Transcriptomes And TCR Characteristics Of PTregs And In Vitro Expanded ITregs

Purpose:Regulatory T cells（Tregs）are a special subgroup of T cells and an important part of tumor microenvironment,which suppress CD4⁺and CD8⁺T cell-mediated immune responses in vivo and in vitro through a variety of mechanisms,and play a critical role in the maintenance of immune tolerance and tumor evasion.However,the relative low proportion of these cells in peripheral blood and tissues has hindered the development of many related studies.In this study,we sought to explore and establish a method in vitro of Tregs expansion,and understand their phenotype and function,and compare the transcriptome and TCR characteristics of peripheral regulatory T cells（p Tregs）and in vitro induced regulatory T cells（i Tregs）in patients with colon cancer by single cell sequencing technology,so as to better understand Tregs cells,To explore new targets for immune-based tumor therhapy.Methods:Firstly,we isolated peripheral blood mononuclear cells（PBMC）from patients with colon cancer by density gradient centrifugation.And then,CD25⁺cells enriched from PBMC by CD25⁺microbeads were cultured in x-vivo 15 medium,supplemented with 5%human AB serum,L-glutamine,rapamycin,interleukin-2（IL-2）,and anti-CD3/CD28 microbeads for 21 days to expand Tregs in vitro.The phenotype of i Tregs were determined by flow cytometry during culture,And the inhibitory function of itregs were determined by co-incubation experiment of CFSE labeled CD25 cells and itregs.Then,we compared the similarities and differences between peripheral regulatory T cells（p Tregs）and induced regulatory T cells（i Tregs）by single cell transcriptome/TCR sequencing.We used cell Ranger（v2.2.2,10x genomics）and Seurat（v2.3.0）for bioinformatics and statistical analysis.David and Metascape were used to perform functional enrichment analysis.Monocle2 was used to analyze the pseudo time development trajectory.SPSS（version 25.0,IBM）and Graphpad prism（version 8.0.1,USA）were used for statistical analysis.Spearman correlation analysis was used to analyze the correlation of different biomarkers（P<0.05）.Kaplan Meier log rank test was used for univariate survival analysis.Results:A total of 11 culture procedures were performed.The median expansion fold was75（range,20–105 fold）.During expasion,the proportion of CD4⁺CD25⁺CD127^dim/-cells increased gradually and was more than 90.0%on day 21.CD4⁺CD25⁺Foxp3⁺cells accounted for more than 60%of the harvested cells on day 21.Compared to ptregs,in vitro expanded i Tregs highly expressed the immune checkpoint molecules PD-1and CTLA-4.CFSE based coincubation test showed that i Tregs significantly inhibited the proliferation of CD8⁺T cells in vitro.Single cell sequencing integrated data analysis showed that the transcriptome of p Tregs and i Tregs were interlaced.p Tregs exhibited enhanced suppressive function,whereas i Tregs exhibited increased proliferative capacity.TCR repertoire analysis indicated minimal overlap between p Tregs and i Tregs.Pseudo-time trajectory analysis of Tregs revealed that p Tregs were a continuum composed of three main branches:activated/effector,resting and proliferative Tregs.In contrast,in vitro expanded i Tregs were a mixture of proliferating and activated/effector cells.The expression of trafficking receptors was also different in p Tregs and i Tregs.Various chemokine receptors were upregulated in p Tregs.Activated effector p Tregs overexpressed the chemokine receptor CCR10,which was not expressed in i Tregs.Multiple fluorescence immunohistochemistry showed that CCL28,the ligand of CCR10,was overexpressed in colorectal cancer and was associated with poor prognosis.Pearson correlation analysis showed that the expression of CCL28 in colorectal cancer was significantly correlated with CCR10⁺Treg（r=0.515,P<0.00）.Kaplan Meier univariate survival analysis showed that both CCL28 and CCR10⁺Tregs were poor predictors of progression-free survival（PFS）in colorectal patients（49.9 vs 38.6 months,log rank P=0.007;49.4 vs 14.8months,log rank P<0.0001）.Conclusion:Treg cells with satisfactory phenotype and function were successfully expanded from CD4⁺CD25⁺cells in patients with colorectal cancer.The observation that p Treg and i Treg cells were functionally similar and transcriptionally overlapped.But,TCR repertoire analysis indicated minimal overlap between p Tregs and i Tregs.Chemokine receptor CCR10 is highly expressed in p Tregs.CCR10 interacts with chemokine CCL28,to mediate the recruitment of Treg to tumor and accelerate tumor progression,which is associated with poor prognosis in colon cancer patients.Our data distinguished the transcriptomic and TCR characteristics of different subsets of Treg cells and revealed the context-dependent functions of different populations of Treg cells,which was crucial to the development of alternative therapeutic strategies for Treg cells in autoimmune disease and cancer.