Resolving unknown nucleotides in the IPD-IMGT/HLA database by extended and full-length sequencing of HLA class I and II alleles.

In the past, identification of HLA alleles was limited to sequencing the region of the gene coding for the peptide binding groove, resulting in a lack of sequence information in the HLA database, challenging HLA allele assignment software programs. We investigated full-length sequences of 19 HLA class I and 7 HLA class II alleles, and we extended another 47 HLA class I alleles with sequences of 5' and 3' UTR regions that were all not yet available in the IPD-IMGT/HLA database. We resolved 8638 unknown nucleotides in the coding sequence of HLA class I and 2139 of HLA class II. Furthermore, with full-length sequencing of the 26 alleles, more than 90 kb of sequence information was added to the non-coding sequences, whereas extension of the 47 alleles resulted in the addition of 5.5 kb unknown nucleotides to the 5' UTR and > 31.7 kb to the 3' UTR region. With this information, some interesting features were observed, like possible recombination events and lineage evolutionary origins. The continuing increase in the availability of full-length sequences in the HLA database will enable the identification of the evolutionary origin and will help the community to improve the alignment and assignment accuracy of HLA alleles.

Ellipro scores of donor epitope specific HLA antibodies are not associated with kidney graft survival.

In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants.

Determination of the clinical relevance of donor epitope-specific HLA-antibodies in kidney transplantation.

In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level.

Report from the extended HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype of the 18th international HLA and immunogenetics workshop.

The primary goal of the HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype component of the 18th IHIWS was to characterise the extended haplotypes within the HLA-DP region and survey the extent of genetic diversity in this region across human populations. In this report, we analysed single-nucleotide polymorphisms (SNPs) in 255 subjects from 6 different cohorts. The results from the HLA-DP haplotype component have validated findings from the initial pilot study. SNPs in this region were inherited in strong linkage, particularly HLA-DPA1, SNP-linked promoter haplotypes and motifs in exon 2 of HLA-DPB1. We reported 17 SNP-linked haplotypes in the promoter region. Together with HLA-DPA1 and HLA-DPB1 alleles, they formed 74 distinct extended HLA-DP haplotypes in 438 sequences. We also observed the presence of region-specific alleles and promoter haplotypes. Our approach involved phasing extended SNPs including promoter SNPs, HLA-DPA1 and HLA-DPB1 alleles, in a 22 kb region, GRCh38/hg38 (chr6:33,064,111-33,086,679), followed by clustering of these SNPs as one extended haplotype. This hierarchical clustering revealed four major clades, suggesting that haplotypes within each clade may have diverged from a common ancestral haplotype and undergone similar evolutionary processes. The correlation between HLA-DPA1 and the promoter region raises questions about the role of HLA-DPA1 antigen in the heterodimer. This finding requires validation on a larger sample size specifically designed for anthropological analysis. Nevertheless, the results from this study highlight the clinical potential of selecting better-matched donors for patients awaiting haematopoietic stem cell transplants from genetically overlapping groups that share common ancestral haplotypes.

Polymorphic differences within HLA-C alleles contribute to alternatively spliced transcripts lacking exon 5.

The HLA genes are amongst the most polymorphic in the human genome. Alternative splicing could add an extra layer of complexity, but has not been studied extensively. Here, we applied an RNA based approach to study the influence of allele polymorphism on alternative splicing of HLA-C in peripheral blood. RNA was isolated from these peripheral cells, converted into cDNA and amplified specifically for 12 common HLA-C allele groups. Through subsequent sequencing of HLA-C, we observed alternative splicing variants of HLA-C*04 and *16 that resulted in exon 5 skipping and were co-expressed with the mature transcript. Investigation of intron 4 sequences of HLA-C*04 and *16 compared with other HLA-C alleles demonstrated no effect on predicted splice sites and branch point. To further investigate if the unique polymorphic positions in exon 5 of HLA-C*04 or *16 may facilitate alternative splicing by acting on splicing regulatory elements (SRE), in-silico splicing analysis was performed. While the HLA-C*04 specific SNP in exon 5 had no effect on predicted exonic SRE, the HLA-C*16 specific exon 5 SNP did alter exonic SRE. Our findings provide experimental and theoretical support for the concept that polymorphisms within the HLA-C alleles influence the alternative splicing of HLA-C.

