Genetic immune escape landscape in primary and metastatic cancer.

Studies have characterized the immune escape landscape across primary tumors. However, whether late-stage metastatic tumors present differences in genetic immune escape (GIE) prevalence and dynamics remains unclear. We performed a pan-cancer characterization of GIE prevalence across six immune escape pathways in 6,319 uniformly processed tumor samples. To address the complexity of the HLA-I locus in the germline and in tumors, we developed LILAC, an open-source integrative framework. One in four tumors harbors GIE alterations, with high mechanistic and frequency variability across cancer types. GIE prevalence is generally consistent between primary and metastatic tumors. We reveal that GIE alterations are selected for in tumor evolution and focal loss of heterozygosity of HLA-I tends to eliminate the HLA allele, presenting the largest neoepitope repertoire. Finally, high mutational burden tumors showed a tendency toward focal loss of heterozygosity of HLA-I as the immune evasion mechanism, whereas, in hypermutated tumors, other immune evasion strategies prevail.

Characterization of a novel HLA-A*11:335 allele resulting from a rare interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles with nanopore sequencing, in a volunteer from the China Marrow Donor Program.

BACKGROUND: The major histocompatibility complex (MHC) in humans includes three classical class I loci (A, B, and C), which are important biomarkers for the transplantation of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing. METHODS: In the sample, the genotypes of HLA-A, B, C, DRB1, and DQB1 were firstly determined using the methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger's sequencing, and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele. RESULTS: Finally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing. CONCLUSIONS: The identification of the interlocus recombination indicated that nanopore sequencing can be helpful in the characterization of novel alleles with complex rearrangements. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.

Coronavirus hemagglutinin-esterase and spike proteins coevolve for functional balance and optimal virion avidity.

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.

HLA-G whole gene amplification reveals linkage disequilibrium between the HLA-G 3'UTR and coding sequence.

Polymorphic sites in the HLA-G gene may influence expression and function of the protein. Knowledge of the association between high-resolution HLA-G alleles and 3-prime untranslated (3'UTR) haplotypes is useful for studies on the role of HLA-G in transplantation, pregnancy, and cancer. We developed a next generation sequencing (NGS)-based typing assay enabling full phasing over the whole HLA-G gene sequence with inclusion of the 3'UTR region. DNA from 171 mother-child pairs (342 samples) was studied for: (a) HLA-G allele information by the NGSgo-AmpX HLA-G assay, (b) 3'UTR haplotype information by an in-house developed sequence-based typing method of a 699/713 base pair region in the 3'UTR, and (c) the full phase HLA-G gene sequence, by combining primers from both assays. The mother to child inheritance allowed internal verification of newly identified alleles and of association between coding and UTR regions. The NGSgo workflow compatible with Illumina platforms was employed. Data was interpreted using NGSengine software. In 99.4% of all alleles analyzed, the extended typing was consistent with the separate allele and 3'UTR typing methods. After repeated analysis of four samples that showed discrepancy, consistency reached 100%. A high-linkage disequilibrium between IPD-IMGT/HLA Database-defined HLA-G alleles and the extended 3'UTR region was identified (D' = 0.994, P < .0001). Strong associations were found particularly between HLA-G*01:04 and UTR-3, between HLA-G*01:01:03 and UTR-7, and between HLA-G*01:03:01 and UTR-5 (for all: r = 1). Six novel HLA-G alleles and three novel 3'UTR haplotype variants were identified, of which three and one, respectively, were verified in the offspring.

Activating KIRs exert a crucial role on relapse and overall survival after HLA-identical sibling transplantation.

Recognition of HLA-C molecules by killer cell immunoglobulin-like receptors (KIRs) is an important mechanism in the regulation of natural killer (NK) cell activity. Eradication of residual leukaemic cells by alloreactive donor NK cells after haematopoietic stem cell transplantation (HSCT) fulfils a crucial role in the control of relapse. This retrospective study evaluates 83 patients and their related donors. All individuals were typed at low-resolution level to determine their HLA repertoire. KIR genotyping data were obtained by the use of sequence-specific oligonucleotide (SSO) analysis. All data were combined with patient and donor characteristics and post-transplant clinical data. A higher overall survival was seen when KIR2DS1 in the donor was mismatched with the HLA-C group 2 ligand in the patient (p=0.03). The number of activating KIRs either in the patient or in the donor was significantly correlated with the occurrence of relapse (p=0.003 and p=0.02, respectively). In addition, the presence of KIR2DS5 in the patient alone or in both the patient and donor was significantly correlated with the occurrence of relapse (p=0.004 and p=0.005, respectively). In conclusion, significant correlations were found for activating KIRs with overall survival and relapse.

Patients benefit from the addition of KIR repertoire data to the donor selection procedure for unrelated haematopoietic stem cell transplantation.

Killer cell immunoglobulin-like receptors (KIRs) expressed on donor natural killer (NK) cells are important for induction of NK cell alloreactivity in haematopoietic stem cell transplantation (HSCT). Current criteria in the selection procedure of an unrelated donor do not account for this potential NK alloresponse. In this study the KIR gene repertoire of 21 HSCT patients and all their potential, unrelated donors (N=64) has been identified by the sequence-specific priming (SSP) procedure. KIR genotype characteristics are correlated with HLA and clinical data. These data show that for 16 cases an HLA compatible alternative donor was available. Among those 16 were 8 donors with a favourable predicted NK alloreactivity directed against the leukaemic cells. In conclusion, it is feasible and clinically relevant to add the KIR repertoire to the unrelated donor selection procedure.

The elucidation of KIR2DL4 gene polymorphism.

