Mapping The Spatial Dimensionality Of The T Cell Receptor Repertoire

T cells,also known as T lymphocytes,are an essential part of the immune system.T cells can be distributed to tissues and organs throughout the body through the lymphatic and circulatory systems,constitute a widespread immune surveillance network,and play an important role in adaptive immune responses.Antigen recognition by T cells depends on the expression of highly polymorphic surface receptors called T cell receptors(TCRs).TCRs are cell specific,and a T cell expresses only one TCR.It is increasingly appreciated that the T cell pool is unevenly distributed within an organism,with substantial spatiotemporal heterogeneity.Notably,a substantial portion of T cell clones reside at barrier sites such as the intestine and skin and are generally restricted from circulation.Yet,despite this knowledge,many basic questions remain unanswered,such as how the highly diverse TCR repertoire is spatially distributed in tissues and organs,whether the TCR repertoire diversity differs across tissues,whether the TCRs in tissues are commonly shared across individuals,and what is the life history of a single TCR clone.Therefore,this thesis has carried out the following research work aiming at the above problems.We investigated the TCR repertoires of CD3+T cells(about 5×10~5 cells)from sixteen tissues of healthy C57BL/6 mice by high-throughput TCRseq.We found that TCR repertoires generally were classified into three categories(lymph nodes,non-lymph node tissues and small intestine)based on sequence similarity.Clonal distribution and diversity analyses showed that small intestine compartment had a more skewed repertoire as compared to lymph nodes and non-lymph node tissues.However,analysis of TRBV and TRBJ gene usage across tissue compartments,as well as comparison of CDR3 length distributions,showed no significant tissue-dependent differences.Interestingly,analysis of clonotype sharing between mice showed that although non-redundant public clonotypes were found more easily in lymph nodes,CD4+T cells in small intestine harbored more abundant public clonotypes.In order to overcome the bias in library construction,we developed a two spike-in T cell receptor sequencing(TS-TCRseq)method,wherein fixed amounts of barcoded PCR templates and T cell hybridoma cells were included as key points of reference.TS-TCRseq could correct for PCR amplification bias and sequencing errors and normalize sequencing depth between samples by external spike-ins,and could also accurately estimate the number of T cells in a sample.This technique was not only applicable to cell samples,but also to complex solid tissue samples.Using TS-TCRseq,we estimated the number of T cells in solid tissues and found that cell isolation underestimated the number of T cells in the tissue.During the cell isolation process,most of the high-frequency T cell clones were retained,while some low-frequency rare T cell clones would be lost.To gain a more comprehensive and accurate understanding of the TCR repertoire of tissues and organs of the organism,we analyzed the TCR sequences of 30 tissues in healthy wild-type C57BL/6 mice using TS-TCRseq technology.It was found that the total number of T cells in 30 tissues was about 1.0×10~8,the number of unique clonotypes was as high as4.4×10~6,and the TCR diversity varies widely between tissues.Heat map clustering analysis further showed that the peripheral TCR repertoires showed regional distribution characteristics.Moreover,we sought to recapitulated our mapping effort with CD4+and CD8+T cell populations in 19 tissues,and found that the TCR repertoires of CD4+and CD8+T cells had similar spatial distribution and diversity characteristics.Having obtained the general result of high overlapping between CD8+T cells in several major organs(kidney,liver and lung),we further combined the single-cell RNA sequencing(scRNAseq)and single-cell T cell receptor sequencing(scTCRseq)to analyze the single-cell gene expression profile and TCR from the lung,liver,kidney,and spleen of healthy wild-type C57BL/6 mice,and identified CD8+T cells sharing TCRs between tissues were mainly effector memory-like T cells,and there were also differential and tissue-specific expression.The pseudo-time trajectory analysis further revealed the tissue differentiation and development trajectory of a single T cell clone.Among them,lung tissue-derived T cells that highly express Klrg1 and Cx3cr1 are at the beginning of the trajectory,indicating the initial state of T cells;And kidney-derived T cells that highly express Cd69 and Cxcr6 occupies the end,marking the ultimate fate of T cells.Finally,the trajectory inference of pathway significance(TIPS)uncovers the mechanism of tissue-specific differential differentiation of T cell clones driven by chemokine receptors.In summary,using the traditional TCRseq technology and our newly established TS-TCRseq technology,combined with bioinformatics analysis methods,we comprehensively analyzed the spatial distribution and diversity characteristics of the TCR repertoire in peripheral tissues of healthy mice.Combined use of scRNAseq and scTCRseq for paired single-cell gene expression profiling and TCR analysis further confirmed that the CD8+T cells sharing TCR between different tissues were mainly Tems,but there were also chemokine receptor-driven T cells clonal tissue-specific differential differentiation.These findings under healthy physiological conditions offer an important reference dataset,which may contribute to our ability to better manipulate T cell responses against infection and vaccination.Moreover,our findings helped to understand the defense mechanisms of the immune system in tissues and organs,and lays the foundation for future research on tissue and organ-related autoimmune diseases and tumor immune characteristics.