David M. Turner

Genotypic variation in the transforming growth factor-β1 gene : Association with transforming growth factor-β1 production, fibrotic lung disease, and graft fibrosis after lung transplantation

BACKGROUND Transforming growth factor (TGF)-beta1 is a profibrogenetic cytokine that has been implicated in the development of fibrosis in transplanted tissues. In this study, we have analyzed the genetic regulation of TGF-beta1 production in lung transplant recipients. METHOD A polymerase chain reaction-single-stranded conformational polymorphism technique was used to detect polymorphisms in the TGF-beta1 gene from genomic DNA. Polymorphisms were shown to correlate with in vitro TGF-beta1 production by stimulated lymphocytes. A single-specific oligonucleotide probe hybridization method was devised to screen for these polymorphisms in lung transplant groups and controls. RESULTS We have identified five polymorphisms in the TGF-beta1 gene: two in the promoter region at positions -800 and -509, one at position +72 in a nontranslated region, and two in the signal sequence at positions +869 and +915. The polymorphism at position +915 in the signal sequence, which changes codon 25 (arginine-->proline), is associated with interindividual variation in levels of TGF-beta1 production. Stimulated lymphocytes of homozygous genotype (arginine/arginine) from control individuals produced significantly more TGF-beta1 in vitro (10037+/-745 pg/ml) compared with heterozygous (arginine/proline) individuals (6729+/-883 pg/ml; P<0.02). In patients requiring lung transplantation for a fibrotic lung condition, there was an increase in the frequency of the high-producer TGF-beta1 allele (arginine). This allele was significantly associated with pretransplant fibrotic pathology (P<0.02) (n=45) when compared with controls (n=107) and with pretransplant nonfibrotic pathology (P<0.004) (n=50). This allele was also associated with allograft fibrosis in transbronchial biopsies when compared with controls (P<0.03) and with nonallograft fibrosis (P<0.01). CONCLUSION The production of TGF-beta1 is under genetic control, and this in turn influences the development of lung fibrosis. Hence, the TGF-beta1 genotype has prognostic significance in transplant recipients.

Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease

Epstein‐Barr virus (EBV) is associated with several malignancies, including post‐transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV‐specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV‐specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV‐associated non‐haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre‐terminal. Thus, this third party donor‐derived EBV‐specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

Status report from 'double agent HLA': health and disease.

The human major histocompatibility complex is a multi-gene ~4 Mb of DNA including the highly polymorphic HLA genes which are fundamental in effecting a healthy immune response. Detailed knowledge of the structure and function of HLA genes and their alleles and HLA proteins and their allotypes has rapidly evolved because of their role in clinical transplantation. A significant additional finding is the association or linkage of a wide range of diseases with HLA. We review the function of HLA and the history of disease association studies and focus on specific informative examples in the context of recent genome-wide screening analyses.

Electrostatic Potential Change in a Paired Epitope: A Novel Explanation for Bw4 Antibodies in Patients With B13 (Bw4) Antigens.

It is established that HLA antibodies are specific for epitopes as opposed to antigens. An epitope may be present on a small number of HLAmolecules, with shared epitopes now established as the basis for cross-reacting groups, or be widely expressed. The Bw4/6 epitopes are mutually exclusive “public epitopes” present on all HLA-B antigens with Bw4 also existing on a number of HLA-A molecules. The characterization of epitopes as 3 Å regions of polymorphic residues and in relation to their electrostatic potential (EP) has led to an improved definition of antibody reactivity. In addition, HLA antibodies have been described that require 2 sites for stable binding; these are thought to consist of a self-epitope and non–self-epitope on the antigen surface. In this model, it is possible that the non–self-epitope determines antibody specificity and that the self-epitope confers stability to the antigenantibody complex. We report 2 cases of individuals awaiting kidney transplantation who have HLA-B13 as their only Bw4 antigen and have developed HLA antibodies to all other Bw4 antigens present within the Luminex single antigen bead panel (Figure 1). The first patient is a 38-year-old man with HLA class I type A2, B13, B62, Bw4, Bw6, Cw6, Cw9 who received a kidney transplant mismatched at B27. After allograft failure, the patient demonstrated HLA antibodies to all Bw4 antigens with the exception of B13. The second patient, a 33-year-old woman, with HLA class I type A2, A11, B13, B62, Bw4, Bw6, Cw1, Cw6, was exposed to

