Rhonda Holdsworth

Human Leukocyte Antigen Class I-Restricted Activation of CD8+ T Cells Provides the Immunogenetic Basis of a Systemic Drug Hypersensitivity

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçets disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

A Naturally Selected Dimorphism within the HLA-B44 Supertype Alters Class I Structure, Peptide Repertoire, and T Cell Recognition

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the α2 helix (B*4402 Asp156→B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this “minimal” mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Natural HLA class I polymorphism controls the pathway of antigen presentation and susceptibility to viral evasion

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion.

Review article: Luminex technology for HLA antibody detection in organ transplantation.

Since its inception in the early 1960s, the serologically based complement‐dependent cytotoxicity (CDC) assay has been the cornerstone technique for the detection of human leucocyte antigen (HLA) antibodies, not only in pre‐transplant renal patients, but also in other forms of organ transplantation. Recently, solid phase assays have been developed and introduced for this purpose, and in particular the Flow‐based bead assays such as the Luminex system. This latter assay has proved to be far more sensitive than the CDC assay and has revealed pre‐sensitization in potential transplant recipients not detected by other methods of HLA antibody detection. However, the clinical implications of this increased sensitivity have not been convincingly demonstrated until recently. This technology for HLA antibody detection permits the evaluation of the clinical importance of antibodies directed at, for example, HLA‐DPB1 and HLA‐DQA1, which has not been possible to date. There are Luminex issues, however, requiring resolution such as the ability to distinguish between complement fixing and non‐complement fixing antibodies and determination of their relative clinical significance. Luminex technology will permit a re‐evaluation of the role of HLA antibodies in both early and late antibody‐mediated rejection.

Solid phase HLA antibody detection technology – challenges in interpretation

The introduction into routine diagnostic laboratories of solid phase assays for human leukocyte antigen (HLA) antibody detection has resulted in the application of new laboratory matching algorithms in clinical organ transplantation which have improved pre-transplant detection of immunization, in turn resulting in avoidance of rejection in many cases which until their introduction would not have been possible using the historical complement dependent serological techniques. There have been two generations of solid phase assays introduced into routine practice, namely, the enzyme-linked immunosorbent assay (ELISA) technique and the use of fluorescent beads with HLA molecules bound to their surface which can either be used in conventional flow cytometry or in conjunction with Luminex instrumentation, the latter having become the most popular approach. The use of the fluorescent bead techniques has raised interesting questions both with respect to technical performance and the interpretation of the results obtained. The advantages of bead technology for HLA antibody determination and the technical issues requiring resolution are the subject of this review.

Virtual Crossmatch Approach to Maximize Matching in Paired Kidney Donation

We developed and tested a new computer program to match maximal sets of incompatible live donor/recipient pairs from a national paired kidney donation (PKD) registry. Data of 32 incompatible pairs included ABO and 4 digit‐high‐resolution donor and recipient HLA antigens and recipients HLA antibodies. Three test runs were compared, in which donors were excluded from matching to recipients with either donor‐specific antibodies (DSA) >8000MFI (mean fluorescent intensity) at low‐resolution (Run 1) or >8000MFI at high‐resolution (Run 2) or >2000MFI and high‐resolution (Run 3). Run 1 identified 22 703 possible combinations, with 20 pairs in the top ranked, Run 2 identified 24 113 combinations, with 19 pairs in the top ranked and Run 3 identified 8843 combinations, with 17 pairs in the top ranked. Review of DSA in Run 1 revealed that six recipients had DSA 2000–8000MFI causing a possible positive crossmatch resulting in breakdown of two 3‐way and three 2‐way chains. In Run 2, four recipients had DSA 2000–8000MFI, also potentially causing breakdown of three 2‐way chains. The more prudent approach of excluding from matching recipients with DSA with >2000MFI reduces the probability of matched pairs having a positive crossmatch without significantly decreasing the number of possible transplants.

Donor methylenetetrahydrofolate reductase genotype is associated with graft-versus-host disease in hematopoietic stem cell transplant patients treated with methotrexate

Methotrexate (MTX), used as a graft-versus-host disease (GvHD) prophylactic agent in hematopoietic stem cell transplantation (HSCT), exerts its effect via folate cycle inhibition. A critical enzyme involved in folate metabolism is 5,10-methylenetetrahydrofolate reductase (MTHFR). We examined the association of a single nucleotide polymorphism (SNP) at position 677 in the MTHFR gene on GvHD outcomes in allogeneic HSCT patients administered MTX. MTHFR genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 193 HSCT patients and donors. A total of 140 patients were transplanted with an HLA-matched related donor and 53 with an unrelated donor. GvHD outcomes were compared between genotypes by univariate and multivariate analysis. The combined donor 677CT and TT genotypes were associated with a decreased incidence of GvHD (acute and chronic combined) in HSCT recipients with an HLA-matched related donor (75% at 1 year in the CT and TT group compared with 91% in the wild type CC group, P=0.01), increased time to onset of first GvHD (P=0.001) and time to first GvHD treated with systemic therapy (P=0.022). Unrelated donor MTHFR genotype was not associated with outcome parameters and no associations of recipient genotype in either related or unrelated donor cohorts were observed.