T-Cell Epitopes Shared Between Immunizing HLA and Donor HLA Associate With Graft Failure After Kidney Transplantation.

CD4+ T-helper cells play an important role in alloimmune reactions following transplantation by stimulating humoral as well as cellular responses, which might lead to failure of the allograft. CD4+ memory T-helper cells from a previous immunizing event can potentially be reactivated by exposure to HLA mismatches that share T-cell epitopes with the initial immunizing HLA. Consequently, reactivity of CD4+ memory T-helper cells toward T-cell epitopes that are shared between immunizing HLA and donor HLA could increase the risk of alloimmunity following transplantation, thus affecting transplant outcome. In this study, the amount of T-cell epitopes shared between immunizing and donor HLA was used as a surrogate marker to evaluate the effect of donor-reactive CD4+ memory T-helper cells on the 10-year risk of death-censored kidney graft failure in 190 donor/recipient combinations using the PIRCHE-II algorithm. The T-cell epitopes of the initial theoretical immunizing HLA and the donor HLA were estimated and the number of shared PIRCHE-II epitopes was calculated. We show that the natural logarithm-transformed PIRCHE-II overlap score, or Shared T-cell EPitopes (STEP) score, significantly associates with the 10-year risk of death-censored kidney graft failure, suggesting that the presence of pre-transplant donor-reactive CD4+ memory T-helper cells might be a strong indicator for the risk of graft failure following kidney transplantation.

Polymorphism clustering of the 21.5 kb DPA-promoter-DPB region reveals novel extended full-length haplotypes.

DPB1 and DPA1 genes share the same promoter region. Single-nucleotide polymorphisms (SNPs) within the regulatory regions of DP have been reported. This study hypothesizes that by including the SNPs in the promoter region of DP, extended haplotypes are defined, and promoter polymorphism is more extensive than what is currently reported. To identify the SNPs in the region of interest, the DP region spanning 21.5 kb was amplified in three separate long-ranged polymerase chain reactions. A DNA panel consisting of 100 samples was selected to represent a broad range of DPB1 alleles. The panel was amplified and sequenced using a dual sequencing strategy. Binary alignment map (BAM) alignments were generated and the mapped sequence alignments were analyzed using Integrative Genomics Viewer. A total of 76 SNPs were identified, and SNPs were clustered into 12 SNP-linked haplotypes. Multiple sequence alignments of promoter sequences indicated four distinct lineages within the connective region (CR) between two genes. The relationship between DPA1, CR, DPB1, and amino acid motifs was found to be correlated with HV1 and HV6. Of the 12 promoter haplotypes, DPB1 alleles observed with ProDP-4 were in complete linkage with HV1/2/5/6, the rs9277534G SNP, and the highly immunogenic T-cell epitope group. Multiple extended haplotypes of different intronic subtypes of the same DPB1 alleles were also identified. This new view of the full DP haplotype shows the relation of polymorphism, genes, and alleles, and provides a basis for future functionality related nomenclature. The novel clustering of the DP-extended haplotype warrants future investigations of DP haplotype matching in the outcome of haematopoietic stem cell transplantation (HSCT).

Specific amino acid patterns define split specificities of HLA-B15 antigens enabling conversion from DNA-based typing to serological equivalents.

