Modeling the benefits and costs of integrating an acceptable HLA mismatch allocation model for highly sensitized patients.

BACKGROUND: The Eurotransplant acceptable mismatch program has improved transplantation access for highly sensitized recipients. However, the benefits and costs of implementing such a program remain unknown. METHODS: Using decision analytical modeling, we compared the average waiting time for transplantation, overall survival gains (in life-years and quality-adjusted life-years gained), and costs of integrating an acceptable mismatch allocation model compared with the current deceased-donor kidney allocation model in Australia. RESULTS: Acceptable mismatches were identified in 12 of 28 (43%) highly sensitized recipients using HLAMatchmaker. Inclusion of acceptable mismatches in the current allocation model improved the transplantation access for four (14%) highly sensitized recipients, with an average reduction in waiting time of 34 months (from 86 to 52 months). Compared with the current allocation model, incorporating an acceptable mismatch allocation model achieved an overall lifetime gain of 0.034 quality-adjusted life-years and savings of over $4,000 per highly sensitized patient, with a small consequential loss of 0.005 quality-adjusted life-years and extra costs of $800 for every reallocated patient. CONCLUSIONS: Despite modest overall health gains, application of an acceptable mismatch allocation model is an equitable approach to improve transplantation access for highly sensitized transplant candidates without compromising the overall health benefits among the other patients on the deceased-donor waitlist in Australia.

Killer Ig-like receptor 2DL4 does not mediate NK cell IFN-γ responses to soluble HLA-G preparations.

The MHC class Ib molecule HLA-G has previously been reported to be the ligand for the NK cell receptor killer Ig-like receptor (KIR)2DL4, but this remains controversial. In this study, we investigated IFN-γ production by freshly isolated NK cells in response to both soluble and solid-phase ligands, including anti-KIR2DL4 mAbs and rHLA-G. Although freshly isolated CD56(bright) NK cells produced IFN-γ in response to soluble HLA-G preparations, the response was found to be absolutely dependent on the presence of small numbers of contaminating CD56(-), CD14(-), CD11c(+) myeloid dendritic cells (mDCs). HLA-G tetramers bound only to the contaminating mDCs in the NK preparations, and Abs to KIR2DL4 and HLA-G did not block NK cell IFN-γ production. NK cells did not respond to plate-bound HLA-G. Freshly isolated NK cells also produced IFN-γ in response to unpurified soluble anti-KIR2DL4 mAb but not to low endotoxin affinity-purified Ab. The data suggest that previous reports of functional interactions between KIR2DL4 and HLA-G may have resulted from the use of purified NK cells that were contaminated with mDCs and HLA-G preparations that were contaminated with material capable of stimulating mDCs to produce cytokines that stimulate NK cells to produce IFN-γ.

Peri-operative third party red blood cell transfusion in renal transplantation and the risk of antibody-mediated rejection and graft loss.

Historic red blood cell transfusion (RBCT) may induce anti-HLA antibody which, if donor specific (DSA), is associated with increased antibody-mediated rejection (AMR). Whether post-operative RBCT influences this risk is unknown. We examined the RBCT history in 258 renal transplant recipients stratified according to prevalent recipient HLA antibody (DSA, Non-DSA or No Antibody). AMR occurred more frequently in patients who received RBCT both pre and post transplant compared with all other groups (Pre+Post-RBCT 21%, Pre-RBCT 4%, Post-RBCT 6%, No-RBCT 6%, HR 4.1 p=0.004). In the 63 patients who received Pre+Post-RBCT, 65% (13/20) with DSA developed AMR compared with 0/6 in the Non-DSA group and 2/37 (5%) in the No-Antibody group (HR 13.9 p<0.001). In patients who received No-RBCT, Pre-RBCT or Post-RBCT there was no difference in AMR between patients with DSA, Non-DSA or No-Antibody. Graft loss was independently associated with Pre+Post-RBCT (HR 6.5, p=0.001) AMR (HR 23.9 p<0.001) and Non-AMR (6.0 p=0.003) after adjusting for DSA and delayed graft function. Re-exposure to RBCT at the time of transplant is associated with increased AMR only in patients with preformed DSA, suggesting that RBCT provides additional allostimulation. Patients receiving Pre+Post-RBCT also had an increased risk of graft loss independently of AMR or DSA. Both pre and post procedural RBCT in renal transplantation is associated with modification of immunological risk and warrants additional study.

The influence of non-HLA gene polymorphisms and interactions on disease risk in a Western Australian multiple sclerosis cohort.

