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.

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.

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.

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.

[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.

[Lymph node pathology - an update]. / Lymphknotenpathologie - ein Update.

Immune suppression is a risk factor for malignant lymphoma development. Progress in medical science has increased the numbers of immunosuppressed patients due to organ transplantations or successful treatment of autoimmune diseases. Different forms of immune suppression and the respective lymphoma entities are discussed in this article. Another issue treated are gray zone lymphomas between Hodgkin's lymphoma and diffuse large B cell lymphoma. This category not only represents a diagnostic challenge but also represents more a true biological continuum.

Modulation of immune responses following solid organ transplantation by microRNA.

Organ transplantation, an accepted treatment for end stage organ failure, is often complicated by allograft rejection and disease recurrence. In this review we will discuss the potential role of microRNAs in allograft immunity especially leading to rejection of the transplanted organ. microRNAs (miRNAs), originally identified in C. elegans, are short non-coding 21-24 nucleotide sequences that bind to its complementary sequences in functional messenger RNAs and inhibits post-translational processes through RNA duplex formation resulting in gene silencing (Lau et al., 2001). Gene specific translational silencing by miRNAs regulates pathways for immune responses such as development of innate immunity, inflammation, T-cell and B-cell differentiation and signaling that are implicated in various stages of allograft rejection. miRNAs also play a role in development of post-transplant complicacies like fibrosis, cirrhosis, carcinogenesis often leading to graft loss and poor patient outcome. Recent advancements in the methods for detecting and quantifying miRNA in tissue biopsies, as well as in serum and urine samples, has led to identification of specific miRNA signatures in patients with allograft rejection and have been utilized to predict allograft status and survival. Therefore, miRNAs play a significant role in post-transplant events including allograft rejection, disease recurrence and tumor development impacting patient outcome.

Haploidentical hematopoietic transplantation: current status and future perspectives.

For patients with hematologic malignancies at high risk of relapse who do not have matched donors, a suitable alternative stem cell source is the HLA-haploidentical 2 or 3-loci mismatched family donor who is readily available for nearly all patients. Transplantation across the major HLA barrier is associated with strong T-cell alloreactions, which were originally manifested as a high incidence of severe GVHD and graft rejection. The present review shows how these obstacles to successful transplantation were overcome in the last 15 years, making full haplotype-mismatched transplantation a clinical reality that provides similar outcomes to transplantation from matched unrelated donors. The review also discusses the advantages and drawbacks of current options for full haplotype-mismatched transplantation and highlights innovative approaches for re-building immunity after transplantation and improving survival.

Histocompatibility and immunogenetics in cord blood transplantation.

This review of the immunogenetics of cord blood transplantation attempts to highlight the connections between classical studies and conclusions of the tissue transplantation field as a scholarly endeavor, exemplified by the work of Professor Hoecker, with the motivations and some recent and key results of clinical cord blood transplantation. The authors review the evolution of understanding of transplantation biology and find that the results of the application of cord blood stem cells to Transplantation Medicine are consistent with the careful experiments of the pioneers in the field, from the results of tumor and normal tissue transplants, histocompatibility immunogenetics, to cell and molecular biology. Recent results of the National Cord Blood Program of the New York Blood Center describe the functioning in cord blood transplantation of factors, well known in transplantation immunogenetics, like the Fl anti-parent effect and the tolerance-like status of donors produced by non-inherited maternal HLA antigens. Consideration of these factors in donor selection strategies can improve the prognosis of transplantation by characterizing "permissibility" in HLA-incompatible transplantation thereby increasing the probability of survival and reducing the likelihood of leukemic relapse.

Histocompatibility and Immunogenetics in Cord Blood Transplantation

This review of the immunogenetics of cord blood transplantation attempts to highlight the connections between classical studies and conclusions of the tissue transplantation field as a scholarly endeavor, exemplified by the work of Professor Hoecker, with the motivations and some recent and key results of clinical cord blood transplantation. The authors review the evolution of understanding of transplantation biology and find that the results of the application of cord blood stem cells to Transplantation Medicine are consistent with the careful experiments of the pioneers in the field, from the results of tumor and normal tissue transplants, histocompatibility immunogenetics, to cell and molecular biology. Recent results of the National Cord Blood Program of the New York Blood Center describe the functioning in cord blood transplantation of factors, well known in transplantation immunogenetics, like the Fl anti-parent effect and the tolerance-like status of donors produced by non-inherited maternal HLA antigens. Consideration of these factors in donor selection strategies can improve the prognosis of transplantation by characterizing "permissibility" in HLA-incompatible transplantation thereby increasing the probability of survival and reducing the likelihood of leukemic relapse.