The killer cell immunoglobulin-like receptors (KIRs) on NK cells recognize defined groups of HLA class I alleles. By this mechanism the NK cells fulfil a significant role in the first line of defense against infectious agents and cancer. For the treatment of leukaemia this NK cell allorecognition is of great importance. Still, an appropriate effect against the leukaemic cells requires sufficient expression of both KIR and HLA proteins. KIR gene polymorphism influence membrane expression of the KIR protein. We addressed KIR2DL4 gene polymorphism by a newly developed DNA and cDNA based direct sequencing based typing (SBT) and cloning approach. A panel of 44 individuals revealed a variety of KIR2DL4 alleles. Three new alleles have been identified, among those one allele showed alternatively spliced products. In conclusion, this approach is applicable for routine KIR2DL4 allele typing and enables the characterisation of new KIR2DL4 alleles.

HLA and MICA associations with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive tumour arising from the epithelial lining of the upper aerodigestive tract. The precise mechanisms involved in the pathogenesis of HNSCC have not been elucidated. Previous studies observed aberrant HLA expression patterns on HNSCC tumour cells and this study focused on the allelic polymorphism of HLA genes and the MHC class I chain related gene A (MICA) and HNSCC. We investigated whether associations with HLA and/or MIC alleles or haplotypes are involved in the pathogenesis of HNSCC and could explain the observed HLA expression patterns. Patients and controls were typed for HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 with sequence specific priming (SSP), supplemented with sequencing based typing (SBT). MICA allelic polymorphism was included and MICA allele assignment was based upon the combination of high resolution SBT of exons 2-4 in combination with repeat analysis and nucleotide polymorphism of exon 5. HLA-B *35 (p=0.014, OR=0.31) and HLA-B *40 (p=0.013, OR=2.9) were significantly associated in respectively the metastasized patients and the oral cavity patients. In addition, the HLA-B *40-DRB1 *13 haplotype (p=0.016, OR=4.1) was more often observed in the oral cavity patient group. The biological significance of the prevalence of specific HLA haplotypes in patients with oral cavity HNSCC and metastasizing HNSCC requires further investigation.

Genes in the HLA region indicative for head and neck squamous cell carcinoma.

The majority of genes in the HLA region are directly or indirectly involved in immunological functions. They comprise HLA, HLA-related and non-HLA-related genes. Aberrant HLA expression patterns, including heterogeneous and negative HLA expression, are observed in specimens from head and neck squamous cell carcinoma (HNSCC). To explore the possible role of genes in the HLA region other than the classical HLA genes, susceptibility regions within the HLA region for HNSCC were defined in this study. Microsatellite analysis for 49 microsatellites dispersed throughout the HLA region, in combination with the DNA pooling approach of respectively one control DNA pool and three patient DNA pools, based upon the tumour location, offered an efficient method to define susceptibility regions. In the oral cavity three significant susceptibility regions were localized, one in the class I region (330 kb), and two in the class II region (170 and 210 kb). Eighteen genes from these regions were tested for their RNA expression in oral cavity tumour tissue and compared to expression in the surrounding healthy tissue. A significant increased MICA RNA expression in tumour tissues compared to healthy surrounding tissue and a significant decreased HSD17B8 RNA in tumour tissues compared to surrounding healthy tissue, particular in those tumours without lymph node metastasis, were observed. A trend for decreased RXRbeta and NOTCH4 RNA expression was observed in tumour tissue. In addition to the classical HLA genes, other genes within the HLA region define susceptibility for oral squamous cell carcinoma of the head and neck.

MHC class I chain-related gene a diversity in head and neck squamous cell carcinoma.

Many immune-related genes are located within the human leukocyte antigen (HLA) region on chromosome 6. The MHC class I chain-related gene A (MICA), located centromeric of HLA-B, is involved in the innate and adaptive immune response through activation of NK and T cells. Differences of MICA transmembrane repeat lengths have been associated with diseases and expression is observed on epithelial tumors. Head and neck squamous cell carcinoma (HNSCC) is an epithelial tumor. In the present study we evaluated the MICA repeat length diversity in relation to MICA expression in Dutch HNSCC patients. MICA short tandem repeat analysis indicated a significant decrease in the frequency for the MICA-A9 repeat in patients diagnosed with oral cavity squamous cell carcinoma (SCC) but not in patients with SCC in the hypoharynx, larynx, or oropharynx. Interestingly, the majority of patients expressed MICA as observed with immunohistochemical staining whereas no soluble MICA was detected in patients' sera by enzyme-linked immunosorbent assay. In conclusion, the length of the MICA transmembrane repeats in Dutch HNSCC patients does not influence the MICA expression on tumor cells.

Identification of HLA-A*0111N: a synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele.

A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient's cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing.

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5' untranslated region (UTR) through the 3'-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5' and 3'-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.

HLA-DP4 presents an immunodominant peptide from the RSV G protein to CD4 T cells.

CD4 T cells play a crucial role during virus infections by producing antiviral cytokines and by regulating humoral and cellular immune responses. Unfortunately however, exaggerated CD4 T cell responses can cause significant immune-mediated disease as was observed during RSV infections in children previously vaccinated with a formalin-inactivated virus in the 1960s. It has been observed that vaccination with the G protein of RSV tends to prime mice for a similar Th2-mediated enhanced disease. Whether the G protein may play a role in enhanced disease in man is unclear. In the present study, we identified an immunodominant epitope in the conserved region of the G protein encompassing amino acid residues 162-175. This epitope is presented in the context of HLA-DPB1*0401 and DPB1*0402, the most prevalent HLA class II alleles. Importantly, in some patients, a mixed Th1/Th2 response against this epitope was found in bronchoalveolar lavage samples during primary RSV infections.