Non-Human Leukocyte Antigen (HLA) genes have concomitant, although modest, effects on multiple sclerosis (MS) susceptibility; however findings have varied in different populations. Here we present the results of an association study of 16 single nucleotide polymorphisms (SNPs) in 10 non-HLA genes (IL7R, IL2RA, CLEC-16A, TYK2, CD58, IRF5, STAT3, CTLA-4, APOE, ICAM-1) in a Western Australian cohort of 350 MS patients and 498 population control subjects. Our results indicate that in this population, SNPs in IL7R, TYK2, IRF5 and APOE have modifying effects on MS susceptibility. We also found evidence of interactive protective effects between polymorphisms in the IL7R/CD58, CLEC-16A/CTLA-4, and TYK2/IRF5 genes, which in some instances are restricted within HLA- or gender-defined groups.

Pre-transplant donor specific anti-HLA antibody is associated with antibody-mediated rejection, progressive graft dysfunction and patient death.

BACKGROUND: The long term effect of donor specific antibodies (DSA) detected by Luminex Single Antigen Bead (SAB) assay in the absence of a positive complement-dependant cytotoxicity (CDC) crossmatch is unclear. DSA at the time of transplant were determined retrospectively in 258 renal transplant recipients from 2003 to 2007 and their relationship with rejection and graft function prospectively evaluated. After a median of 5.6 years follow-up 9% of patients had antibody mediated rejection (AMR) (DSA 11/37 (30%), DSA-Neg 13/221 (6%), HR 6.6, p<0.001). Patients with anti-HLA class II (HR 6.1) or both class I+II (HR 10.1) DSA had the greatest risk for AMR. The Mean Fluorescent Intensity (MFI) of the DSA was significantly higher in patients with AMR than those with no rejection (p=0.006). Moreover, the strength of the antibody was shown to be important, with the risk of AMR significantly greater in those with DSA >8000 MFI than those with DSA <8000 MFI (HR 23, p<0.001). eGFR progressively declined in patients with DSA but was stable in those without DSA (35.7 ± 20.4 mls/min vs 48.5 ± 22.7) and composite patient and graft survival was significantly worse in those with class II (HR 2.9) or both class I+II (HR 3.7) but not class I DSA. Class II DSA alone, or in combination with class I DSA had the strongest association with graft loss and patient death. Patients with DSA not only have increased rates of acute AMR, but also chronic graft dysfunction, graft loss and death. Antibody burden quantified by SAB assay may identify patients at highest immunological risk and therefore influence patient management and improve long-term patient outcome.

Transnational validation of the Australian algorithm for virtual crossmatch allocation in kidney paired donation.

An independent pool of 16 incompatible live donor-recipient pairs registered at the Vienna transplant unit was applied to test whether virtual crossmatch allocation used in the Australian kidney paired donation (KPD) program reliably predicts negative crossmatches. High resolution HLA data were entered into the computer-matching algorithm and allocation was performed excluding any DSA>2000MFI. CDC and flow crossmatch data of recipients against any of the donors were available for 112 crossmatch combinations. The computer program identified 19 possible pairings in 2-way or 3-way chains in multiple combinations. The top ranked combination included one 3-way and two 2-way ABO-compatible chains. Where crossmatches were available all recipients were CDC crossmatch negative with the computer-matched donor. Excluding allocation of KPD donors in the presence of DSA>2000MFI had a negative predictive of 99.9% for CDC and 96.4% for flow crossmatch. In the 12 pairings with ⩾1 DSA against crossmatched donors there was a negative CDC and flow crossmatch. These results show excellent correlation between matching using virtual crossmatch and actual crossmatch results. Using the 2000MFI cut-off the number of potentially unacceptable CDC and flow crossmatch positive pairings identified by virtual crossmatching is low, but some potential crossmatch negative pairings are missed.

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.

Contributions of vitamin D response elements and HLA promoters to multiple sclerosis risk.