Rapid screening for the detection of HLA-B57 and HLA-B58 in prevention of drug hypersensitivity

HLA-B57 and HLA-B58 are major histocompatibility class (MHC)-I allotypes that are potentially predictive of important clinical immune phenotypes. HLA-B*5701 is strongly associated with hypersensitivity to the HIV drug abacavir, liver toxicity from the antibiotic flucloxacillin and is a marker for slow progression of HIV AIDS. HLA-B*5801 is associated with hypersensitivity to allopurinol used to treat hyperuricaemia and recurrent gout. Here we describe a monoclonal antibody (mAb) specific for HLA-B57 and HLA-B58 that provides an inexpensive and sensitive screen for these MHC-I allotypes. The usefulness of HLA-B57 screening for prediction of abacavir hypersensitivity was shown in three independent laboratories, including confirmation of the mAb sensitivity and specificity in a cohort of patients enrolled in the PREDICT-1 trial. Our data show that patients who test negative by mAb screening comprise 90%-95% of all individuals in most human populations and require no further human leukocyte antigen (HLA) typing. Patients who test positive by mAb screening should proceed to high-resolution typing to ascertain the presence of HLA-B*5701 or HLA-B*5801. Hence, mAb screening provides a low-cost alternative to high-resolution typing of all patients and lends itself to point-of-care diagnostics and rapid ascertainment of low-risk patients who can begin immediate therapy with abacavir, flucloxacillin or allopurinol.

High transplant rates of highly sensitized recipients with virtual crossmatching in kidney paired donation

Background In kidney paired donation (KPD), flexibility in the allocation of incompatible pairs is required if a critical mass of pairs to efficiently find matches cannot be reached. Methods In the Australian KPD program, virtual crossmatch is used for the allocation of suitable donors to registered recipients. Matching is based on acceptable mismatches, and donors are excluded from matching to recipients with donor-specific antibodies (DSAs) greater than 2000 mean fluorescence intensity (MFI). Match and transplant rates in the first year of the program were reviewed with respect to recipient and donor characteristics, including blood group distribution, level of recipient’s sensitization, and postallocation crossmatches. Results Four quarterly match runs were performed, which included 53 pairs and 2 altruistic donors. Human leukocyte antigen incompatibility accounted for 90% of the listed pairs. In the second run, the DSA threshold was increased to greater than 8000 MFI, because no matches were found with standard allocation. Optional ABO-incompatible matching was introduced from run 3. Matches were identified in 37 (70%) patients, of whom 92% had a negative crossmatch with their matched donor. Crossmatch positive results were found only in recipients with DSAs greater than 2000 MFI in the second run. In 4 cases immunological reasons and in 4 cases other reasons resulted in breakdown of chains and 17 patients not progressing to transplantation. Eventually, 20 (38%) patients received a KPD transplant, and 35% of these had a calculated panel-reactive antibody greater than 90%. Conclusions KPD using virtual crossmatch is a valid and effective solution for patients with immunologically incompatible donors even in the context of highly sensitized recipients.

Immunodominance Hierarchies and Gender Bias in Direct TCD8‐Cell Alloreactivity

Allogeneic solid organ transplantation often occurs across multiple donor–recipient HLA mismatches with consequent risk of allograft rejection. However, there is growing evidence that not all HLA mismatches are equivalent in their stimulation of allogeneic T cells making it important to determine which of these might be more significant as predictors of allograft rejection. To this end, we used defined antigen‐presenting cell (APC) transfectants expressing single MHC‐I allotypes as target cells that could discriminate the relative contribution of individual mismatched MHC‐I allotypes to direct T‐cell alloreactivity. We demonstrate remarkably reproducible patterns of immunodominance in reactivity across mismatched MHC‐I allotypes. These patterns are HLA context‐dependent, partly reflecting alloantigenic competition in responder cell responses. In strong alloresponses, we also observed an increased percentage of alloreactive TCD8 cells in female responders, regardless of the stimulator gender, highlighting HLA‐independent factors in the potency of the alloresponse. This approach provides a potential measure of specific alloreactive T cells that could be used in clinical practice for selection of donors, assessment of posttransplant outcomes, modulation of immunosuppression and detection of rejection episodes.