pharmacogenetics and immunosuppressive drugs in solid organ transplantation.

The transplantation literature includes numerous papers that report associations between polymorphisms in genes encoding metabolizing enzymes and drug transporters, and pharmacokinetic data on immunosuppressive drugs. Most of these studies are retrospective in design, and although a substantial number report significant associations, pharmacogenetic tests are hardly used in clinical practice. One of the reasons for this poor implementation is the current lack of evidence of improved clinical outcome with pharmacogenetic testing. Furthermore, with efficient therapeutic drug monitoring it is possible to rapidly correct for the effect of genotypic deviations on pharmacokinetics, thereby decreasing the utility of genotype-based dosing. The future of pharmacogenetics will be in treatment models in which patient characteristics are combined with data on polymorphisms in multiple genes. These models should focus on pharmacodynamic parameters, variations in the expression of drug transporter proteins, and predictors of toxicity. Such models will provide more information than the relatively small candidate gene studies performed so far. For implementation of these models into clinical practice, linkage of genotype data to medication prescription systems within electronic health records will be crucial.

HLA-G as a tolerogenic molecule in transplantation and pregnancy.

HLA-G is a nonclassical HLA class I molecule. In allogeneic situations such as pregnancy or allograft transplantation, the expression of HLA-G has been related to a better acceptance of the fetus or the allograft. Thus, it seems that HLA-G is crucially involved in mechanisms shaping an allogeneic immune response into tolerance. In this contribution we focus on (i) how HLA-G is involved in transplantation and human reproduction, (ii) how HLA-G is regulated by genetic and microenvironmental factors, and (iii) how HLA-G can offer novel perspectives with respect to therapy.

[Post-transplant cell immune reconstitution in humanized NOD/SCID mice].

OBJECTIVE: To investigate the cell immune reconstitution in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice by the transplantation of human umbilical cord blood (HUCB) CD34⁺; cells. Methods CD34⁺; cells were isolated from HUCB by magnetic activated cell sorting (MACS), and then were transplanted into NOD/SCID mice following the irradiation of sublethal doses via the lateral tail vein. Human CD45⁺; CD3⁺; CD56⁺; cell populations in the peripheral blood of mice were dynamically analyzed by flow cytometry (FCM) 4, 6, 8 and 10 weeks after transplantation. After 10 weeks, the expression of human ALU gene was detected by PCR in the bone marrow of mice, and the expressions of human CD3⁺; CD56⁺; cells were examined by immunohistochemical staining in the spleen tissues. RESULTS: After irradiation, the nucleated cells and giant cells in the marrow cavity of NOD/SCID mice were reduced significantly or completely demolished. The effect of myeloablative pretreatment was ideal. Human CD45⁺; CD3⁺; CD56⁺; cells were found by FCM in the peripheral blood of all surviving mice in transplantation group 4, 6, 8, and 10 weeks after the transplantation. The population of the human lymphocytes varied over time, peaked at the 8th week, and remained at a high level later. At the 10th week, the human ALU sequence could be detected in the bone marrow of all surviving mice in transplantation group, and human CD3⁺; CD56⁺; cells could be observed in the spleen tissues. All mice which received no transplantation died within 2 weeks after irradiation. CONCLUSION: The hu-SRC-NOD/SCID model was successfully established in irradiation-induced NOD/SCID mice by the transplantation of HUCB CD34⁺; cells, and its cell immune system was effectively rebuilt.

Tolerogenic dendritic cells: molecular and cellular mechanisms in transplantation.

During the discovery of mechanisms that govern immune activation and suppression, immune tolerance always came second in the scientific timeline. This has subsequently shaped the advances in the clinical translation of DC therapy protocols used for immunostimulation or immunosuppression. With several hundred clinical trials already registered within the U.S. National Institutes of Health for the use of DCs in cancer vaccination, only a few involve TolDCs for use as negative vaccines. However, as a result of the strong scientific rationale from preclinical and clinical trials, the use of negative vaccination in organ transplantation is likely on its way to reach the extent of the use of positive cancer vaccines in the future. As the underlying mechanisms emerge, the role of DCs in the induction of transplant tolerance is recognized unambiguously as central in the bidirectional communication with various types of immune cells. This is achieved by a complex interplay of numerous tolerogenic signals involving regulatory cytokines and other surface-bound or soluble inhibitory molecules associated with corresponding inhibitory signaling cascades. A detailed understanding of these processes will accelerate the advances of clinical immunologists in translating their knowledge from bench to bedside. In this review, we present the role of TolDCs as well as the most recent findings concerning associated molecular and cellular mechanisms that shape the balance between regulatory and effector immune responses during organ transplantation.

Alteration of innate immunity by donor IL-6 deficiency in a presensitized heart transplant model.

Engraftment of IL-6 deficient donor into wild-type recipient could significantly improve allograft survival through T cell lineage particularly regulatory T cells (Tregs) in non-sensitized transplant host. However, its effect on innate immune responses remains uncertain. Our data revealed that donor IL-6 deficiency significantly increased infiltration of two subsets of MDSCs (CD11b+Gr1+myeloid-derived suppressor cells), CD11b+Gr1(-low) and CD11b+Gr1(-int) with strong immunosuppression activity in the transplanted graft. It resulted in a dramatic increase of CD11b+Gr1(-low) frequency and a significant decrease of the frequency of CD11b+Gr1(-high) and CD4-CD8-NK1.1+ cells in the recipient's spleen. Unexpectedly, donor IL-6 deficiency could not significantly reduce macrophage frequency irrespective of in the host's spleen or graft. Taken together, suppression of innate immune effector cells and enhanced activity of regulatory MDSCs contributed to tolerance induction by blockade of IL-6 signaling pathway. The unveiled novel mechanism of targeting IL-6 might shed light on clinical therapeutic application in preventing accelerated allograft rejection for those pre-sensitized transplant recipients.

Primate models in organ transplantation.

Large animal models have long served as the proving grounds for advances in transplantation, bridging the gap between inbred mouse experimentation and human clinical trials. Although a variety of species have been and continue to be used, the emergence of highly targeted biologic- and antibody-based therapies has required models to have a high degree of homology with humans. Thus, the nonhuman primate has become the model of choice in many settings. This article will provide an overview of nonhuman primate models of transplantation. Issues of primate genetics and care will be introduced, and a brief overview of technical aspects for various transplant models will be discussed. Finally, several prominent immunosuppressive and tolerance strategies used in primates will be reviewed.