OBJECTIVE: The identification of a vitamin D-responsive (VDRE) motif within the HLA-DRB1*15:01 promoter region provides an attractive explanation for the combined effects of HLA-DR inheritance and vitamin D exposure on multiple sclerosis (MS) risk. We therefore sought to incorporate HLA-DRB1 promoter variation, including the VDRE motif, in an assessment of HLA-DRB1-associated MS risk. METHODS: We utilized 32 homozygous HLA cell lines (covering 17 DRB1 alleles) and 53 heterozygote MS samples (20 DRB1 alleles) for HLA-DRB1 promoter sequencing. The influence of HLA-DRB1 variation on MS risk was then assessed among 466 MS cases and 498 controls. RESULTS: The majority of HLA*DRB1 alleles (including HLA-DRB1*15:01) express the functional VDRE motif, apart from HLA-DRB1*04, *07, and *09 alleles that comprise the HLA-DR53 serologic group. Allele-specific variation within functional X-box and Y-box motifs was also associated with serologically defined HLA-DR haplotypes. Incorporating these results in an analysis of MS risk, we identified a strong protective effect of HLA-DRB1*04, *07, and *09 (DR53) alleles (p = 10(-12)) and elevated risk associated with DRB1*15 and *16 (DR51) and *08 (DR8) alleles (p < 10(-18)). CONCLUSIONS: HLA-DRB1 groups corresponding to serologic HLA-DR profiles as well as promoter polymorphism haplotypes effectively stratified MS risk over an 11-fold range, suggesting functional relationships between risk-modifying HLA-DRB1 alleles. An independent contribution of VDRE motif variation to increase MS risk was not discernible, although vitamin D-dependent regulation of HLA-DR expression may still play an important role given that HLA-DRB1*04/*07/*09 (DR53) alleles that express the "nonresponsive" VDRE motif were associated with significantly reduced risk of MS.

The detection of NK cell alloreactivity by flow cytometric CD107a assay.

Natural killer (NK) cell alloreactivity can be exploited in haploidentical (one haplotype mismatched) haematopoietic stem cell transplantation (HSCT) to prevent leukaemia relapse, rejection, and graft-vs-host disease (GVHD) (Blood 94:333-339; Science 295:2097-2100). If NK cell alloreactivity is to be exploited in HSCT, it is important to be able to reliably select donors who have NK alloreactivity towards the patient. The detection of donor NK alloreactivity towards patient target cells has traditionally been evaluated by NK cell cloning and (51)Cr-release cytotoxicity assay. This approach is complex and time consuming with results taking up to 6 weeks. Here, we detail a novel flow cytometric CD107a-based assay capable of detecting NK cell alloreactivity in 14 days.

Spinal cord involvement in multiple sclerosis: a correlative MRI and high-resolution HLA-DRB1 genotyping study.

BACKGROUND: There have been few magnetic resonance imaging (MRI) studies of the spinal cord in large multiple sclerosis (MS) patient cohorts and little is known about correlations between cord lesions and human leukocyte antigen (HLA) alleles. OBJECTIVE: To investigate the spectrum of MRI changes in the spinal cord in MS and associations with the HLA-DRB1 genotype. METHODS: Two hundred and fifty two consecutive MS patients from the Perth Demyelinating Diseases Database had MRI of the spinal cord and brain and high-resolution HLA-DRB1 genotyping. The numbers, locations, shape and segmental extent of cord lesions were analysed and were correlated with carriage of individual HLA-DRB1 alleles and diplotypes. RESULTS: Focal cord lesions were present in 82.9% of cases, with numbers being maximal in the cervical cord and increasing with disease duration. Focal lesions were usually round or oval in shape but in 35% of cases subpial wedge-shaped lesions were present. Diffuse cord involvement was present in 10% of cases and correlated with carriage of HLA-DRB1*1501 and with higher disability. Carriage of the minor allele HLA-DRB1*0701 was significantly associated with numbers of wedge-shaped lesions and lesions in the cervical cord, while HLA-DRB1*1104 and DRB1*0103 were significantly associated respectively with higher and lower numbers of thoracic cord lesions. HLA-DRB1*1501 and the HLA-DRB1*11 sub-alleles DRB1*1101 and DRB1*1104 were significantly associated with the segmental length of cord lesions. CONCLUSIONS: Our study is the first to investigate the frequency of subpial wedge-shaped lesions in the cord in vivo and has provided preliminary evidence that HLA-DRB1 alleles may play a role in determining the severity and extent of spinal cord involvement in MS.

Clinical profile and HLA-DRB1 genotype of late onset multiple sclerosis in Western Australia.

