膜分子VSTM1-v1在CD4+ T细胞中的功能和机制研究

VSTM1-v1 is a potential leukocyte differentiation antigen selected by the strategy of Immunogenomics, which is a type I transmembrane molecule, containing an extracellular IgV-like domain and two ITIMs in its cytoplasmic region. It is widely expressed on normal human peripheral blood leukocytes. Preliminary studies show that it is down-regulated upon activation of CD4+ T cells, and is undetectable in CD4+ lymphoid leukemia cell line Jurkat with promoter hypermethylation. Overexpression of VSTM1-v1 inhibited Jurkat cell growth and cross-linking of VSTM1-v1 by antibody promoted the inhibitory effect. The interaction of the co-overexpressed VSTM1-v1 and SHP1 was detected in HEK293T cells. Based on these results, this project will focus on the function and mechanism of VSTM1-v1 in CD4+ T cells from normal human peripheral blood. We will employ anti-CD3 and anti-CD28 to stimulate the cells to evoke a similar effect by the engagement of TCR by the antigen presented on MHC molecules. The kinetic expression of VSTM1-v1 on CD4+ T cells during the activation and its association with CD25 will be analyzed. Then the effects of VSTM1-v1 on CD4+ T cell activation, proliferation and apoptosis will be studied by cross-linking the molecules with rabbit anti-VSTM1 polyclonal antibody. The regulatory roles of VSTM1-v1 in the TCR-mediated signaling pathway will be investigated by assessing the impacts of cross-linking VSTM1-v1 on Ca2+ influx and phosphorylation of some pivotal signaling proteins such as ERK, PKC, CD3ζand ZAP-70. As the inhibitory immune receptors often recruit phosphatases (SHP1/SHP2/SHIP) through the ITIMs in their cytoplasmic tails to transmit the inhibitory signals intracellularly, we will employ the lentiviral expression system to deliver VSTM1-v1 or its ITIM mutants into Jurkat cells. Immunoprecipitation will be carried out to analyze the phosphorylation of the ITIMs upon the engagement of VSTM1-v1 and to find the phosphatase(s) interacting with VSTM1-v1. And the importance of ITIMs for the function of VSTM1-v1 will be explored with the ITIM mutants by detecting IL-2 production and the interaction with SHP1/SHP2/SHIP.

VSTM1-v1是我们采用免疫基因组学策略遴选的潜在白细胞分化抗原，为I型膜分子，胞外有一个Ig样结构域，胞内有两个ITIM，在正常人外周血白细胞中广泛表达。初步结果提示，其在CD4+T细胞活化后表达下调；在CD4+T细胞白血病细胞Jurkat中其启动子甲基化导致其表达缺失，恢复其表达能抑制该细胞增殖，使用其抗体交联能够增强抑制效果；在HEK293T细胞中其可与共转染的SHP1相互作用。本项目基于前期工作，分离健康人外周血CD4+T细胞，使用CD3和CD28抗体联合刺激模拟TCR活化，研究VSTM1-v1表达的动态变化；而后通过抗体对其进行交联，研究其对TCR介导的细胞活化增殖和凋亡的影响及其对TCR信号通路的影响；进而包装其野生型及ITIM突变体慢病毒感染Jurkat细胞，使用其抗体交联后检测ITIM磷酸化及其与SHP1/SHP2/SHIP的相互作用，探讨ITIM对其功能的重要性。

VSTM1-v1是我们采用免疫基因组学策略发现的新免疫细胞膜分子，主要表达于骨髓、外周血白细胞和脾脏。与正常供者相比，VSTM1在急慢性髓系白血病和急性淋巴性白血病患者骨髓细胞中表达下调。另一方面，VSTM1在绝大多数白血病细胞系中表达缺失。其启动子区域含有一个典型的CpG岛，在CD4+ T细胞白血病细胞系Jurkat中，该CpG岛的甲基化修饰水平与VSTM1-v1的表达密切相关。在正常培养的Jurkat细胞中该基因表达缺失，而用逆转甲基化药物处理Jurkat细胞后能恢复其表达。超表达VSTM1-v1能够抑制Jurkat细胞的增殖；使用具有交联活性的兔抗VSTM1多克隆抗体进行交联后，其抑制Jurkat细胞增殖的效果增强。我们进一步分离健康供者的外周血CD4+ T细胞并检测细胞膜表面VSTM1-v1的表达，结果在所检测的5例样品中，仅有1例样品中可检测到VSTM1-v1的表达，且用CD3与CD28抗体联合刺激后其表达下调；而在其他4例样品中均未检测到其表达。同时，我们还检测了其在CD8+T细胞、B细胞及NK细胞中的表达情况。结果在5例CD8+T细胞样品中均未检测到VSTM1-v1的表达；在B细胞（3例）表面VSTM1-v1有中等程度的表达；而在NK细胞（5例）中VSTM1-v1的表达分为高低两群，CD56dimCD16+ NK细胞表面表达较低而CD56brightCD16- NK细胞表面表达较高，且随IL-2的刺激表达逐渐上调。抗体交联VSTM1-v1能够显著抑制NK细胞分泌细胞因子和杀伤分子，抑制其杀伤活性。LRRC25是我们采用免疫基因组学策略发现另一个新免疫细胞膜分子，在粒细胞、单核细胞等髓系细胞高表达，而在T、B、NK细胞中低表达；且其在多种髓系细胞系及AML病人骨髓细胞中表达下调，而在ATRA诱导的AML细胞系、APL骨髓细胞分化过程中表达上调；恢复NB4细胞中LRRC25的表达不影响细胞增殖、不能促进细胞自发向粒细胞分化；敲减或敲除LRRC25表达，显著抑制ATRA诱导的粒细胞分化。这些结果表明LRRC25是一个髓系终末分化的重要调节分子，在粒细胞和单核细胞的终末发育阶段发挥重要作用，还可能成为AML的一个新的药物靶点。