Identification and characterization of 122 novel HLA alleles in bone marrow donors recruited to the Ezer Mizion Bone Marrow Donor Registry.

Between January 2018 and June 2021, the Ezer Mizion recruited 223,960 donors. All donors were typed for their HLA class I and II alleles at high resolution by Next Generation Sequencing techniques. Comparison between the sequences obtained from these donors and those in the IPD-IMGT/HLA Database, revealed 122 Novel HLA alleles that were found in 133 donors. Most of the alleles, 94 (77%) were identified in the HLA class I genes (30, 35, and 29 in HLA-A, -B, and -C, respectively), and 28 (23%) were detected in the HLA class II genes (9 in HLA-DRB1 and -DQB1 and 10 in -DPB1). Most of these novel alleles, 106 (86.9%) comprised single nucleotide variation (SNV), 9 (7.4%) present multiple amino acids variation, 4 and 3 were generated because of deletions and insertions, respectively. Ten of these novel alleles were seen to be null alleles.

Soluble HLA peptidome of pleural effusions is a valuable source for tumor antigens.

BACKGROUND: Soluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and tissues. Other body fluids, such as pleural effusions, may also contain sHLA-peptide complexes, and can potentially serve as a source of tumor antigens since these fluids are drained from the tumor microenvironment. We explored this possibility by developing a methodology for purifying and analyzing large pleural effusion sHLA class I peptidomes of patients with malignancies or benign diseases. METHODS: Cleared pleural fluids, cell pellets present in the pleural effusions, and the primary tumor cells cultured from cancer patients' effusions, were used for immunoaffinity purification of the HLA molecules. The recovered HLA peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the resulting LC-MS/MS data were analyzed with the MaxQuant software tool. Selected tumor antigen peptides were tested for their immunogenicity potential with donor peripheral blood mononuclear cells (PBMCs) in an in vitro assay. RESULTS: Mass spectrometry analysis of the pleural effusions revealed 39,669 peptides attributable to 11,305 source proteins. The majority of peptides identified from the pleural effusions were defined as HLA ligands that fit the patients' HLA consensus sequence motifs. The membranal and soluble HLA peptidomes of each individual patient correlated to each other. Additionally, soluble HLA peptidomes from the same patient, obtained at different visits to the clinic, were highly similar. Compared with benign effusions, the soluble HLA peptidomes of malignant pleural effusions were larger and included HLA peptides derived from known tumor-associated antigens, including cancer/testis antigens, lung-related proteins, and vascular endothelial growth factor pathway proteins. Selected tumor-associated antigens that were identified by the immunopeptidomics were able to successfully prime CD8+ T cells. CONCLUSIONS: Pleural effusions contain sHLA-peptide complexes, and the pleural effusion HLA peptidome of patients with malignant tumors can serve as a rich source of biomarkers for tumor diagnosis and potential candidates for personalized immunotherapy.

MHC Haplotyping of SARS-CoV-2 Patients: HLA Subtypes Are Not Associated with the Presence and Severity of COVID-19 in the Israeli Population.

HLA haplotypes were found to be associated with increased risk for viral infections or disease severity in various diseases, including SARS. Several genetic variants are associated with COVID-19 severity. Studies have proposed associations, based on a very small sample and a large number of tested HLA alleles, but no clear association between HLA and COVID-19 incidence or severity has been reported. We conducted a large-scale HLA analysis of Israeli individuals who tested positive for SARS-CoV-2 infection by PCR. Overall, 72,912 individuals with known HLA haplotypes were included in the study, of whom 6413 (8.8%) were found to have SARS-CoV-2 by PCR. A total of 20,937 subjects were of Ashkenazi origin (at least 2/4 grandparents). One hundred eighty-one patients (2.8% of the infected) were hospitalized due to the disease. None of the 66 most common HLA loci (within the five HLA subgroups: A, B, C, DQB1, DRB1) was found to be associated with SARS-CoV-2 infection or hospitalization in the general Israeli population. Similarly, no association was detected in the Ashkenazi Jewish subset. Moreover, no association was found between heterozygosity in any of the HLA loci and either infection or hospitalization. We conclude that HLA haplotypes are not a major risk/protecting factor among the Israeli population for SARS-CoV-2 infection or severity. Our results suggest that if any HLA association exists with the disease it is very weak, and of limited effect on the pandemic.

