Recipients Receiving Better HLA-Matched Hematopoietic Cell Transplantation Grafts, Uncovered by a Novel HLA Typing Method, Have Superior Survival: A Retrospective Study.

HLA matching at an allelic-level resolution for volunteer unrelated donor (VUD) hematopoietic cell transplantation (HCT) results in improved survival and fewer post-transplant complications. Limitations in typing technologies used for the hyperpolymorphic HLA genes have meant that variations outside of the antigen recognition domain (ARD) have not been previously characterized in HCT. Our aim was to explore the extent of diversity outside of the ARD and determine the impact of this diversity on transplant outcome. Eight hundred ninety-one VUD-HCT donors and their recipients transplanted for a hematologic malignancy in the United Kingdom were retrospectively HLA typed at an ultra-high resolution (UHR) for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 using next-generation sequencing technology. Matching was determined at full gene level for HLA class I and at a coding DNA sequence level for HLA class II genes. The HLA matching status changed in 29.1% of pairs after UHR HLA typing. The 12/12 UHR HLA matched patients had significantly improved 5-year overall survival when compared with those believed to be 12/12 HLA matches based on their original HLA typing but were found to be mismatched after UHR HLA typing (54.8% versus 30.1%, P = .022). Survival was also significantly better in 12/12 UHR HLA-matched patients when compared with those with any degree of mismatch at this level of resolution (55.1% versus 40.1%, P = .005). This study shows that better HLA matching, found when typing is done at UHR that includes exons outside of the ARD, introns, and untranslated regions, can significantly improve outcomes for recipients of a VUD-HCT for a hematologic malignancy and should be prospectively performed at donor selection.

Fifty novel HLA-DPB1 alleles identified in a UK cohort of unrelated hematopoietic cell donors and recipients.

HLA-DPB1 is the classical HLA class II genes with the least recorded variation on the IPD-IMGT/HLA Database, suggesting the full extent of its diversity is perhaps yet to be characterized. Here, a full-gene typing strategy was employed to genotype a UK cohort of 1470 HCT recipients (n = 744) and donors (n = 726). In total, 2940 full-length HLA-DPB1 sequences were generated, comprising 193 distinct alleles. Of these, 107 sequences contained novel variation, totaling 49 unique intronic HLA-DPB1 alleles, and one coding variant (HLA-DPB1*1188:01). Full-gene sequencing resulted in zygosity changes for 129 individuals by identifying two distinct intronic variants of the same coding allele. We verified the existence of nine unconfirmed alleles and extended the sequence of two existing alleles on the IPD-IMGT/HLA Database.

The novel HLA-F*01:16 and HLA-F*01:17 alleles identified in hematopoietic cell donors.

Two novel non-classical HLA class I alleles have been characterized, HLA-F*01:16 and -F*01:17.

Characterisation of RAET1E/ULBP4 exon 4 and 3' untranslated region genetic architecture reveals further diversity and allelic polymorphism.

NKG2D is a natural killer cell activating receptor recognising ligands on infected or tumorigenic cells, leading to their cytolysis. There are eight known genes encoding NKG2D ligands: MICA, MICB and ULBP1-6. MICA and MICB are highly polymorphic and well characterised, whilst ULBP ligands are less polymorphic and the functional implication of their diversity is not well understood. Using International HLA and Immunogenetics Workshop (IHIW) cell line DNA, we previously characterised alleles of the RAET1E gene (encoding ULBP4 proteins), including the 5' UTR promoter region and exons 1-3. We found 11 promoter haplotypes associating with alleles based on exons 1-3, revealing 19 alleles overall. The current study extends this analysis using 87 individual DNA samples from IHIW cell lines or cord blood to include RAET1E exon 4 and the 3' UTR, as polymorphism in these regions have not been previously investigated. We found two novel exon 4 polymorphisms encoding amino acid substitutions altering the transmembrane domain. An amino acid substitution at residue 233 was unique to the RAET1E*008 allele whereas the substitution at residue 237 was shared between groups of alleles. Additionally, four haplotypes were found based on 3' UTR sequences, which were unique to certain alleles or shared with allele groups based on exons 1-4 polymorphisms. Furthermore, putative microRNAs were identified that may interact with these polymorphic sites, repressing transcription and potentially affecting expression levels.

