Unrepresented human leucocyte antigen alleles in single-antigen bead assays: A single-centre cohort study.

Human leucocyte antigen (HLA) alleles may generate antibodies that are undetectable by routine single-antigen beads (SABs) assays if their unique epitopes are unrepresented. We aimed to describe the prevalence and explore the potential impact of unrepresented HLA alleles in standard SAB kits in our cohort. All individuals who had undergone two-field HLA typing (HLA-A/B/C/DRB1/DQA1/-DQB1/-DPA1/-DPB1) from February 2021 to July 2023 were included. Two-field HLA-DRB3/4/5 typing was imputed. Each unrepresented allele was compared with the most similar represented allele in the standard LABScreen, LABScreen ExPlex (One Lambda) and the LIFECODES (Immucor) SAB kits. Differences in eplet expression (HLA Eplet Registry) were identified. Differences in three-dimensional molecular structures were visualized using generated models (SWISS-MODEL). Two-field HLA typing was performed for 116 individuals. Overall, 16.7% of all HLA alleles, found in 36.2% of individuals, were unrepresented by all SAB test kits. Four eplets, found in 12.9% of individuals, were unrepresented in at least 1 SAB kit. Non-Chinese individuals were more likely to have unrepresented HLA alleles and eplets than Chinese individuals. There were differences in HLA allele and eplet representation amongst the different SAB test kits. Use of supplementary SAB test kits may improve HLA allele and eplet representation. Although some HLA alleles were unrepresented, most epitopes were represented in current SAB kits. However, some unrepresented alleles may contain epitopes which may generate undetectable antibodies. Further studies may be needed to investigate the potential clinical impact of these unrepresented alleles and eplets, especially in certain ethnic populations or at-risk individuals.

A cost-effective strategy for selection of third-party donors for a virus-specific T-cell bank for an Asian patient population.

BACKGROUND AIMS: Third party virus-specific T cells (VST) has shown efficacy for opportunistic virus infection which do not have effective treatment or are drug-refractory. We describe our preparatory work in setting up a third-party VST bank for a multi-ethnic Asian population. METHODS: Discarded white cells from regular blood bank plateletpheresis donors with known locally prevalent HLA antigens were cultured in small scale to generate VST against Adenovirus, BK virus, Cytomegalovirus, Epstein-Barr virus, and Human Herpes Virus 6. Multi-virus specific T cells (multi-VST) were also generated against all 5 viruses in single cultures. A strategy of allelic typing for donors with good and broad-spectrum cytotoxicity together with consideration on HLA restriction for the virus epitope was used to select combinations of VST lines for a hypothetical third party VST bank. The breadth of coverage based on these selection criteria was validated using our database of 100 post haematopoietic stem cell transplant patients. RESULTS: We show that 50%, 42%, 56%, 56% and 42% of single VST cultures demonstrated specific cytotoxicity against AdV, BKV, CMV, EBV and HHV6 respectively. Twenty four of the 36 multi-VST lines showed activity against at least 2 of the 5 viruses studied. A carefully selected combination of just 6 VST lines can offer VST with at least 1 allelic match to 99% of potential recipients, while 92% can find 2 allelic matches and 79% can find 3 allelic matches. CONCLUSIONS: This preparatory work confirms that a cost-effective strategy recruiting a small number of pre-characterized donors can generate VST lines with broad coverage for a multi-ethnic Asian patient population, thereby laying the foundation for setting up of a third party VST bank for Asian patients.

Large-scale Isolation of Highly Pure "Untouched" Regulatory T Cells in a GMP Environment for Adoptive Cell Therapy.

Adoptive cell therapy is an emerging treatment strategy for a number of serious diseases. Regulatory T (Treg) cells represent 1 cell type of particular interest for therapy of inflammatory conditions, as they are responsible for controlling unwanted immune responses. Initial clinical trials of adoptive transfer of Treg cells in patients with graft-versus-host disease were shown to be safe. However, obtaining sufficient numbers of highly pure and functional Treg cells with minimal contamination remains a challenge. We developed a novel approach to isolate "untouched" human Treg cells from healthy donors on the basis of negative selection using the surface markers CD49d and CD127. This procedure, which uses an antibody cocktail and magnetic beads for separation in an automated system (RoboSep), was scaled up and adapted to be compatible with good manufacturing practice conditions. With this setup we performed 9 Treg isolations from large-scale leukapheresis samples in a good manufacturing practice facility. These runs yielded sufficient numbers of "untouched" Treg cells for immediate use in clinical applications. The cell preparations consisted of viable highly pure FoxP3-positive Treg cells that were functional in suppressing the proliferation of effector T cells. Contamination with CD4 effector T cells was <10%. All other cell types did not exceed 2% in the final product. Remaining isolation reagents were reduced to levels that are considered safe. Treg cells isolated with this procedure will be used in a phase I clinical trial of adoptive transfer into leukemia patients developing graft-versus-host disease after stem cell transplantation.