Examination of Thymic Positive and Negative Selection by Flow Cytometry

Hu, Qian; Nicol, Stephanie A.; Suen, Alexander Y.W.; Baldwin, Troy A.

doi:10.3791/4269-v

Cited by 2 publications

(2 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Owing to the continuous nature of development and technical variations in marker detection, no consensus has emerged on how to define positive selection intermediates by flow cytometry 1 , 2 , 21 , 22 . To address this, we performed in silico flow cytometry on totalVI denoised surface protein expression to distinguish different pseudotime phases.…”

Section: Resultsmentioning

confidence: 99%

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4+ T cell and CD8+ T cell lineage commitment

et al. 2023

View full text Add to dashboard Cite

The development of CD4+ T cells and CD8+ T cells in the thymus is critical to adaptive immunity and is widely studied as a model of lineage commitment. Recognition of self-peptide major histocompatibility complex (MHC) class I or II by the T cell antigen receptor (TCR) determines the CD8+ or CD4+ T cell lineage choice, respectively, but how distinct TCR signals drive transcriptional programs of lineage commitment remains largely unknown. Here we applied CITE-seq to measure RNA and surface proteins in thymocytes from wild-type and T cell lineage-restricted mice to generate a comprehensive timeline of cell states for each T cell lineage. These analyses identified a sequential process whereby all thymocytes initiate CD4+ T cell lineage differentiation during a first wave of TCR signaling, followed by a second TCR signaling wave that coincides with CD8+ T cell lineage specification. CITE-seq and pharmaceutical inhibition experiments implicated a TCR–calcineurin–NFAT–GATA3 axis in driving the CD4+ T cell fate. Our data provide a resource for understanding cell fate decisions and implicate a sequential selection process in guiding lineage choice.

show abstract

Section: Resultsmentioning

confidence: 99%

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4+ T cell and CD8+ T cell lineage commitment

et al. 2023

View full text Add to dashboard Cite

show abstract

“…We next sought to use pseudotime information to clarify the intermediate stages of development in the two lineages. Thymocyte populations have been commonly defined by surface protein expression using flow cytometry, and various marker combinations and gating strategies have been employed to subset thymocyte populations based on maturity and lineage (Germain, 2002; Xiong and Bosselut, 2012; Saini et al, 2010; Hu et al, 2012). However, due to the continuous nature of developmental intermediates, as well as technical variations in marker detection, no uniform consensus has emerged on how to define positive selection intermediates by flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

Single-cell multi-omic analysis of thymocyte development reveals drivers of CD4/CD8 lineage commitment

Steier

Lutes

et al. 2021

Preprint

View full text Add to dashboard Cite

CD4 and CD8 T cells play critical roles in the mammalian immune system. While their development within the thymus from the CD4+CD8+ stage has been widely studied as a model of lineage commitment, the underlying mechanism remains unclear. To deconstruct this process, we apply CITE-seq, measuring the transcriptome and over 100 surface proteins in thymocytes from wild-type and lineage-restricted mice. We jointly analyze the paired measurements to build a comprehensive timeline of RNA and protein expression in each lineage, supporting a sequential model of lineage determination in which both lineages go through an initial phase of CD4 lineage audition, which is followed by divergence and specification of CD8 lineage cells. We identify early differences implicating T cell receptor signaling via calcineurin-NFAT in driving CD4 lineage commitment. Pharmacological inhibition validates the requirement of calcineurin- NFAT for CD4, but not CD8, lineage development, providing insight into the CD4/CD8 commitment mechanism.

show abstract

Examination of Thymic Positive and Negative Selection by Flow Cytometry

Cited by 2 publications

References 0 publications

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4+ T cell and CD8+ T cell lineage commitment

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4+ T cell and CD8+ T cell lineage commitment

Single-cell multi-omic analysis of thymocyte development reveals drivers of CD4/CD8 lineage commitment

Contact Info

Product

Resources

About