Single-Cell Sequencing Reveals the Transcriptome and TCR Characteristics of pTregs and in vitro Expanded iTregs.
Single-Cell Sequencing Reveals the Transcriptome and TCR Characteristics of pTregs and in vitro Expanded iTregs.
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单细胞测序揭示 pTreg 和体外扩增 iTreg 的转录组和 TCR 特征
DOI：
10.3389/fimmu.2021.619932
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发表时间：
2021
影响因子：
7.3
通讯作者：
Ren X
中科院分区：
文献类型：
--
作者：
Hui Z;Zhang J;Zheng Y;Yang L;Yu W;An Y;Wei F;Ren X
调节性T细胞(Tregs)在维持免疫耐受和肿瘤逃逸中起着关键作用。然而，这些细胞在外周血和组织中的比例相对较低，阻碍了许多研究。我们试图在诊断为结直肠癌的患者中建立基于雷帕霉素的Treg体外扩增程序，并进行单细胞测序以探索Treg细胞的特征。从结直肠癌患者外周血单个核细胞中分离纯化CD25+细胞，在X-VIVO15培养液中加入5%人AB血清、L-谷氨酰胺、雷帕霉素、白介素2(IL-2)和戴纳贝兹人Treg扩张器培养21d，扩增Treg细胞。从结直肠癌患者的CD4+CD25+细胞中成功扩增出表型和功能良好的Treg细胞。中位扩增倍数为75倍(20-105倍)，90.0%的收获细胞为CD_4+CD_(25)+CD_(127)/−细胞。CD4+CD25+Foxp3+细胞比例超过60%。功能分析显示，iTregs在体外可显著抑制CD8+T细胞的增殖。单细胞测序显示，pTreg(从结直肠癌患者外周血中分离的CD_4+CD_(25)+CD_(127)dim/−细胞)和iTreg(CD_4+CD_(25)+CD_(127)dim/−细胞按上述方案体外扩增)的转录组相互交错。PTregs的抑制功能增强，而iTregs的增殖能力增强。TCR图谱分析表明，pTregs和iTregs之间的重叠很小。Tregs的伪时间轨迹分析表明，pTregs是由激活/效应型、静止型和增殖型Tregs三个主要分支组成的连续体。相比之下，在体外扩增的iTregs是增殖和激活/效应细胞的混合物。贩运受体在pTregs和iTregs中的表达也不同。多种趋化因子受体在pTregs中表达上调。激活的效应器pTregs过度表达趋化因子受体CCR10，而iTregs不表达该受体。趋化因子CCL28在结直肠癌中高表达，且与预后不良相关。CCR10与CCL28相互作用，介导Treg在肿瘤中的募集，加速肿瘤进展。从肿瘤微环境(TME)中去除CCR10+Treg细胞可作为结直肠癌患者的有效治疗策略。我们的数据区分了不同亚群的Treg细胞的转录特征，并揭示了不同群体的Treg细胞的上下文相关功能，这对于开发Treg细胞在自身免疫性疾病和癌症中的替代治疗策略至关重要。
Regulatory T cells (Tregs) 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 many studies. We sought to establish a rapamycin-based in vitro Treg expansion procedure in patients diagnosed with colorectal cancer and perform single-cell sequencing to explore the characteristics of Treg cells. CD25+ cells enriched from peripheral blood mononuclear cells (PBMC) of colorectal tumor patients were cultured in X-VIVO15 medium, supplemented with 5% human AB serum, L-glutamine, rapamycin, interleukin-2 (IL-2), and Dynabeads human Treg expander for 21 days to expand Tregs. Treg cells with satisfactory phenotype and function were successfully expanded from CD4+CD25+ cells in patients with colorectal cancer. The median expansion fold was 75 (range, 20–105-fold), and >90.0% of the harvest cells were CD4+CD25+CD127dim/− cells. The ratio of CD4+CD25+Foxp3+ cells exceeded 60%. Functional assays showed that iTregs significantly inhibited CD8+T cell proliferation in vitro. Single-cell sequencing showed that the transcriptome of pTreg (CD4+CD25+CD127dim/− cells isolated from PBMC of colorectal cancer patients) and iTreg (CD4+CD25+CD127dim/− cells expanded in vitro according to the above regimen) cells were interlaced. pTregs exhibited enhanced suppressive function, whereas iTregs exhibited increased proliferative capacity. TCR repertoire analysis indicated minimal overlap between pTregs and iTregs. Pseudo-time trajectory analysis of Tregs revealed that pTregs were a continuum composed of three main branches: activated/effector, resting and proliferative Tregs. In contrast, in vitro expanded iTregs were a mixture of proliferating and activated/effector cells. The expression of trafficking receptors was also different in pTregs and iTregs. Various chemokine receptors were upregulated in pTregs. Activated effector pTregs overexpressed the chemokine receptor CCR10, which was not expressed in iTregs. The chemokine CCL28 was overexpressed in colorectal cancer and associated with poor prognosis. CCR10 interacted with CCL28 to mediate the recruitment of Treg into tumors and accelerated tumor progression. Depletion of CCR10+Treg cells from tumor microenvironment (TME) could be used as an effective treatment strategy for colorectal cancer patients. Our data distinguished the transcriptomic 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.
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影响因子：
3.5
作者：
Chen, Linbo;Lu, Dewen;Xu, Feng
通讯作者：
Xu, Feng
影响因子：
30.5
作者：
Kitagawa Y;Ohkura N;Kidani Y;Vandenbon A;Hirota K;Kawakami R;Yasuda K;Motooka D;Nakamura S;Kondo M;Taniuchi I;Kohwi-Shigematsu T;Sakaguchi S
通讯作者：
Sakaguchi S
DOI：
10.1016/j.omtm.2018.01.006
发表时间：
2018-03-16
期刊：
Molecular therapy. Methods & clinical development
影响因子：
--
作者：
Fraser H;Safinia N;Grageda N;Thirkell S;Lowe K;Fry LJ;Scottá C;Hope A;Fisher C;Hilton R;Game D;Harden P;Bushell A;Wood K;Lechler RI;Lombardi G
通讯作者：
Lombardi G
影响因子：
4.5
作者：
Alsuliman, Abdullah;Appel, Stanley H.;Rezvani, Katayoun
通讯作者：
Rezvani, Katayoun
影响因子：
3
作者：
Toby IT;Levin MK;Salinas EA;Christley S;Bhattacharya S;Breden F;Buntzman A;Corrie B;Fonner J;Gupta NT;Hershberg U;Marthandan N;Rosenfeld A;Rounds W;Rubelt F;Scarborough W;Scott JK;Uduman M;Vander Heiden JA;Scheuermann RH;Monson N;Kleinstein SH;Cowell LG
通讯作者：
Cowell LG