How Many Gamma Delta T Cells are There? Calling Strategy, Quality Control and Receptor-locus Specificity in 3-prime Single-nucleus RNA Sequencing of Human Small Intestine
Document Type
Article
Publication Title
Journal of Immunological Methods
Abstract
Public single-nucleus transcriptomes are increasingly reused to count gamma delta T cells in the human small intestine, but how far that count depends on analytical choices has not been quantified. Using a public 3-prime single-nucleus dataset of 33,600 nuclei from six small-intestinal biopsies, we benchmarked three calling strategies across three quality-control levels, tested six module-score cutoff rules, and applied two specificity checks that need no additional assay. The recovered fraction spanned 3.38% to 12.49% on the deposited matrix and 5.46% to 21.62% under strict filtering, a 3.70-fold to 4.08-fold range depending on the filter. Tightening the module-score cutoff corrected the count but not the membership: at a cutoff matched to the gated call, agreement with it reached only 0.408 by Jaccard index. Within one quality-control stratum the call rate rose from 0.51% to 12.17% across deciles of genes per nucleus, so detection sensitivity rather than abundance dominates. Constant-region genes were near-absent from non-T epithelial nuclei (TRGC2 0.25% versus 36.19% of CD3-positive nuclei), indicating that broad TRGC2 detection reflects expression that is not lineage restricted rather than ambient RNA. However, 96.3% of gated calls also carried TRAC or TRBC counts, which is hard to reconcile with excision of the TRD locus during TRA rearrangement and points to unquantified alpha-beta admixture. Variable-region genes were near-undetectable, so subtype assignment was infeasible. We conclude that absolute gamma delta counts from 3-prime nuclear data are not interpretable on their own, and we supply a reporting checklist and open code.
DOI
10.1016/j.jim.2026.114126
Publication Date
9-27-2026
Keywords
Gamma-delta, Single-nucleus, Benchmarking, TRDC, Ambient-RNA, Reproducibility
ISSN
1872-7905
Recommended Citation
Haji-Boloori F, Kubiak A, Powers N, Neary J, Osikoya O. How Many Gamma Delta T Cells are There? Calling Strategy, Quality Control and Receptor-locus Specificity in 3-prime Single-nucleus RNA Sequencing of Human Small Intestine. Journal of Immunological Methods. 2026; . doi: 10.1016/j.jim.2026.114126.