Preformed donor-specific HLA antibodies are associated with increased risk of early mortality after liver transplantation

There is limited evidence for a negative impact of preformed, donor‐specific HLA antibodies (DSA) identified by cross‐matching on outcomes after liver transplantation. Three recent studies have suggested an association between preformed DSA detected by Luminex and reduced graft or recipient survival in liver transplant cohorts with a high prevalence of hepatitis C. This study investigated the impact of preformed DSA identified by Luminex in the Scottish liver transplant population. All recipients of liver transplants in Scotland between 2007 and 2015 with samples available for day of transplant antibody testing and donor HLA typing were included (n=459); 96% of the cohort were white and 19% had a primary diagnosis of hepatitis C. The median follow‐up time was 36 months. Preformed DSA were detected in 88 recipients. In multivariate analysis, preformed DSA with a median fluorescent intensity ≥10 000 were associated with recipient mortality at 1 year. There was no association between DSA and overall graft or recipient survival. This study adds to the growing body of evidence supporting a detrimental impact of preformed, high‐level DSA in a subset of liver transplant recipients by identifying an association in an ethnically and demographically distinct liver transplant population.

Repurposing the Cord Blood Bank for Haplobanking of HLA‐Homozygous iPSCs and Their Usefulness to Multiple Populations

Although autologous induced pluripotent stem cells (iPSCs) can potentially be useful for treating patients without immune rejection, in reality it will be extremely expensive and labor‐intensive to make iPSCs to realize personalized medicine. An alternative approach is to make use of human leukocyte antigen (HLA) haplotype homozygous donors to provide HLA matched iPSC products to significant numbers of patients. To establish a haplobank of iPSCs, we repurposed the cord blood bank by screening ∼4,200 high resolution HLA typed cord blood samples, and selected those homozygous for the 10 most frequent HLA‐A,‐B,‐DRB1 haplotypes in the Korean population. Following the generation of 10 iPSC lines, we conducted a comprehensive characterization, including morphology, expression of pluripotent markers and cell surface antigens, three‐germ layer formation, vector clearance, mycoplasma/microbiological/viral contamination, endotoxin, and short tandem repeat (STR) assays. Various genomic analyses using microarray and comparative genomic hybridization (aCGH)‐based single nucleotide polymorphism (SNP) and copy number variation (CNV) were also conducted. These 10 HLA‐homozygous iPSC lines match 41.07% of the Korean population. Comparative analysis of HLA population data shows that they are also of use in other Asian populations, such as Japan, with some limited utility in ethnically diverse populations, such as the UK. Taken together, the generation of the 10 most frequent Korean HLA‐homozygous iPSC lines serves as a useful pointer for the development of optimal methods for iPSC generation and quality control and indicates the benefits and limitations of collaborative HLA driven selection of donors for future stocking of worldwide iPSC haplobanks. Stem Cells 2018;36:1552–1566

Prozone Effect Can Be Specific to Single Antigen Bead Kit Manufacturers

The practice of diluting patient serum prior to single antigen bead (SAB) testing for HLA antibodies has been reported in the literature, along with mechanisms for removing a variety of potential blocking/interference mechanisms within this assay, such as the use of ethylenediaminetetraacetic acid (EDTA), dithiothreitol, and hypotonic dialysis (1–4). We read with interest the case reported in this journal by Tambur showing that sample dilution can aid in the definition of HLA reactivity in terms of epitope specificity (5). The author highlights that blocking effects, although often termed “prozone,” are probably multifactoral and can be attributed to a variety of mechanisms. Like many histocompatibility and immunogenetics (H&I) laboratories, we incorporate an EDTA step in our HLA antibody detection assays to remove complement blocking of secondary antibody. The addition of EDTA alone, however, does not appear to resolve all interference (1), and sample dilution can reveal additional specificities in some cases. Tambur’s case shows that the use of dilution can reveal distinct reactivity patterns that allow further characterization of antibody reactivity (4). A challenge, however, for routine H&I laboratory testing is to address identified blocking mechanisms by the addition of a dilution/titration step in a cost-efficient manner, to allow improved epitope analysis.