The HLA-B15 typing by serological approaches defined the serological subgroups (or splits) B62, B63, B75, B76, B77 and B70 (B71 and B72). The scarcity of sera with specific anti-HLA antibodies makes the serological typing method difficult to discriminate a high variety of HLA antigens, especially between the B15 antigen subgroups. Advancements in DNA-based technologies have led to a switch from serological typing to high-resolution DNA typing methods. DNA sequencing techniques assign B15 specificity to all alleles in the HLA-B*15 allele group, without distinction of the serological split equivalents. However, the presence of antibodies in the patient defined as split B15 antigens urges the identification of HLA-B*15 allele subtypes of the donor, since the presence of donor-specific antibodies is an important contraindication for organ transplantation. Although the HLA dictionary comprises information regarding the serological subtypes of HLA alleles, there are currently 394 B15 antigens out of 516 in the IPD-IMGT/HLA database (3.38.0) without any assigned serological subtype. In this regard, we aimed to identify specific amino acid patterns for each B*15 serological split, in order to facilitate the assignment of B*15 alleles to serological equivalents after high-resolution molecular typing. As a result, serological specificities of 372/394 not yet assigned alleles could be predicted based on amino acid motifs. Furthermore, two new serological types were identified and added, B62-Bw4 and B71-Bw4.

Long-Read Nanopore Sequencing Validated for Human Leukocyte Antigen Class I Typing in Routine Diagnostics.

Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D2 nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy.

A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing.

The rapid progress of HLA typing techniques has contributed to improving the outcome of haematopoietic stem cell transplantation (HSCT). However, unambiguous HLA typing remains challenging. Next generation sequencing (NGS) has been shown to resolve the HLA typing ambiguity and simplify HLA typing workflows. The aim of this study is to develop a multiplexed full-gene PCR assay for 11 HLA loci that can be used on any NGS platform to provide additional information to the traditionally sequenced regions. The entire gene of HLA-A, HLA-B, HLA-C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1 were amplified in four multiplexed reactions. A DNA reference panel of 47 samples representing the most common allele groups was selected to evaluate this novel assay using the Ion Torrent sequencing platform. The specificity and sensitivity of this assay was confirmed on additional 158 samples from a local Caucasian control cohort. Full gene sequences from start to stop codons including some UTR regions were obtained for all 11 HLA loci with complete gene coverage and sufficient read-depth for 3619 alleles. The whole amplicon was analysed for HLA class I genes, while only exons were analysed for class II genes. All alleles were amplified as expected with 100% concordance at full gene resolution for HLA class I and exon resolution for HLA class II loci when compared with previously used NGS or Sanger sequencing methods. In summary, the novel multiplexed PCR approach for full-gene HLA typing enabled for a large amount of genetic information to be generated in a simple and fast workflow.

Insights into the polymorphism in HLA-DRA and its evolutionary relationship with HLA haplotypes.

HLA-DRA encodes the alpha chain of the HLA-DR protein, one of the classical HLA class II molecules. Reported polymorphism within HLA-DRA is currently limited compared with other HLA genes, as only a single polymorphism encodes an amino acid difference in the translated protein. Since this SNP (rs7192, HLA00662.1:g.4276G>T p.Val217Leu) lies within exon 4, in the region encoding the cytoplasmic tail, the resulting protein is effectively monomorphic. For this reason, in-depth studies on HLA-DRA and its function have been limited. However, analysis of sequences from the 1000 Genomes Project and preliminary data from our lab reveals unrepresented polymorphism within HLA-DRA, suggesting a more complex role within the MHC than previously assumed. This study focuses on elucidating the extent of HLA-DRA polymorphism, and extending our understanding of the gene's role in HLA-DR~HLA-DQ haplotypes. Ninety-eight samples were sequenced for full-length HLA-DRA, and from this analysis, we identified 20 novel SNP positions in the intronic sequences within the 5711 bp region represented in IPD-IMGT/HLA. This polymorphism gives rise to at least 22 novel HLA-DRA alleles, and the patterns of intronic and 3' UTR polymorphism correspond to HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes. The current understanding of the organization of the genes within the HLA-DR region assumes a single lineage for the HLA-DRA gene, as opposed to multiple gene lineages, such as in HLA-DRB. This study suggests that the intron and 3' UTR polymorphism of HLA-DRA indicates different lineages, and represents the HLA-DRA~HLA-DRB345~HLA-DRB1~HLA-DQB1 haplotypes.

