Brendan Clark

Immunologic Human Renal Allograft Injury Associates with an Altered IL-10/TNF-α Expression Ratio in Regulatory B Cells

Human B cells with immunoregulatory properties in vitro (Bregs) have been defined by the expression of IL-10 and are enriched in various B-cell subsets. However, proinflammatory cytokine expression in B-cell subsets is largely unexplored. We examined the cytokine profiles of human PBMCs and found that subsets of CD24(hi)CD38(hi) transitional B cells (TrBs), CD24(hi)CD27(+) memory B cells, and naïve B cells express IL-10 and the proinflammatory cytokine TNF-α simultaneously. TrBs had the highest IL-10/TNF-α ratio and suppressed proinflammatory helper T cell 1 (Th1) cytokine expression by autologous T cells in vitro more potently than memory B cells did, despite similar IL-10 expression. Whereas neutralization of IL-10 significantly inhibited TrB-mediated suppression of autologous Th1 cytokine expression, blocking TNF-α increased the suppressive capacity of both memory and naïve B-cell subsets. Thus, the ratio of IL-10/TNF-α expression, a measure of cytokine polarization, may be a better indicator of regulatory function than IL-10 expression alone. Indeed, compared with TrB cells from patients with stable kidney graft function, TrBs from patients with graft rejection displayed similar IL-10 expression levels but increased TNF-α expression (i.e., reduced IL-10/TNF-α ratio), did not inhibit in vitro expression of Th1 cytokines by T cells, and abnormally suppressed expression of Th2 cytokines. In patients with graft dysfunction, a low IL-10/TNF-α ratio in TrBs associated with poor graft outcomes after 3 years of follow-up. In summary, these results indicate that B cell-mediated immune regulation is best characterized by the cytokine polarization profile, a finding that was confirmed in renal transplant patients.

Influence of recipient and donor IL-1α, IL-4, and TNFα genotypes on the incidence of acute renal allograft rejection

Aims: To determine whether polymorphisms of the genes encoding donor or recipient interleukin 1α (IL-1α), tumour necrosis factor α (TNFα), or IL-4 have any impact on the incidence of acute rejection after renal transplantation. Methods: All donors and recipients were genotyped for three polymorphisms in the three cytokine genes: IL1A −889, TNFA −308, and IL4 –590. Results: Statistical analysis of the data obtained revealed no association between the cytokine gene polymorphisms tested and the incidence of post-transplant acute rejection. After stratification for human leucocyte antigen (HLA) matching, it was found that kidneys from donors positive for the TNFA-A allele had a significantly increased incidence of acute rejection in HLA-DR mismatched transplants. Conclusions: This finding argues for prospective TNFA genotyping of renal donors, with avoidance of allocation of kidneys from donors positive for the TNFA-A allele to HLA-DR mismatched recipients.

The HLA system: immunobiology, HLA typing, antibody screening and crossmatching techniques

The Human Leukocyte Antigen (HLA) system plays a critical role in regulating the immune response. As a consequence of its role in immune regulation and exquisite polymorphism, the HLA system also constitutes an immunological barrier which must be avoided or otherwise overcome in clinical transplantation. This introductory review provides a brief summary of the immunobiology of the HLA system and methodology for HLA typing, antibody screening and patient-donor cross-matching. This constitutes a basis for consideration of the importance of these procedures in the system-specific reviews which follow.

Soluble CD30 as a prognostic factor for outcome following renal transplantation

Aims: To determine whether measurement of soluble CD30 (sCD30) levels predicts for early rejection in a cohort of first deceased kidney transplant recipients. Methods: Pre-transplant serum samples were analysed for sCD30 levels using a commercial ELISA kit (Biotest). A 100 U/ml cut-off for “high sCD30” was applied. Clinical outcome parameters were biopsy-proven rejection episodes, creatinine levels and glomerular filtration rate. Results: In the cohort of patients who experienced at least one episode of rejection in the first 6 months post-transplant, levels of pre-transplant sCD30 were significantly higher than in those who did not experience rejection. Despite this association, the occurrence of a high sCD30 level did not predict for rejection on an individual basis. Conclusions: The prognostic value of pre-transplant sCD30 testing is diminished by the large number of patients with high sCD30 levels who do not develop rejection. Although this limits the utility of the test in informing clinical management of individual patients, a high pre-transplant sCD30 level should still be considered a risk factor for poorer outcome.

Intravenous immunoglobulin-induced panel reactive antibody A reduction: Not all preparations are created equal

Background. Use of intravenous (IV) immunoglobulin (Ig) to obtain panel reactive antibody (PRA) A reduction in sensitized patients has been widely reported. Because no IVIg preparation is formulated specifically for this purpose, the authors have sought to determine whether, through laboratory testing, they could guide the rational choice of product for clinical use. Methods. Using a flow cytometric approach, the authors have quantitatively determined the capacity of 22 different IVIg preparations to cause PRA reduction. Results. IVIg preparations showed considerable variability in their individual capacity to reduce serum PRA. Protein-A pretreatment of IVIg preparations was found to reduce their capacity to cause PRA reduction. Conclusion. Laboratory screening of IVIg preparations provides a rational basis for the selection of product for administration to patients in whom the aim is to produce a PRA reduction. Experiments involving protein-A treatment of IVIg preparations indicate that immunoglobulin G is the principal factor involved in the abrogation of serum reactivity.