Fundamentos de genética e inmunología para bancos de sangre y medicina transfusional: [revisión] / Fundamentals of genetics and immunology to blood banks and transfusion medicine: [review]

Más de 100 años han transcurrido desde el descubrimiento de los grupos sanguíneos A, B y O y hasta el presente se han reconocido 308 antígenos, de los cuales 270 están agrupados en 30 sistemas sanguíneos. Su importancia radica en la seguridad con la cual se transfunde la sangre de donantes a pacientes con pruebas tan sencillas en su elaboración, como la clasificación sanguínea ABO y Rh, las pruebas cruzadas y el rastreo de anticuerpos irregulares, con el fin de evitar la aloinmunización de los receptores...

Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

Female-versus-male alloreactivity as a model for minor histocompatibility antigens in hematopoietic stem cell transplantation.

H-Y encoded gene products were the first to be recognized as clinically relevant minor histocompatibility antigens. Compared to other gender combinations, female donor/male recipient (FDMR) transplants are associated with increased graft-versus-host disease (GvHD), increased transplant-related mortality (TRM) and reduced risk of relapse. Still, their relative impact on transplant outcome remains controversial. We analyzed donor/recipient sex combination in 53,988 patients treated with allogeneic hematopoietic stem cell transplantation (HSCT) between 1980 and 2005. We found a strong increase in chronic GvHD and late TRM and decreased survival in FDMR transplants irrespective of underlying disease. Conversely, FDMR patients had lower relapse rates. The negative effect on survival decreased with advancing disease stage as relapse protection became more important. Effects of H-Y alloreactivity were most pronounced in patients transplanted from HLA-matched donors and in those receiving transplants from an adult donor. Adjustment for acute and chronic GvHD only partially corrected the effects of H-Y alloreactivity. Analysis of the FDMR proportion over time indicated that the frequency of this gender combination has declined in unrelated transplants over the last 10 years. These data define the role of H-Y mismatching in allogeneic HSCT and support the current practice of avoiding female donors for male patients, if possible.

Implications of HLA sequence-based typing in transplantation.

Serology-based conventional microlymphocytotoxicity HLA typing method, which has been regarded as the gold standard in organ and hematopoietic stem cell transplantation, has been replaced now by DNA-based typing. Many laboratories all over the world have already switched over to molecular methods. Microlymphocytotoxicity-based tissue typing was done using commercial sera, while the molecular typing by genomic DNA based. DNA quality and its quantity obtained using various DNA extraction protocols was found to be an important factor in the molecular method of tissue typing in transplant outcome. Many polymerase chain reaction-based molecular techniques have been adopted with far reaching clinical outcome. The sequence-based typing (SBT) has been the ultimate technique, which has been of the highest reliability in defining the HLA alleles. The nonavailability of specific HLA antisera from native populations, large number of blank alleles yet to be defined and comparable low resolution of HLA alleles in SSP or SSOP technique, suggests that highly refined DNA-based methods like SBT should be used as an adjunct to HLA serology and/or low/intermediate/high resolution HLA typing in order to achieve a better transplant outcome.

Immunogenetic factors in donors and patients that affect the outcome of hematopoietic stem cell transplantation.