We aimed to characterize the clinical profile and human leukocyte antigen (HLA)-DRB1 genotype of patients with late onset multiple sclerosis (LOMS) in Western Australia. The clinical features, laboratory studies and HLA-DRB1 alleles were analysed in patients with multiple sclerosis (MS) with onset over 50years of age and compared with 100 patients with early onset MS (EOMS). Of a cohort of 829 patients with MS, 73 (8.8%) presented at over 50years of age, including 14 (1.7%) over 60years. Patients with LOMS had a lower female to male ratio, more frequent initial motor dysfunction, less frequent sensory symptoms and optic neuritis, a more frequent primary-progressive course and shorter time to reach Extended Disability Status Scale (EDSS) scores of 3.0 and 6.0. More LOMS patients were initially misdiagnosed compared to patients with EOMS. HLA-DRB1 *1501 was strongly associated with both LOMS and EOMS compared to the Control subjects, while HLA-DRB1 *0801 was over-represented in patients with LOMS. We concluded that patients with LOMS have a different clinical profile when compared to those with EOMS. Carriers of HLA-DRB1 *0801 may be more prone to develop MS at a later age.

Influence of HLA-DRB1 allele heterogeneity on disease risk and clinical course in a West Australian MS cohort: a high-resolution genotyping study.

BACKGROUND: Previous studies on the influence of HLA-DRB1 alleles on multiple sclerosis (MS) susceptibility and clinical course have mostly employed the 2-point genotyping method. OBJECTIVE: To assess the influence of HLA-DRB1 alleles and allele interactions on disease risk and clinical course in a large West Australian MS patient cohort using high-resolution genotyping. METHODS: Four digit HLA-DRB1 genotyping was performed on a group of 466 clinically definite or probable MS patients from the Perth Demyelinating Diseases Database and 189 healthy Caucasian controls from the Busselton Community Health Study. RESULTS: In addition to the known risk allele HLA-DRB1*1501, evidence of increased susceptibility to MS was found for three additional alleles, DRB1*0405, DRB1*1104 and DRB1*1303, though the power was insufficient to sustain significance for these when crudely Bonferroni corrected over all alleles considered. DRB1*0701 was found to be protective even after correction for multiple comparisons. In addition we found evidence that the DRB1*04 sub-allele HLA-DRB1*0407 and HLA-DRB1*0901 may be protective. Among the diplotypes, the highest estimated risk was in HLA-DRB1*1501/*0801 heterozygotes and DRB1*1501 homozygotes and the lowest in HLA-DRB1*0701/*0101 heterozygotes. There was no significant gender association with HLA-DRB1*1501 overall, but the HLA-DRB1*1501/*1104 risk genotype was significantly associated with female gender. HLA-DRB1*1501 was the strongest risk allele in both primary progressive MS and relapsing-remitting MS. CONCLUSION: Our results demonstrate the advantages of high-resolution HLA genotyping in recognizing risk-modifying alleles and allele combinations in this patient cohort and in recognizing the differential effects of HLA-DRB1*04 and DRB1*11 sub-alleles.

Presence of CSF oligoclonal bands (OCB) is associated with the HLA-DRB1 genotype in a West Australian multiple sclerosis cohort.

High-resolution HLA-DRB1 genotyping was performed in 97 OCB-positive and 68 OCB-negative cases with demyelinating disease to determine the influence of HLA-DRB1 alleles on the presence of OCB in a West Australian multiple sclerosis (MS) cohort. Carriage of the HLA-DRB1*1501 allele was associated with both OCB-positive and OCB-negative MS compared with controls, but more strongly with the OCB-positive group, and increased the likelihood of having OCB 2.1-fold with evidence of a dominant dose-effect. The HLA-DRB1*0301 allele was negatively correlated with OCB, with all homozygotes OCB-negative, suggesting a possible recessive protective effect of HLA-DRB1*0301. There was no significant correlation between OCB and the DRB1*04 alleles which have been associated with OCB-negative MS in previous Swedish and Japanese studies. Evidence of allelic interactions was found with HLA-DRB1*1501/*1301 heterozygotes having a reduced frequency of OCB and HLA-DRB1*0301/*0401 heterozygotes all being OCB-negative. These findings confirm the strong association between HLA-DRB1*1501 and OCB which has been found in other populations but indicate that the influence of other HLA-DRB1 alleles varies in different populations. Our study is the first to show that HLA-DRB1 allele interactions and dose-effects influence the frequency of OCB.

Rapid, flow cytometric assay for NK alloreactivity reveals exceptions to rules governing alloreactivity.

