High resolution HLA genotyping with third generation sequencing technology-A multicentre study.

Molecular HLA typing techniques are currently undergoing a rapid evolution. While real-time PCR is established as the standard method in tissue typing laboratories regarding allocation of solid organs, next generation sequencing (NGS) for high-resolution HLA typing is becoming indispensable but is not yet suitable for deceased donors. By contrast, high-resolution typing is essential for stem cell transplantation and is increasingly required for questions relating to various disease associations. In this multicentre clinical study, the TGS technique using nanopore sequencing is investigated applying NanoTYPE™ kit and NanoTYPER™ software (Omixon Biocomputing Ltd., Budapest, Hungary) regarding the concordance of the results with NGS and its practicability in diagnostic laboratories. The results of 381 samples show a concordance of 99.58% for 11 HLA loci, HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. The quality control (QC) data shows a very high quality of the sequencing performed in each laboratory, 34,926 (97.15%) QC values were returned as 'passed', 862 (2.4%) as 'inspect' and 162 (0.45%) as 'failed'. We show that an 'inspect' or 'failed' QC warning does not automatically lead to incorrect HLA typing. The advantages of nanopore sequencing are speed, flexibility, reusability of the flow cells and easy implementation in the laboratory. There are challenges, such as exon coverage and the handling of large amounts of data. Finally, nanopore sequencing presents potential for applications in basic research within the field of epigenetics and genomics and holds significance for clinical concerns.

NK and T cell repertoire is established early after allogeneic HSCT and is profoundly imprinted by CMV reactivation.

Besides genetic influences, non-genetic factors such as graft-versus-host disease (GvHD) and viral infections have been shown as important shapers of the immune reconstitution and diversification processes after hematopoietic stem cell transplantation (HSCT). However, the differential susceptibility to immune modulation by non-genetic factors is not fully understood. We determined to follow the reconstitution of the T cell receptor (TCR) repertoire through immune-sequencing, of natural killer (NK) cells using a 35-marker spectral flow cytometry panel, and in relation to clinical events. Longitudinal investigation was performed on samples derived from 54 HSCT recipients during the first-year post-HSCT. We confirmed a significant contraction in TCR repertoire diversity with a remarkable stability over time. CMV reactivation had the ability to significantly change TCR repertoire clonality and composition, with a long-lasting imprint. Our data further revealed skewing of NK cell reconstitution in CMV reactivated recipients, with an increased frequency of KIR2DL2L3S2+ adaptive, cytolytic and functional CD107a+ NK cells concomitant with a reduced pool of NKG2A+ NK cells. We provided support that CMV might act as one of the more important driver of peripheral homeostatic proliferation of circulating specific T and NK cells, which can be viewed as a compensatory mechanism to establish a new peripheral repertoire.