Common, intermediate and well-documented HLA alleles in world populations: CIWD version 3.0.0.

A catalog of common, intermediate and well-documented (CIWD) HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQB1 and -DPB1 alleles has been compiled from over 8 million individuals using data from 20 unrelated hematopoietic stem cell volunteer donor registries. Individuals are divided into seven geographic/ancestral/ethnic groups and data are summarized for each group and for the total population. P (two-field) and G group assignments are divided into one of four frequency categories: common (≥1 in 10 000), intermediate (≥1 in 100 000), well-documented (≥5 occurrences) or not-CIWD. Overall 26% of alleles in IPD-IMGT/HLA version 3.31.0 at P group resolution fall into the three CIWD categories. The two-field catalog includes 18% (n = 545) common, 17% (n = 513) intermediate, and 65% (n = 1997) well-documented alleles. Full-field allele frequency data are provided but are limited in value by the variations in resolution used by the registries. A recommended CIWD list is based on the most frequent category in the total or any of the seven geographic/ancestral/ethnic groups. Data are also provided so users can compile a catalog specific to the population groups that they serve. Comparisons are made to three previous CWD reports representing more limited population groups. This catalog, CIWD version 3.0.0, is a step closer to the collection of global HLA frequencies and to a clearer view of HLA diversity in the human population as a whole.

Audits of collection and apheresis centers: guidelines by the World Marrow Donor Association Working Group Quality and Regulation.

According to the Standards of the World Marrow Donor Association (WMDA), unrelated stem cell donor registries and donor centers are responsible for compliance of their collection and apheresis centers with these Standards. To ensure high stem cell product quality and high standards for safety and satisfaction of voluntary unrelated stem cell donors, we here present guidelines for audits of collection and apheresis centers that can be used by new and established donor registries, as well as by collection centers in preparation of audits. We define the general requirements and recommendations for collaboration with the collection and apheresis centers and define critical procedures for the collection of the stem cell product, such as information session, medical assessment, product collection, quality controls, product handover for transportation, and donor follow-up. The specific guidelines are accompanied by detailed checklists and forms that can be found in Supplementary Information and may be used during an initial or follow-up on-site or paper-based audit.

East Meets West-Impact of Ethnicity on Donor Match Rates in the Ezer Mizion Bone Marrow Donor Registry.

HLA haplotype frequencies in a volunteer bone marrow donor registry should reflect the frequencies of potential transplant recipients served by that registry, a challenge in a country with diverse subethnicities of immigrants from Eastern and Western cultures, such as Israel. We evaluated the likelihood of finding suitable donors for hypothetical patients drawn from defined subethnicities in the Ezer Mizion Bone Marrow Donor Registry (EM BMDR) from donors both within and outside the registry now and during the coming decade. On average, bioinformatics modeling predicts that, given current donor recruitment trends, 6/6 high-resolution HLA match rates for Israelis, which currently stand at 40% to 55% for most subethnicities, will rise by up to 1% per year over the next decade. Subethnicities with historically lower rates of interethnic admixture are less likely to find matches outside of their designated group but will benefit from expansion of the registry, whereas ethnically directed drives will enhance matching rates for currently underrepresented subethnicities. Donor searches for the same cohort using a large extramural registry was of only slight benefit for most of the 19 EM BMDR subethnicities evaluated, confirming that local donor registries that reflect the ethnic diversity of the community being served are best equipped to serve the needs of their respective communities. Contemporary trends of an increasingly multiethnic admixture in Israel may impact the effect of ethnic profiling in assessing future match rates for EM BMDR.