Alloreactive NK cells lyse target cells lacking self-HLA-C or the HLA-B-Bw4 epitope. Prior to haploidentical stem cell transplants, donor alloreactivity toward the patient is evaluated by natural killer (NK) cloning followed by testing of the clones in the (51)Cr-release assay. As only a few percent of NK clones are alloreactive, a large number of NK clones must be established and evaluated. This approach is laborious and time consuming, with a complete evaluation taking up to 6 weeks. We developed a flow cytometry-based cytotoxicity assay utilizing CD107a expression on 12-day polyclonally expanded NK cells and showed that NK alloreactivity mediated by inhibitory and activating KIR can be detected by measuring CD107a expression following incubation with targets lacking the appropriate class I epitope. The percentage of alloreactive NK cells varied greatly between individuals and was easily estimated by the CD107a assay. For each epitope (C1, C2, Bw4), donors were found who did not have alloreactivity, although alloreactivity was predicted by the current rules thought to govern alloreactivity. The data emphasize the importance of demonstrating alloreactivity in a functional assay.

HLA-DRB1 allele heterogeneity influences multiple sclerosis severity as well as risk in Western Australia.

Susceptibility to multiple sclerosis (MS) has been consistently associated with the Human Leukocyte Antigen (HLA)-DRB11501 genotype, however effects on disease severity and clinical outcome have varied in different populations. We present the results of a high-resolution HLA-DRB1 genotyping and genotype-phenotype correlation study in a large West Australian MS cohort. Our findings indicate that in this population, which is of largely Anglo-Celtic and Northern European origin, HLA-DRB11501 is not only a strong determinant of disease risk but may also be associated with disease severity as measured by the Multiple Sclerosis Severity Score (MSSS), with the MSSS increasing by an estimated 0.51 per DRB11501 allele. We also found evidence that the HLA-DRB11201 allele is associated with less severe disease.

Modifying effects of HLA-DRB1 allele interactions on age at onset of multiple sclerosis in Western Australia.

The contribution of genetic factors to the age at onset in multiple sclerosis is poorly understood. Our objective was to investigate the disease modifying effects of HLA-DRB1 alleles and allele interactions on age at onset of multiple sclerosis. High-resolution four-digit HLA-DRB1 genotyping was performed in a cohort of 461 multiple sclerosis patients from the Perth Demyelinating Diseases Database. Carriage of the HLA-DRB1*1501 risk allele was not significantly associated with age at onset but HLA-DRB1*0801 was associated with a later onset of the disease. The HLA-DRB1*0401 allele was associated with a reduced age at onset when combined with DRB1*1501 but may delay age at onset when combined with DRB1*0801. These findings indicate that epistatic interactions at the HLA-DRB1 locus have significant modifying effects on age at onset of multiple sclerosis and demonstrate the value of high-resolution genotyping in detecting such associations.

Sporadic inclusion body myositis: HLA-DRB1 allele interactions influence disease risk and clinical phenotype.

Susceptibility to sIBM is strongly associated with the HLA-DRB1*03 allele and the 8.1 MHC ancestral haplotype (HLA-A1, B8, DRB1*03) but little is known about the effects of allelic interactions at the DRB1 locus or disease-modifying effects of HLA alleles. HLA-A, B and DRB1 genotyping was performed in 80 Australian sIBM cases and the frequencies of different alleles and allele combinations were compared with those in a group of 190 healthy controls. Genotype-phenotype correlations were also investigated. Amongst carriers of the HLA-DRB1*03 allele, DRB1*03/*01 heterozygotes were over-represented in the sIBM group (p<0.003) while. DRB1*03/*04 heterozygotes were under-represented (p<0.008). The mean age-at-onset (AAO) was 6.5 years earlier in DRB1*03/*01 heterozygotes who also had more severe quadriceps muscle weakness than the rest of the cohort. The findings indicate that interactions between the HLA-DRB1*03 allele and other alleles at the DRB1 locus can influence disease susceptibility and the clinical phenotype in sIBM.

Natural killer cell allorecognition of missing self in allogeneic hematopoietic transplantation: a tool for immunotherapy of leukemia.

Donor-versus-recipient natural killer (NK) cell alloreactivity has been established as a key therapeutic element in HLA haplotype mismatched hematopoietic transplants in adult AML and pediatric ALL and as a possible beneficial effector in cord blood transplant for AML. It is effected by functional NK cells which express inhibitory killer cell immunoglobulin-like receptor(s) (KIR) for self-class I ligand(s), sense missing expression of donor KIR ligand(s) in the recipient and mediate alloreactions. At present NK cell allotherapy for leukemia is deployed through stem cell transplantation (and ensuing NK cell reconstitution) across KIR ligand mismatches. Studies have been performed to infuse NK cells for immunotherapy outside the fields of transplantation and/or harness the function of endogenous NK cells in patients with hematological malignancies.