Antibodies against ARHGDIB are associated with long-term kidney graft loss.

The clinical significance of non-HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large-scale studies incorporating analysis of multiple non-HLA antibodies simultaneously. We developed a multiplex non-HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non-HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP-dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased-donor kidney (N = 3276) but not in recipients of a living-donor kidney (N = 1496). At 10 years after deceased-donor transplantation, recipients with anti-ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death-censored covariate-adjusted graft survival compared to the anti-ARHGDIB-negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32-2.53; P = .0003). These antibodies occur independently from donor-specific anti-HLA antibodies (DSA) or other non-HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non-HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti-ARHGDIB antibodies in all patients awaiting deceased-donor transplantation.

Allocation to highly sensitized patients based on acceptable mismatches results in low rejection rates comparable to nonsensitized patients.

Whereas regular allocation avoids unacceptable mismatches on the donor organ, allocation to highly sensitized patients within the Eurotransplant Acceptable Mismatch (AM) program is based on the patient's HLA phenotype plus acceptable antigens. These are HLA antigens to which the patient never made antibodies, as determined by extensive laboratory testing. AM patients have superior long-term graft survival compared with highly sensitized patients in regular allocation. Here, we questioned whether the AM program also results in lower rejection rates. From the PROCARE cohort, consisting of all Dutch kidney transplants in 1995-2005, we selected deceased donor single transplants with a minimum of 1 HLA mismatch and determined the cumulative 6-month rejection incidence for patients in AM or regular allocation. Additionally, we determined the effect of minimal matching criteria of 1 HLA-B plus 1 HLA-DR, or 2 HLA-DR antigens on rejection incidence. AM patients showed significantly lower rejection rates than highly immunized patients in regular allocation, comparable to nonsensitized patients, independent of other risk factors for rejection. In contrast to highly sensitized patients in regular allocation, minimal matching criteria did not affect rejection rates in AM patients. Allocation based on acceptable antigens leads to relatively low-risk transplants for highly sensitized patients with rejection rates similar to those of nonimmunized individuals.

Toward a Sensible Single-antigen Bead Cutoff Based on Kidney Graft Survival.

BACKGROUND: There is no consensus in the literature on the interpretation of single-antigen bead positive for a specific HLA antibody. METHODS: To inform the debate, we studied the relationship between various single-antigen bead positivity algorithms and the impact of resulting donor-specific HLA antibody (DSA) positivity on long-term kidney graft survival in 3237 deceased-donor transplants. RESULTS: First, we showed that the interassay variability can be greatly reduced when working with signal-to-background ratios instead of absolute median fluorescence intensities (MFIs). Next, we determined pretransplant DSA using various MFI cutoffs, signal-to-background ratios, and combinations thereof. The impact of the various cutoffs was studied by comparing the graft survival between the DSA-positive and DSA-negative groups. We did not observe a strong impact of various cutoff levels on 10-year graft survival. A stronger relationship between the cutoff level and 1-year graft survival for DSA-positive transplants was found when using signal-to-background ratios, most pronounced for the bead of the same HLA locus with lowest MFI taken as background. CONCLUSIONS: With respect to pretransplant risk stratification, we propose a signal-to-background ratio-6 (using the bead of the same HLA-locus with lowest MFI as background) cutoff of 15 combined with an MFI cutoff of 500, resulting in 8% and 21% lower 1- and 10-year graft survivals, respectively, for 8% DSA-positive transplants.

A paired kidney analysis on the impact of pre-transplant anti-HLA antibodies on graft survival.