The novel alleles, HLA-G*01:04:09 and HLA-DPB1*04:01:01:136, detected in a hematopoietic cell donor.

Two novel alleles, HLA-G*01:04:09 and HLA-DPB1*04:01:01:136, were identified in a single healthy individual.

KIR2DS2+ NK cells in cancer patients demonstrate high activation in response to tumour-targeting antibodies.

Strategies to mobilise natural killer (NK) cells against cancer include tumour-targeting antibodies, NK cell engagers (NKCEs) and the adoptive transfer of ex vivo expanded healthy donor-derived NK cells. Genetic and functional studies have revealed that expression of the activating killer immunoglobulin-like receptor KIR2DS2 is associated with enhanced function in NK cells from healthy donors and improved outcome in several different malignancies. The optimal strategy to leverage KIR2DS2+ NK cells therapeutically is however currently unclear. In this study, we therefore evaluated the response of KIR2DS2-expressing NK cells to activation against cancer with clinically relevant tumour-targeting antibodies and following ex vivo expansion. We identified that KIR2DS2high NK cells from patients with chronic lymphocytic leukaemia and hepatocellular carcinoma had enhanced activation in response to tumour-targeting antibodies compared to KIR2DS2- NK cells. However, the superior function of healthy donor derived KIR2DS2high NK cells was lost following ex vivo expansion which is required for adoptive transfer-based therapeutic strategies. These data provide evidence that targeting KIR2DS2 directly in cancer patients may allow for the utilisation of their enhanced effector function, however such activity may be lost following their ex vivo expansion.

MICA and MICB allele assortment in Finland.

Genetic variation in the MICA and MICB genes located within the major histocompatibility complex region has been reported to be associated with transplantation outcome and susceptibility to autoimmune diseases and infections. Only limited data of polymorphism in these genes in different populations are available. We here report allelic variation at 2-field resolution and the haplotypes of the MICA and MICB genes in Finland (n = 1032 individuals), a north European population with historical bottleneck and founder effects. Altogether 24 MICA and 18 MICB alleles were found, forming 70 estimated MICA-MICB haplotypes. As compared to other populations frequency differences were found, for example, MICA*010:01 was found to be at an allele frequency of 0.133 in Finland which is higher than in other European populations (0.021-0.077), but close to Asian populations (0.151-0.220). Three novel alleles with amino acid change are described. The results demonstrate a relatively high level of polymorphism and population differences in MICA and MICB allele distribution.

Description of a novel allele HLA-DRB1*16:02:10, identified in a bone marrow donor.

The new allele HLA-DRB1*16:02:10 showed one synonymous nucleotide difference with HLA-DRB1*16:02:01:01 in codon 58.

The novel HLA-DRB1*03:01:01:05 and -DPB1*04:02:01:21 alleles identified in patients with acute leukemia.

Two novel HLA class II alleles were characterized, HLA-DRB1*03:01:01:05 and HLA-DPB1*04:02:01:21.

Widespread non-coding polymorphism in HLA class II genes of International HLA and Immunogenetics Workshop cell lines.

As the primary genetic determinant of immune recognition of self and non-self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real-Time (SMRT®) DNA sequencing to perform four-field resolution HLA typing of HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 from a panel of 181 B-lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD-IMGT/HLA database. We observed widespread non-coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD-IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full-length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full-length sequencing with long, phased reads. The fourth-field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under-appreciated non-coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies.

Relative Traffic Tolerance of Warm-Season Grasses and Suitability for Grazing by Equine.