We have correlated the clinical outcome with the level of HLA matching in 423 patients who received a transplant from a volunteer unrelated donor in the United Kingdom. HLA matching was performed at the allelic level (i.e. high-resolution) using reference strand mediated conformation analysis (RSCA) at HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1. The three-year probability of overall survival (OS) was 45% (median survival of 593 days; six-year overall survival probability was 40%). The mean follow-up was 1013 days (range 89-2697). Those matched for their HLA loci had a significantly better overall survival than the mismatched pairs (47% versus 40%, p=0.040). This result could be refined based on the number of alleles that were mismatched. In patients with a single HLA mismatch, the overall survival was 43%, compared to 30% in those with multiple mismatches; however, there was no statistically significant difference between matched pairs or those with one mismatch. Although there was no significant difference in the overall survival dependent on DPB1 matching in the group overall, in acute lymphocytic leukemia, DPB1-matched pairs had a significantly worse overall survival (log rank; p=0.025). Thus, a match for DPB1 is associated with a significantly increased risk of disease relapse, irrespective of the matching status for the other HLA molecules. In a multivariate analysis, a high pre-transplant levels of Tregs resulted in worse overall survival (relative risk (RR), 2.74; p=0.01), a trend to reduce disease-free survival (RR, 2.05; p=0.060) and to increase disease relapse (RR, 3.36; p=0.006). Residual patient CD4(+)CD25(hi) regulatory T cells may suppress graft-versus-tumour responses, decreasing the overall survival by increasing the rates of relapse. In acute leukemia, the presence of NOD2/CARD15 SNPs in the genotype of unrelated donor hematopoietic stem cell transplant pairs results in significant increases in disease relapse and consequently in death. These data show an important role for NOD2/CARD15 genotyping in transplantation and suggest a possible effect of the NOD2 protein in alloreactivity and tumour surveillance. Genotyping recipients and donors prior to transplant may present valuable information for planning and management of pre-transplant conditioning regimens, and for prognosis of the outcome.

A novel method for KIR-ligand typing by pyrosequencing to predict NK cell alloreactivity.

Studies have shown that KIR-ligand mismatching to predict NK cell alloreactivity may result in less relapse and better survival in patients with AML. KIR-ligands are distinguished by single nucleotide polymorphisms (SNPs) from HLA-B and HLA-C sequences. We hypothesized that pyrosequencing to determine KIR-ligand status by direct sequencing of the ligand epitope can be done as an alternative to high-resolution HLA-typing. Pyrosequencing is rapid and would be particularly useful in analysis of retrospective cohorts where high-resolution HLA-typing is unavailable or too expensive. To validate this assay, RNA and DNA from 70 clinical samples were tested for KIR-ligand by pyrosequencing. Primer binding to invariant regions without known SNPs was critical for KIR-ligand assignment by pyrosequencing to be in full concordance with high-resolution HLA-typing. Pyrosequencing is sensitive, specific, high-throughput, inexpensive, and can rapidly screen KIR-ligand status to evaluate potential alloreactive NK cell or transplant donors.

Fas ligand enhances hematopoietic cell engraftment through abrogation of alloimmune responses and nonimmunogenic interactions.

Early after transplantation, donor lineage-negative bone marrow cells (lin(-) BMC) constitutively upregulated their expression of Fas ligand (FasL), suggesting an involvement of the Fas/FasL axis in engraftment. Following the observation of impaired engraftment in the presence of a dysfunctional Fas/FasL axis in FasL-defective (gld) donors or Fas-defective (lpr) recipients, we expressed a noncleavable FasL chimeric protein on the surface of donor lin(-) BMC. Despite a short life span of the protein in vivo, expression of FasL on the surface of all the donor lin(-) BMC improved the efficiency of engraftment twofold. The FasL-coated donor cells efficiently blunted the host alloimmune responses in primary recipients and retained their hematopoietic reconstituting potential in secondary transplants. Surprisingly, FasL protein improved the efficiency of engraftment in syngeneic transplants. The deficient engraftment in lpr recipients was not reversed in chimeric mice with Fas(-) stroma and Fas(+) BMC, demonstrating that the host marrow stroma was also a target of donor cell FasL. Hematopoietic stem and progenitor cells are insensitive to Fas-mediated apoptosis and thus can exploit the constitutive expression of FasL to exert potent veto activities in the early stages of engraftment. Manipulation of the donor cells using ectopic FasL protein accentuated the immunogenic and nonimmunogenic interactions between the donor cells and the host, alleviating the requirement for a megadose of transplanted cells to achieve a potent veto effect. Disclosure of potential conflicts of interest is found at the end of this article.