BACKGROUND: Pre-transplant donor-specific anti-human leucocyte antigen (HLA) antibodies (DSAs) are associated with impaired kidney graft survival while the clinical relevance of non-donor-specific anti-HLA antibodies (nDSAs) is more controversial. The aim of the present paired kidney graft study was to compare the clinical relevance of DSAs and nDSAs. METHODS: To eliminate donor and era-dependent factors, a post hoc paired kidney graft analysis was performed as part of a Dutch multicentre study evaluating all transplantations between 1995 and 2005 with available pre-transplant serum samples. Anti-HLA antibodies were detected with a Luminex single-antigen bead assay. RESULTS: Among 3237 deceased donor transplantations, we identified 115 recipient pairs receiving a kidney from the same donor with one recipient being DSA positive and the other without anti-HLA antibodies. Patients with pre-transplant DSAs had a significantly lower 10-year death-censored graft survival (55% versus 82%, P=0.0001). We identified 192 pairs with one recipient as nDSA positive (against Class I and/or II) and the other without anti-HLA antibodies. For the patients with nDSAs against either Class I or II, graft survival did not significantly differ compared with patients without anti-HLA antibodies (74% versus 77%, P = 0.79). Only in patients with both nDSAs Class I and II was there a trend towards a lower graft survival (58%, P = 0.06). Lastly, in a small group of 42 recipient pairs, 10-year graft survival in recipients with DSAs was 49% compared with 68% in recipients with nDSAs (P=0.11). CONCLUSION: This paired kidney analysis confirms that the presence of pre-transplant DSAs in deceased donor transplantations is a risk marker for graft loss, whereas nDSAs in general are not associated with a lower graft survival. Subgroup analysis indicated that only in broadly sensitized patients with nDSAs against Class I and II, nDSAs may be a risk marker for graft loss in the long term.

Effect of initial immunosuppression on long-term kidney transplant outcome in immunological low-risk patients.

BACKGROUND: Few studies have evaluated the effect of different immunosuppressive strategies on long-term kidney transplant outcomes. Moreover, as they were usually based on historical data, it was not possible to account for the presence of pretransplant donor-specific human-leukocyte antigen antibodies (DSA), a currently recognized risk marker for impaired graft survival. The aim of this study was to evaluate to what extent frequently used initial immunosuppressive therapies increase graft survival in immunological low-risk patients. METHODS: We performed an analysis on the PROCARE cohort, a Dutch multicentre study including all transplantations performed in the Netherlands between 1995 and 2005 with available pretransplant serum (n = 4724). All sera were assessed for the presence of DSA by a luminex single-antigen bead assay. Patients with a previous kidney transplantation, pretransplant DSA or receiving induction therapy were excluded from the analysis. RESULTS: Three regimes were used in over 200 patients: cyclosporine (CsA)/prednisolone (Pred) (n = 542), CsA/mycophenolate mofetil (MMF)/Pred (n = 857) and tacrolimus (TAC)/MMF/Pred (n = 811). Covariate-adjusted analysis revealed no significant differences in 10-year death-censored graft survival between patients on TAC/MMF/Pred therapy (79%) compared with patients on CsA/MMF/Pred (82%, P = 0.88) or CsA/Pred (79%, P = 0.21). However, 1-year rejection-free survival censored for death and failure unrelated to rejection was significantly higher for TAC/MMF/Pred (81%) when compared with CsA/MMF/Pred (67%, P < 0.0001) and CsA/Pred (64%, P < 0.0001). CONCLUSION: These results suggest that in immunological low-risk patients excellent long-term kidney graft survival can be achieved irrespective of the type of initial immunosuppressive therapy (CsA or TAC; with or without MMF), despite differences in 1-year rejection-free survival.

A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism.

