Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.

Laboratory of Adaptive Immunity Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

Zobrazit více v PubMed

Papalexi E, Satija R. Single-cell RNA sequencing to explore immune cell heterogeneity. Nat Rev Immunol. (2018) 18:35–45. doi: 10.1038/nri.2017.76 PubMed DOI

Wang X, Shen X, Chen S, Liu H, Hong N, Zhong H, et al. . Reinvestigation of classic T cell subsets and identification of novel cell subpopulations by single-cell RNA sequencing. J Immunol. (2022) 208:396–406. doi: 10.4049/jimmunol.2100581 PubMed DOI

Verstegen NJM, Pollastro S, Unger PA, Marsman C, Elias G, Jorritsma T, et al. . Single-cell analysis reveals dynamics of human B cell differentiation and identifies novel B and antibody-secreting cell intermediates. Elife. (2023) 12. doi: 10.7554/eLife.83578.sa2 PubMed DOI PMC

Prince HE, York J, Jensen ER. Phenotypic comparison of the three populations of human lymphocytes defined by CD45RO and CD45RA expression. Cell Immunol. (1992) 145:254–62. doi: 10.1016/0008-8749(92)90329-N PubMed DOI

Dawes R, Petrova S, Liu Z, Wraith D, Beverley PC, Tchilian EZ. Combinations of CD45 isoforms are crucial for immune function and disease. J Immunol. (2006) 176:3417–25. doi: 10.4049/jimmunol.176.6.3417 PubMed DOI PMC

Machura E, Mazur B, Pieniazek W, Karczewska K. Expression of naive/memory (CD45RA/CD45RO) markers by peripheral blood CD4+ and CD8 + T cells in children with asthma. Arch Immunol Ther Exp (Warsz). (2008) 56:55–62. doi: 10.1007/s00005-008-0005-6 PubMed DOI PMC

Desai DM, Sap J, Silvennoinen O, Schlessinger J, Weiss A. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. EMBO J. (1994) 13:4002–10. doi: 10.1002/j.1460-2075.1994.tb06716.x PubMed DOI PMC

Streuli M, Matsuyama T, Morimoto C, Schlossman SF, Saito H. Identification of the sequence required for expression of the 2H4 epitope on the human leukocyte common antigens. J Exp Med. (1987) 166:1567–72. doi: 10.1084/jem.166.5.1567 PubMed DOI PMC

Johnson P, Greenbaum L, Bottomly K, Trowbridge IS. Identification of the alternatively spliced exons of murine CD45 (T200) required for reactivity with B220 and other T200-restricted antibodies. J Exp Med. (1989) 169:1179–84. doi: 10.1084/jem.169.3.1179 PubMed DOI PMC

Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol. (2004) 22:745–63. doi: 10.1146/annurev.immunol.22.012703.104702 PubMed DOI

Birkeland ML, Johnson P, Trowbridge IS, Pure E. Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation. Proc Natl Acad Sci U S A. (1989) 86:6734–8. doi: 10.1073/pnas.86.17.6734 PubMed DOI PMC

Coffman RL, Weissman IL. A monoclonal antibody that recognizes B cells and B cell precursors in mice. J Exp Med. (1981) 153:269–79. doi: 10.1084/jem.153.2.269 PubMed DOI PMC

Coffman RL, Weissman IL. B220: a B cell-specific member of th T200 glycoprotein family. Nature. (1981) 289:681–3. doi: 10.1038/289681a0 PubMed DOI

Stoeckius M, Hafemeister C, Stephenson W, Houck-Loomis B, Chattopadhyay PK, Swerdlow H, et al. . Simultaneous epitope and transcriptome measurement in single cells. Nat Methods. (2017) 14:865–8. doi: 10.1038/nmeth.4380 PubMed DOI PMC

Zheng GX, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R, et al. . Massively parallel digital transcriptional profiling of single cells. Nat Commun. (2017) 8:14049. doi: 10.1038/ncomms14049 PubMed DOI PMC

Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, Pollard MO, et al. . Twelve years of SAMtools and BCFtools. Gigascience. (2021) 10. doi: 10.1093/gigascience/giab008 PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. . The sequence alignment/map format and SAMtools. Bioinformatics. (2009) 25:2078–9. doi: 10.1093/bioinformatics/btp352 PubMed DOI PMC

Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. (2017) 14:417–9. doi: 10.1038/nmeth.4197 PubMed DOI PMC

Team RC . R: A Language and Environment for Statistical Computing: R Foundation for Statistical Computing (2022). Available online at: https://www.R-project.org/ (Accessed December 8, 2023).

Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, et al. . NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res. (2013) 41:D991–5. doi: 10.1093/nar/gks1193 PubMed DOI PMC

Collora JA, Liu R, Pinto-Santini D, Ravindra N, Ganoza C, Lama JR, et al. . Single-cell multiomics reveals persistence of HIV-1 in expanded cytotoxic T cell clones. Immunity. (2022) 55:1013–31 e7. doi: 10.1016/j.immuni.2022.03.004 PubMed DOI PMC

Yu B, Shi Q, Belk JA, Yost KE, Parker KR, Li R, et al. . Engineered cell entry links receptor biology with single-cell genomics. Cell. (2022) 185:4904–20 e22. doi: 10.1016/j.cell.2022.11.016 PubMed DOI PMC

Mogilenko DA, Shpynov O, Andhey PS, Arthur L, Swain A, Esaulova E, et al. . Comprehensive profiling of an aging immune system reveals clonal GZMK(+) CD8(+) T cells as conserved hallmark of inflammaging. Immunity. (2021) 54:99–115 e12. doi: 10.1016/j.immuni.2020.11.005 PubMed DOI

Lawlor N, Nehar-Belaid D, Grassmann JDS, Stoeckius M, Smibert P, Stitzel ML, et al. . Single cell analysis of blood mononuclear cells stimulated through either LPS or anti-CD3 and anti-CD28. Front Immunol. (2021) 12:636720. doi: 10.3389/fimmu.2021.636720 PubMed DOI PMC

Cunningham F, Allen JE, Allen J, Alvarez-Jarreta J, Amode MR, Armean IM, et al. . Ensembl 2022. Nucleic Acids Res. (2022) 50:D988–D95. doi: 10.1093/nar/gkab1049 PubMed DOI PMC

Haradhvala NJ, Leick MB, Maurer K, Gohil SH, Larson RC, Yao N, et al. . Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nat Med. (2022) 28:1848–59. doi: 10.1038/s41591-022-01959-0 PubMed DOI PMC

Aitchison J. The statistical-analysis of compositional data. J Roy Stat Soc B. (1982) 44:139–77. doi: 10.1111/j.2517-6161.1982.tb01195.x DOI

Andreatta M, Carmona SJ. STACAS: Sub-Type Anchor Correction for Alignment in Seurat to integrate single-cell RNA-seq data. Bioinformatics. (2021) 37:882–4. doi: 10.1093/bioinformatics/btaa755 PubMed DOI PMC

Alioto TS, Buchhalter I, Derdak S, Hutter B, Eldridge MD, Hovig E, et al. . A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing. Nat Commun. (2015) 6:10001. doi: 10.1038/ncomms10001 PubMed DOI PMC

Jivanjee T, Ibrahim S, Nyquist SK, Gatter GJ, Bromley JD, Jaiswal S, et al. . Enriching and characterizing T cell repertoires from 3' Barcoded single-cell whole transcriptome amplification products. Methods Mol Biol. (2022) 2574:159–82. doi: 10.1007/978-1-0716-2712-9_7 PubMed DOI PMC