Concomitant loss of TET2 and TET3 results in T cell expansion and genomic instability [WGS]

TET proteins are tumor suppressors that through their catalytic activity oxidize 5-methylcytosine to 5-hydroxymethylcytosine, to promote DNA demethylation and to regulate gene expression. Notably, TET2 is one of the most frequently mutated genes in hematological malignancies, including T cell lymphomas. However, murine models with deletion of TET2 do not exhibit T cell expansion, presumably due to redundancy with other members of the TET family of proteins. In order to gain insight on the TET mediated molecular events that safeguard T cells from aberrant proliferation we performed serial adoptive transfers of murine CD4 T cells that lack concomitantly TET2 and TET3 to fully immunocompetent congenic mice. Our data reveal a progressive acquisition of malignant traits upon loss of TET2 and TET3 that is characterized by loss of genomic integrity, acquisition of aneuploidy and upregulation of the protooncogene Myc. Overall design: To test whether loss of TET2 and TET3 could endow Tet2/3 DKO CD4 cells with hyperproliferative capacity, we performed serial transplantations of sorted, highly pure Tet2/3 DKO CD4 cells in congenic CD45.1+ recipients, that have not received any irradiation. After 16 weeks, we witnessed that the transferred CD45.2+ Tet2/3 DKO cells have evaded the immune surveillance of the recipients and have expanded. At this point, we isolated CD45.2+ cells by FACS sorting and re-transplanted them in new CD45.1+ immunocompetent recipients. The second transplantation resulted in significant acceleration of the expansion of the transplanted cells which became apparent within 2-4 weeks. To assess chromosomal copy number we performed whole genome sequencing. Genomic DNA was isolated from sorted wild type CD4 cells, or sorted expanded CD45.2+ Tet2/3 DKO cells after one or two serial transplantations in congenic CD45.1+ recipient mice.

TET蛋白（TET proteins）是一类肿瘤抑制因子，可通过催化活性将5-甲基胞嘧啶（5-methylcytosine）氧化为5-羟甲基胞嘧啶（5-hydroxymethylcytosine），从而促进DNA去甲基化并调控基因表达。值得注意的是，TET2是包括T细胞淋巴瘤在内的血液系统恶性肿瘤中最常发生突变的基因之一。然而，仅缺失TET2的小鼠模型并未出现T细胞扩增现象，推测原因是TET家族其他蛋白存在功能冗余。为深入探究TET介导的、保护T细胞免受异常增殖的分子事件，我们对同时缺失TET2和TET3的小鼠CD4 T细胞进行连续过继转移，将其植入完全免疫健全的同源基因（congenic）小鼠体内。本研究数据显示，在同时缺失TET2和TET3后，细胞会逐渐获得恶性表型，其特征为基因组完整性丧失、非整倍体获得以及原癌基因Myc的上调。

实验设计：为验证同时缺失TET2和TET3是否可使Tet2/3双敲除（DKO, double knockout）CD4 T细胞获得过度增殖能力，我们将分选得到的高纯度Tet2/3 DKO CD4 T细胞移植至未接受任何辐照的同源基因CD45.1+受体小鼠体内，开展连续移植实验。16周后，我们观察到移植的CD45.2+ Tet2/3 DKO细胞逃脱了受体的免疫监视并发生扩增。此时，我们通过荧光激活细胞分选（FACS, fluorescence-activated cell sorting）分离得到CD45.2+细胞，并将其重新移植至新的CD45.1+免疫健全受体小鼠体内。第二次移植后，移植细胞的扩增速度显著加快，该现象在2-4周内即可显现。为评估染色体拷贝数变异情况，我们开展了全基因组测序（whole genome sequencing）实验。我们从分选得到的野生型CD4 T细胞，或是经1次或2次连续移植后扩增得到的CD45.2+ Tet2/3 DKO细胞中提取基因组DNA。