Can a combined screening/treatment programme prevent premature failure of renal transplants due to chronic rejection in patients with HLA antibodies: study protocol for the multicentre randomised controlled OuTSMART trial

BackgroundRenal transplantation is the best treatment for kidney failure, in terms of length and quality of life and cost-effectiveness. However, most transplants fail after 10 to 12 years, consigning patients back onto dialysis. Damage by the immune system accounts for approximately 50% of failing transplants and it is possible to identify patients at risk by screening for the presence of antibodies against human leukocyte antigens. However, it is not clear how best to treat patients with antibodies. This trial will test a combined screening and treatment protocol in renal transplant recipients.Methods/DesignRecipients >1 year post-transplantation, aged 18 to 70 with an estimated glomerular filtration rate >30 mL/min will be randomly allocated to blinded or unblinded screening arms, before being screened for the presence of antibodies. In the unblinded arm, test results will be revealed. Those with antibodies will have biomarker-led care, consisting of a change in their anti-rejection drugs to prednisone, tacrolimus and mycophenolate mofetil. In the blinded arm, screening results will be double blinded and all recruits will remain on current therapy (standard care). In both arms, those without antibodies will be retested every 8 months for 3 years. The primary outcome is the 3-year kidney failure rate for the antibody-positive recruits, as measured by initiation of long-term dialysis or re-transplantation, predicted to be approximately 20% in the standard care group but <10% in biomarker-led care. The secondary outcomes include the rate of transplant dysfunction, incidence of infection, cancer and diabetes mellitus, an analysis of adherence with medication and a health economic analysis of the combined screening and treatment protocol. Blood samples will be collected and stored every 4 months and will form the basis of separately funded studies to identify new biomarkers associated with the outcomes.DiscussionWe have evidence that the biomarker-led care regime will be effective at preventing graft dysfunction and expect this to feed through to graft survival. This trial will confirm the benefit of routine screening and lead to a greater understanding of how to keep kidney transplants working longer.Trial registrationCurrent Controlled TrialsISRCTN46157828.

Molecular characterisation of the monocytic cell line THP-1 demonstrates a discrepancy with the documented HLA type.

Dear Editor, The use of the acute monocytic leukemia cell line, THP-1, has been well documented since its establishment was reported more than 30 years ago in the IJC. Since then, the THP-1 cell line has been used in countless research papers and is now a widely recognized resource as a model of monocyte cells within the immunology and oncology research communities. Routinely applied good cell culture practice of THP-1, as well as all other cell lines, requires quality assurance of the cell line, including assessment of relevant phenotypes and genotypes, state of differentiation and the assessment of any possible crosscontamination. In line with these requirements, our recent use of the THP-1 cell line revealed an inconsistency with the reported Human Leukocyte Antigen (HLA) type which may have potential consequences for some researchers. Initially THP-1 was recovered from our bank of frozen cell lines and cultured in RPMI 1640 medium (5% Fetal Calf Serum (FCS), 200 mM L-Glut, 100 lg/ml Pen/Strep) before HLA typing for HLA class I A and B, and the HLA class II DR and DQ antigens, via commercially available serological typing trays. These serological typing trays represent the contemporary equivalent of the assay used to determine the HLA type of THP-1 when the cell line was established. The results of these assays conclusively identified HLA-A2. ‘‘Weak’’ reactions against other class I antigens and crossreactivity, particularly between HLA-B5 and B15 antisera, confounded the identification of a full HLA-A and B type. The HLA class II antigens were entirely inconclusive. Since the THP-1 cell line was established in 1980, there have been marked advances within the detection methodologies used to determine HLA types, most notably the development of molecular biology-based techniques, such as the polymerase chain reaction (PCR). Therefore, to determine a conclusive HLA type for the THP-1 cell line, an ‘‘in-house’’ PCR using sequence specific primers (PCR-SSP) was performed for HLA-A*, B*, C*, DRB1*, DRB3*, DRB4*, DRB5* and DQB1* genes. In addition, an ABO group was determined using a similar method. Both of these in-house genotyping systems are based on methods which have been previously published. Using this assay, we identified the following HLA type for THP-1. HLA-A*02; B*15; C*03; DRB1*01, DRB1*15; DRB5*01/02; DQB1*05 and DQB1*06 (and ABO blood group genotype BB). The reported HLA type for THP-1 is HLA-A2, A9, B5, DRw1 and DRw2. (The HLA nomenclature system used here corresponds to the contemporary system at the time THP-1 was established in 1980). Clearly the HLA class I type did not correspond to the reported HLA type for THP-1, differing at the HLA class I A and B loci (to our knowledge HLA-C* has not previously been reported for THP-1). The HLA class II type corresponded to the original HLA type of HLA-DRw1 and DRw2, being DRB1*01 and DRB1*15. The expressed product of the HLA-DRB1*15 allele represents a split of the DR2 antigen and demonstrates the increased resolving capabilities of the PCR-SSP assay in comparison to the serological technique. (again to our knowledge HLA-DQB1* has not previously been reported). In contrast to the reported type, THP-1 appeared to be homozygous at the HLA-A, B and C loci by PCR-SSP. Alterations in the HLA class I phenotype of malignant cells is a frequent event during cancer progression, allowing tumor cells to evade the immune system. Indeed, loss of one major histocompatibility complex class I haplotype in human melanoma cells has been shown not only to allow evasion of immunosurveillance but also to increase their intrinsic oncogenic potential. The mechanisms which lead to HLA class I alterations can occur at any step required for HLA synthesis. Most commonly, the alteration represents a structural defect or a regulatory defect on the transcriptional level. Such alterations would not interfere with the PCR-SSP method used here, although a rarer deletion event upon chromosome 6 would be undetectable. In addition the ‘‘missing’’ class I HLA antigens from the original report show strong crossreactivity with the confirmed HLA type, and most likely account for this discrepancy, indeed our serological assay also demonstrated crossreactivity at the HLA-B5 and B15 antigens, which were proved erroneous by the PCR-SSP. Interestingly, the HLA-B*15 identified within the haplotype HLA-A*02; B*15; C*03, is unusual in itself, corresponding to the serological equivalent of B75 (15). The PCR-SSP was capable of resolving the B*15 allele to B*15:08 or B*15:11. A search on the Allele* Frequencies in World Populations website for each of these alleles revealed their frequency within specific populations. Of the two alleles B*15:11 appeared to be the more common, as, although extremely rare within the majority of populations, it is seen in the Le tt er to th e E di to r