Warm season wear-tolerant turfgrasses, such as those used on golf courses and athletic fields, may be valuable forages on equine operations because of their potential to remain viable during heavy hoof traffic. Crabgrass may also be suitable as it thrives in conditions where other grasses have limited success. The objective of this study was to assess the relative traffic tolerance and nutritional composition of five warm-season (WS) turfgrass cultivars of bermudagrass and zoysiagrass and one WS forage-type crabgrass. All cultivars were established by seed in replicated monoculture plots. Simulated hoof traffic treatments consisted of either none, one, or two passes of a Baldree Traffic Simulator. Traffic was applied weekly for 6 weeks in the summer of 2016 and 2017, with each treatment period followed by a 4-week rest period. Plots were assessed for compaction, biomass, and persistence before and after treatment and rest periods. Nutritional composition was assessed throughout the growing seasons. Soil compaction increased as treatment level increased for all cultivars (P < .0001). There was no effect of treatment on cultivar persistence. Biomass available for grazing was increased in year 1 by the application of LOW traffic treatment (P = .0193). Both bermudagrass and zoysiagrass cultivars showed promise for use in areas of heavy traffic on equine operations, however, zoysiagrass cultivars were more suitable as they were highest ranking in relative traffic tolerance, moderate in yield, and low nonstructural carbohydrates (<12% NSC). Future on-farm studies evaluating bermudagrass and zoysiagrass to determine ideal stocking rate, management methods, and persistence under grazing are warranted.

The novel HLA-DPB1 allele, HLA-DPB1*04:01:51, first described in a Brazilian individual.

The novel HLA-DPB1*04:01:51 allele first described in a potential bone marrow donor from Brazil.

Identification of a novel allele, HLA-DPB1*18:01:01:04, in an African American renal transplant candidate.

HLA-DPB1*18:01:01:04 differs from HLA-DPB1*18:01:01:01 by four substitutions within intron 1.

The novel HLA-C*03:04:01:47 allele sequence identified using Pacific biosciences SMRT sequencing.

A novel variant of HLA-C*03:04:01:47 was identified using Pacific Biosciences SMRT sequencing platform.

Agricultural Selection of Wheat Has Been Shaped by Plant-Microbe Interactions.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

Single molecule real-time DNA sequencing of the full HLA-E gene for 212 reference cell lines.

We have developed a genotyping assay that produces fully phased, unambiguous HLA-E genotyping using Pacific Biosciences' single molecule real-time DNA sequencing. In total 212 cell lines were genotyped, including the panel of 107 established at the 10th International Histocompatibility Workshop. Our results matched the previously known HLA-E genotype in 94 (44.3%) cell lines, in all cases either improving or equalling previous genotyping resolution. Three (1.4%) cells had discrepant HLA-E genotyping data and 115 (54.2%) had no previous HLA-E data. The HLA-E genotypes for four (1.9%) cell lines resulted in a change of zygosity by identifying two distinct haplotypes. We discovered eight novel HLA-E alleles, extended the known reference sequence of seven and confirmed the existence of a further 10.

Relative Traffic Tolerance of Cool-Season Turfgrasses and Suitability for Grazing by Equine.

Unlike traditional forage grasses, turfgrasses, which were developed to be tolerant of foot traffic and close mowing, may be suitable as alternative ground cover in areas of high hoof traffic such as dry lots. The objective of this study was to evaluate the potential of eight cool-season turfgrasses as alternative ground cover in heavy use areas. Cultivars were established via seeding in four replicated plots. To simulate horse traffic at a trot, a Baldree traffic simulator was driven over a section of the plot, either 0 (CON), 1 (LOW), or 2 (HIGH) times per week for 6 weeks followed by 4 weeks of rest in the spring, summer, and fall over a 2-year period. Variables assessed include compaction, biomass available for grazing, vegetative cover (persistence), and nutrient composition. Soil compaction increased with the application of traffic treatments (P < .0001). Traffic treatment reduced persistence following traffic by 19%-36% across all trials in both years (P = .0003). For most trials, biomass available for grazing was reduced after traffic treatment by 19% and 43% (P = .02). Overall, tall fescue cultivars were most traffic tolerant followed by hard fescue. Cultivars considered "low" in nonstructural carbohydrate (<15%) included creeping bentgrass in Year 1 and hard fescue and chewings fescue in Year 2. In Year 2, the Ca:P ratio declined beyond what is recommended for horses. Tall fescue, hard fescue, and creeping bentgrass cultivars are recommended for further evaluation as suitable ground cover in areas of heavy use on equine operations.

A genomic extension to the sequence of HLA-A*02:13, identified using third-generation sequencing.

Pacific Biosciences' SMRT sequencing method was used to extend the sequence of HLA-A*02:13.

A novel HLA allele, HLA-B*56:67, identified in a Melanesian individual from New Caledonia.

Novel allele HLA-B*56:67 potentially formed by recombination between B*56:01:01:03 and B*40:01:01.