Allosensitization following bone graft.

It is recognized that patients may become sensitized to donor‐specific HLA antigens as a result of previous antigenic exposures, classically through previous transplantation, pregnancy, or blood transfusion. We present an unusual case of a patient who unexpectedly developed a range of anti‐HLA antibodies following orthopedic surgery where a bone graft was deployed intraoperatively. We describe the case of a 52‐year‐old man awaiting a renal transplantation, undergoing elective orthopedic surgery requiring a small‐volume bone graft. His postoperative antibody profile was found to be substantially changed compared to his previous negative samples, with the presence of HLA‐DR, DQ, and DP specificities, at levels that would be likely to give a positive flow cytometry crossmatch and therefore according to local procedures required listing as unacceptable antigens for organ allocation. We perform a literature review of all previous cases of allosensitization following bone graft. This case is the first to demonstrate allosensitization following minor surgery with ;low‐volume bone graft. Previous evidence is very limited and pertains only to massive osteochondral surgery for trauma or malignancy, and is confounded by potential concomitant blood transfusion. Clinicians should be aware of the risk of allosensitization where bone grafts are used.

Combined linkage and association analysis of classical Hodgkin lymphoma

The heritability of classical Hodgkin lymphoma (cHL) has yet to be fully deciphered. We report a family with five members diagnosed with nodular sclerosis cHL. Genetic analysis of the family provided evidence of linkage at chromosomes 2q35-37, 3p14-22 and 21q22, with logarithm of odds score >2. We excluded the possibility of common genetic variation influencing cHL risk at regions of linkage, by analysing GWAS data from 2,201 cHL cases and 12,460 controls. Whole exome sequencing of affected family members identified the shared missense mutations p.(Arg76Gln) in FAM107A and p.(Thr220Ala) in SLC26A6 at 3p21 as being predicted to impact on protein function. FAM107A expression was shown to be low or absent in lymphoblastoid cell lines and SLC26A6 expression lower in lymphoblastoid cell lines derived from p.(Thr220Ala) mutation carriers. Expression of FAM107A and SLC26A6 was low or absent in Hodgkin Reed-Sternberg (HRS) cell lines and in HRS cells in Hodgkin lymphoma tissue. No sequence variants were detected in KLHDC8B, a gene previously suggested as a cause of familial cHL linked to 3p21. Our findings provide evidence for candidate gene susceptibility to familial cHL.

HLA-DR Genotyping and Mitochondrial DNA Analysis Reveal the Presence of Family Burials in a Fourth Century Romano-British Christian Cemetery

In Colchester, Britains oldest recorded town, during the Roman period there were areas which were clearly used solely as cemeteries. One of the most significant is at Butt Road, which includes a late Roman probable Christian cemetery with an associated building, apparently a church, that overlies and developed from a pagan inhumation cemetery. DNA was extracted from the long bones (femurs) of 29 individuals, mostly from a large complex of burials centered on two timber vaults. These were thought to comprise a number of family groupings, deduced from osteological analysis, stratigraphical and other considerations. The use of a modified version of the silica-based purification method recovered nanogram quantities of DNA/gram of bone. Two-stage amplification, incorporating primer-extension preamplification-polymerase chain reaction, permitted simultaneous amplification of both mitochondrial and nuclear DNA. Sequence-specific oligonucleotide probes yielded human leukocyte antigen (HLA)-DR typing of seven samples, with four revealing the infrequent HLA-DR10 genotype. Examination of the control region of mitochondrial DNA (mtDNA) by direct sequencing revealed polymorphisms yet to be reported in the modern population. HLA-DRB typing and mtDNA analysis affirmatively supported kinship among some, if not all, individuals in the “vault complex” and demonstrate a continental European origin of the individuals investigated.