Re-Investigation And Re-Classification Of T Cells Subsets Based On Single Cell Sequencing

T lymphocytes are one of the main kinds of immune cells that mediate adaptive cellular immune responses and play an important role in adaptive humoral immune responses.Currently,T lymphocyte subtypes are usually distinguished according to the phenotypes such as T lymphocyte specific surface markers and transcription factors by fluorescence-activated cell sorting.This simplified classification technique cannot directly provide more information on the overall differences between different T cell subtypes and the differences within each subtype.The rapid development of single-cell transcriptome sequencing technology in recent years has provided us with powerful tools for studying the classification of T lymphocytes.Firstly,we reanalyzed peripheral blood mononuclear cells scRNA-seq data,and found that T lymphocytes were clustered independently in the peripheral blood mononuclear cell population,but no obvious subpopulations were formed inside the T lymphocytes.This indicates that T lymphocytes are significantly different from other monocytes such as B cells,NK cells,monocytes,and dendritic cells in the transcriptome landscape,but this dataset still cannot tell us the difference between T lymphocyte subtypes.Then by merging the single-cell transcriptome datasets from six common T-lymphocyte subtypes,we re-clustered the T cells into sub groups and identified the marker genes that are specific to each group and redefined the clusters based on their marker genes,furthermore,we explored the relationship between different T cell subpopulations and their possible differentiation trajectories.As result,for all the merged T lymphocytes,we found that CD4+ T cells and CD8+ T cells are clustered independently but lack clear boundary between them.Re-clustering of CD4+ T cells alone revealed no significant demarcation between CD4+ T cell subsets,indicating some continuity between CD4+ T cell subsets.Through the hierarchy clustering analysis,we found one subset with highly expression of cytotoxic molecules in CD4+ T cells and defined it as “sc-CD4+ CTL”,which was rarely reported before.In addition,we found a special type in the CD8+ CTL sample,which expresses both T cell specific receptors and NK cell specific receptors,which we defined as “sc-CD8+ NKT”,which was also rarely reported before.Finally,by tracing the differentiation trajectories,we found that the differences between different T cell subtypes were not discrete but rather a continuous state in the expression profile.The re-investigation of T cell subsets by single-cell transcriptome sequencing is of great significance for re-recognizing the Tcell subtypes,and will help us to further understand the mechanism of the human adaptive immune response,which also has important implications for the ongoing immunotherapy.