High-resolution HLA A∼B∼DRB1 haplotype frequencies from the Ezer Mizion Bone Marrow Donor Registry in Israel.

We have investigated HLA population alleles and haplotype frequencies for the ethnicities that comprise the contemporary population of Israel, using a large data set from the Ezer Mizion Bone Barrow Donor Registry. We genotyped 275,699 individuals at the HLA-A, -B and -DRB1 loci using HLA genotyping methods. HLA A∼B∼DRB1 haplotype frequencies were estimated from 19 sub-ethnic Jewish populations and other non-Jewish minorities using the maximum likelihood model, which accommodates typing ambiguities. We present overall and sub-ethnicity specific HLA diversity results of the registry, which will help guide a data-driven strategy for future registry expansion.

Bone marrow minimal disseminated disease (MDD) and minimal residual disease (MRD) in childhood T-cell lymphoblastic lymphoma stage III, detected by flow cytometry (FC) and real-time quantitative polymerase chain reaction (RQ-PCR).

BACKGROUND: Despite overlapping features of T-cell lymphoblastic lymphoma (T-LLy) and T-cell acute lymphoblastic leukemia (T-ALL), which respond favorably to T-ALL treatment, clinical and biological differences exist. We retrospectively assessed the prevalence of submicroscopic bone marrow (BM) minimal disseminated disease (MDD) at diagnosis and the early response to treatment (minimal residual disease--MRD) and their prognostic significance in 17 children with stage III T-LLy treated according to Berlin-Frankfurt-Munster (BFM) non-Hodgkin lymphoma protocols. PROCEDURE: Four-color flow cytometry (FC) was used for lymphoma associated immunophenotype and real-time quantitative polymerase chain reaction (RQ-PCR) for T-cell receptor (TCR beta/delta/gamma) gene rearrangements with at least 0.01% sensitivity. RESULTS: Two markers per patient were identified in all cases using FC and in 80% using RQ-PCR. BM MDD at diagnosis of >or=0.01% was detected by FC and RQ-PCR in 88% and 80% of patients, respectively, and by at least one of the methods in all patients. A significant correlation was achieved between the methods by Pearson correlation analysis (P = 0.004). MRD levels significantly decreased to very low levels on day 33 in 9 out of 10 patients studied. The only patient that remained positive relapsed. CONCLUSIONS: MDD was prevalent in stage III T-LLy, for which we could not prove a prognostic significance in the context of ALL-like treatment. This study shows that both FC and RQ-PCR methods are efficient for MDD and MRD analyses in T-LLy.

Hepatitis C virus infection in dialysis and chronic liver patients: Viraemia dependent anti-E2-antibody response.

Cryptic hepatitis C virus (HCV) infection relates to patients infected chronically with HCV that are seronegative but have HCV-RNA. These patients are not identified by the standard serological tests for HCV, which are based on detection of antibodies to core, NS3 and NS5 antigens. They will, therefore, be wrongly diagnosed as non-infected, and are considered as a potential risk for others. Cryptic HCV infection in dialysis units occurs frequently and, due to medical procedures, is a major factor for contracting the virus when unrecognised. This study was conducted in order to assess the humoral immune responses to E2-antigen in sera of patients infected chronically with HCV. Recombinant E2 protein in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) were used to test the occurrence of anti-E2 antibodies in the sera of patients from the liver clinic and of dialysis patients. The presence of E2 antibodies was found to be correlated with the presence of HCV-RNA and with viral load. Antibodies to the E2 protein could be detected in as many as 30% of the sera from dialysis patients with cryptic HCV infection (HCV-RNA only). The results suggest that detection of anti-E2 antibodies may enhance significantly HCV serological standard testing; especially among patients on dialysis, and that antibodies to envelope E2 protein appear to depend on and correlate with the presence of HCV particles.