The FCGR3A gene encodes for the receptor important for antibody-dependent natural killer cell-mediated cytotoxicity. FCGR3A gene polymorphisms could affect the success of monoclonal antibody therapy. Although polymorphisms, such as the FcγRIIIA-V158F and -48L/R/H, have been studied extensively, an overview of other polymorphisms within this gene is lacking. To provide an overview of FCGR3A polymorphisms, we analysed the 1000 Genomes project database and found a total of 234 polymorphisms within the FCGR3A gene, of which 69%, 16%, and 15% occur in the intron, UTR, and exon regions respectively. Additionally, only 16% of all polymorphisms had a minor allele frequency (MAF) > 0.01. To facilitate (full-length) analysis of FCGR3A gene polymorphism, we developed a FCGR3A gene-specific amplification and sequencing protocol for Sanger sequencing and MinION (Nanopore Technologies). First, we used the Sanger sequencing protocol to study the presence of the V158F polymorphism in 76 individuals resulting in frequencies of 38% homozygous T/T, 7% homozygous G/G and 55% heterozygous. Next, we performed a pilot with both Sanger sequencing and MinION based sequencing of 14 DNA samples which showed a good concordance between Sanger- and MinION sequencing. Additionally, we detected 13 SNPs listed in the 1000 Genome Project, from which 11 had MAF > 0.01, and 10 SNPs were not listed in 1000 Genome Project. In summary, we demonstrated that FCGR3A gene is more polymorphic than previously described. As most novel polymorphisms are located in non-coding regions, their functional relevance needs to be studied in future functional studies.

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component "Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing" we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.

Pretransplant C3d-Fixing Donor-Specific Anti-HLA Antibodies Are Not Associated with Increased Risk for Kidney Graft Failure.

Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients.Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay.Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P=0.93). Patients without DSA had a 10-year graft survival of 78%.Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure.

NKG2A Expression Is Not per se Detrimental for the Anti-Multiple Myeloma Activity of Activated Natural Killer Cells in an In Vitro System Mimicking the Tumor Microenvironment.

Natural killer (NK) cell-based immunotherapy is a promising therapy for cancer patients. Inhibitory killer immunoglobulin-like receptors (KIRs) and NKG2A are required for NK cell licensing, but can also inhibit NK cell effector function. Upon reconstitution in a stem cell transplantation setting or after ex vivo NK expansion with IL-2, NKG2A is expressed on a large percentage of NK cells. Since the functional consequences of NKG2A co-expression for activated NK cells are not well known, we compared NKG2A+ vs NKG2A- NK cell subsets in response to K562 cells, multiple myeloma (MM) cell lines and primary MM cells. NK cells were isolated from healthy donors (HLA-C1+C2+Bw4+) and activated overnight with 1,000 U/ml IL-2. NK cell degranulation in subsets expressing KIRs and/or NKG2A was assessed at 21 or 0.6% O2. Activated NKG2A+ NK cell subsets degranulated more vigorously than NKG2A- subsets both at 21 and 0.6% O2. This was irrespective of the presence of KIR and occurred in response to HLA-deficient K562 cells as well as HLA competent, lowly expressing HLA-E MM cell lines. In response to primary MM cells, no inhibitory effects of NKG2A were observed, and NKG2A blockade did not enhance degranulation of NKG2A+ subsets. KIR- NK cells expressing NKG2A degranulated less than their NKG2A- counterparts in response to MM cells having high levels of peptide-induced membrane HLA-E, suggesting that high surface HLA-E levels are required for NKG2A to inhibit activated NK cells. Addition of daratumumab, an anti-CD38 to trigger antibody-dependent cell-mediated cytotoxicity, improved the anti-MM response for all subsets and degranulation of the KIR-NKG2A- "unlicensed" subset was comparable to KIR+ or NKG2A+ licensed subsets. This demonstrates that with potent activation, all subsets can contribute to tumor clearance. Additionally, subsets expressing KIRs mismatched with the HLA ligands on the target cell had the highest level of activation in response to MM cell lines as well as against primary MM. Our current study demonstrated that if NK cells are sufficiently activated, e.g., via cytokine or antibody activation, the (co-)expression of NKG2A receptor may not necessarily be a disadvantage for NK cell-based therapy.