Among many factors known to alter the outcomes of T cell receptor(TCR)-induced proximal signaling,the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained c...Among many factors known to alter the outcomes of T cell receptor(TCR)-induced proximal signaling,the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address.Here,we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations(pTyr-SNVs)that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways.We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin(OVA)antigens and retrovirally engineered syngeneic major histocompatibility complex(MHC)Class I restricted OVA TCR-transgenic CD8 T cells(OT-I).Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4(ITGA4)p.S1027I variant as a prototype,we show that under identical TCR stimulation conditions,genetically determined membrane-proximal immunoreceptor tyrosin activation motif(ITAM)results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein(ZAP70)and the levels of cytotoxic effector molecule granzyme B(GZMB),which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line.This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.展开更多
目的通过应用孟德尔随机化(Mendelian randomization,MR)方法,系统探究10种尿毒症相关毒素及代谢物与CD8^(+)T细胞衰老表型之间的双向因果关系。方法采用双向MR方法,基于全基因组关联研究(genome-wide association studies,GWAS)数据库...目的通过应用孟德尔随机化(Mendelian randomization,MR)方法,系统探究10种尿毒症相关毒素及代谢物与CD8^(+)T细胞衰老表型之间的双向因果关系。方法采用双向MR方法,基于全基因组关联研究(genome-wide association studies,GWAS)数据库汇总数据,提取与10种尿毒症相关毒素及代谢物以及4种CD8^(+)T细胞衰老表型显著相关的单核苷酸多态性位点作为工具变量。逆方差加权法(inverse variance weighting,IVW)作为主要分析方法,同时采用MR-Egger、加权中位数法、简单众数法及加权众数法进行补充分析。通过Cochran’s Q检验、MR-Egger截距检验、MR-PRESSO及留一法等敏感性分析验证结果的稳健性与潜在多效性。结果MR分析显示,肉碱水平与终末分化CD8^(+)T细胞比例(IVW:β=1.71,95%CI:0.59~2.83,P=0.003)、CD45RA^(+)CD28^(-)CD8^(+)T细胞比例(IVW:β=2.23,95%CI:0.65~3.80,P=0.006)、CD28^(-)CD8^(+)T细胞比例(IVW:β=1.13,95%CI:0.11~2.15,P=0.030)正相关。犬尿氨酸水平与终末分化CD8^(+)T细胞比例正相关(IVW:β=1.69,95%CI:0.57~2.82,P=0.0003),而白介素-6水平与CD28^(-)CD8^(+)T细胞比例(IVW:β=0.28,95%CI:0.03~0.53,P=0.026)和CD45RA^(+)CD28^(-)CD8^(+)T细胞比例(IVW:β=0.41,95%CI:0.02~0.79,P=0.04)正相关。N^(2),N^(2)-二甲基鸟苷水平与CD45RA^(+)CD28^(-)CD8^(+)T细胞绝对计数(IVW:β=2.53,95%CI:0.51~4.55,P=0.014)正相关。反向MR分析显示,CD45RA^(+)CD28^(-)CD8^(+)T细胞绝对计数增加可降低肉碱水平(IVW:β=-0.00003,95%CI:-0.000062~-0.000004,P=0.024)。所有敏感性分析均支持主要结果的稳健性。结论部分尿毒症相关毒素及代谢物与CD8^(+)T细胞衰老之间存在双向因果关联,为尿毒症患者免疫功能紊乱的机制提供了新的因果证据与潜在干预靶点。展开更多
文摘Among many factors known to alter the outcomes of T cell receptor(TCR)-induced proximal signaling,the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address.Here,we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations(pTyr-SNVs)that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways.We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin(OVA)antigens and retrovirally engineered syngeneic major histocompatibility complex(MHC)Class I restricted OVA TCR-transgenic CD8 T cells(OT-I).Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4(ITGA4)p.S1027I variant as a prototype,we show that under identical TCR stimulation conditions,genetically determined membrane-proximal immunoreceptor tyrosin activation motif(ITAM)results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein(ZAP70)and the levels of cytotoxic effector molecule granzyme B(GZMB),which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line.This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.