Investigations into the lack of consensus in the reporting of HLA antibody specificities in the UK

Aims: Lack of consensus in HLA antibody reporting in proficiency schemes has previously been attributed to a number of differing factors. This study was set up to eliminate the majority of these factors by reducing analysis to a pure data handling exercise. Methods: Anonymised raw data files for LABScreen Single Antigen class I and II and related patient information were provided to seven participating centres. The centres reported back the HLA antibody specificities according to their single antigen bead reporting policy. Details of the reporting policy of each centre were retrospectively requested by questionnaire. Results: The number of HLA antibody specificities reported by the different centres varied widely. Software analysis called more HLA antibody specificities than any of the centres. None of the centres matched consensus for reported HLA class I specificities on any of the datasets, and no two centres reported the exact same HLA class I antibody profile; consensus was reached by one centre for HLA class II antibody specificities reported from two of the datasets. Retrospective review found data handling practice between centres to vary widely. Conclusions: Lack of agreement exists between UK centres in regard to HLA antibody specificity analysis. The fact that the required analysis was limited to interrogation of supplied data files makes the observation more concerning. The root cause of this variation is differences in data handling practice between the participating centres.

The UK National Registry of ABO and HLA Antibody Incompatible Renal Transplantation: Pretransplant Factors Associated With Outcome in 879 Transplants

Background ABO and HLA antibody incompatible (HLAi) renal transplants (AIT) now comprise around 10% of living donor kidney transplants. However, the relationship between pretransplant factors and medium-term outcomes are not fully understood, especially in relation to factors that may vary between centers. Methods The comprehensive national registry of AIT in the United Kingdom was investigated to describe the donor, recipient and transplant characteristics of AIT. Kaplan-Meier analysis was used to compare survival of AIT to all other compatible kidney transplants performed in the United Kingdom. Cox proportional hazards regression modeling was used to determine which pretransplant factors were associated with transplant survival in HLAi and ABOi separately. The primary outcome was transplant survival, taking account of death and graft failure. Results For 522 HLAi and 357 ABO incompatible (ABOi) transplants, 5-year transplant survival rates were 71% (95% confidence interval [CI], 66-75%) for HLAi and 83% (95% CI, 78-87%) for ABOi, compared with 88% (95% CI, 87-89%) for 7290 standard living donor transplants, and 78% (95% CI, 77-79%) for 15 322 standard deceased donor transplants (P < 0.0001). Increased chance of transplant loss in HLAi was associated with increasing number of donor specific HLA antibodies, center performing the transplant, antibody level at the time of transplant, and an interaction between donor age and dialysis status. In ABOi, transplant loss was associated with no use of IVIg, cytomegalovirus seronegative recipient, 000 HLA donor-recipient mismatch; and increasing recipient age. Conclusions Results of AIT were acceptable, certainly in the context of a choice between living donor AIT and an antibody compatible deceased donor transplant. Several factors were associated with increased chance of transplant loss, and these can lead to testable hypotheses for further improving therapy.

Successful kidney transplantation with a well-matched donor despite a positive crossmatch; detection and management of sensitization secondary to an alternate allelic variant of 'self' HLA.

Under current UK standards of deceased donor typing, the formation by the recipient of HLA antibody against an allelic variant of a ‘self’ antigen creates a particular problem for organ allocation. In the reported case, the decision to transplant was taken in the situation of a positive flow crossmatch result attributed to allelic antibody. The potential that target antigen density contributed to this patient’s subsequent good outcome is discussed.