In 2020, we reported MICA allele frequencies from a cohort of over one million German individuals. This study identified MICA*008 (42%), MICA*002 (12%), and MICA*009 (9%) as the most common MICA alleles at protein resolution. Additionally, we discovered novel alleles with a cumulative frequency of 0.3%. To reduce this fraction of unnamed sequences, we aimed to fully characterize the most frequent novel alleles using both long- and short-read sequencing. As a result, we submitted 603 sequences to the IPD-IMGT/HLA Database: 406 novel alleles and 197 sequence extensions and confirmations. Among the novel alleles, 199 encoded for distinct novel MICA proteins. Following the inclusion of these sequences into the IPD-IMGT/HLA Database, we genotyped 93,814 individuals from an independent cohort. In the German subset (n=48,618), our previous findings on MICA allele frequencies were confirmed. As anticipated, the cumulative frequency of novel alleles decreased significantly from 0.3% to 0.03%, reflecting the expanded reference database. The most frequent of the previously novel alleles were MICA*107N (0.02%), MICA*141 (0.01%), MICA*119 (0.01%), MICA*089 (0.01%), and MICA*247 (0.01%). While allele frequencies in other European and the South African White population were similar to those in Germany, greater variation was observed in the South African Black, non-indigenous Chilean, and Turkish populations. Notably, some of the novel alleles appeared to be population-specific; for example, MICA*258 was exclusively identified in samples from the Black or Colored populations of South Africa. In conclusion, the extensive characterization of novel MICA alleles has substantially reduced the fraction of unknown sequences in MICA donor genotyping, which will support future biomedical and population genetic studies.
Background & Aims FOXP3+ regulatory T cells (Tregs) constitute 4–7% of circulating CD4+ T cells and are central to immune tolerance and homeostasis, making them attractive candidates for preventing transplant rejection and treating autoimmune and inflammatory diseases, including graft-versus-host disease (GvHD). However, clinical trials have reported highly variable in vivo persistence and efficacy, likely reflecting differences in Treg source, ex vivo expansion strategies, and final product phenotype. This study aims to systematically compare Tregs derived from three clinically relevant sources—adult peripheral blood (AB), umbilical cord blood (CB), and pediatric thymus (Thy)—across functional, phenotypic, and manufacturing-relevant parameters to define their relative therapeutic potential for future allogeneic applications. Methodology Highly pure Tregs were isolated using a combination of published magnetic and flow-based sorting strategies, including the MACSQuant Tyto cell sorter. Cells were expanded ex vivo for 14 days using polymer-based anti-CD3/CD28 stimulation in the presence of high-dose IL-2. Post-expansion products were phenotyped by flow cytometry. Bona fide Treg identity was confirmed by TSDR demethylation analysis and absence of IL-2 production upon activation. Suppressive function was assessed using a proliferation-based in vitro suppression assay, and lineage stability was evaluated under Th1- and Th17-polarizing inflammatory cytokine conditions. Results Isolation of highly pure FOXP3+ Tregs from CB was the most cost-effective among all sources, requiring only a single-step FACS isolation using the MACSQuant Tyto due to low starting cell numbers. CB-derived Tregs showed robust ex vivo expansion (median 2678-fold; range 1035–5989; n=4) while maintaining a stable Treg identity, with 99.9% TSDR demethylation across all donors. Consistently, IL-2 expression remained minimal within CB-Tregs after expansion (median 1.13%; range 1.03–2.74%; n=4). Functionally, CB-Tregs exhibited the strongest suppressive capacity against both CD4+ and CD8+ allogeneic effector T cells and retained phenotypic stability after repeated restimulation and exposure to pro-inflammatory cytokines compared to AB- and Thy- Tregs. Conclusion Among the three clinically relevant sources evaluated, CB emerged as the most promising for future allogeneic Treg therapies.
