Single-cell RNA sequencing has transformed immunological research by enabling high-resolution transcriptional profiling of individual immune cells. Despite its transformative impact, annotating immune cells based solely on transcriptomic data remains challenging. These difficulties arise from biological factors, including gene expression heterogeneity and post-transcriptional regulation, as well as technical limitations that contribute to mismatches between mRNA and protein expression. Such discrepancies may lead to cell misclassification and obscure functional insights, particularly in heterogeneous populations such as peripheral blood mononuclear cells. This review highlights the major challenges in immune cell annotation by detailing the mechanisms underlying mRNA-protein discrepancies, examining both the biological factors and technical artifacts that drive this divergence, and emphasizing their implications for accurate cell classification. A critical overview of current single-cell profiling technologies follows, with evaluation of the respective advantages and limitations of transcriptomic, proteomic, and multimodal approaches. In particular, technologies such as Cellular Indexing of Transcriptomes and Epitopes by Sequencing integrate transcriptomic and proteomic data, addressing the shortcomings of single-modality analyses. Further examination focuses on computational strategies for immune cell annotation, with emphasis on automated methods and bioinformatics frameworks tailored to multi-omics datasets. The unique computational challenges of integrating mRNA and protein data, together with solutions for improved annotation accuracy, are discussed. This review integrates key challenges, technologies, and computational tools, highlighting the need for standardized multimodal profiling of immune cells. Such integration enhances annotation reliability and advances disease understanding and therapy discovery.
George et al. (Fri,) studied this question.