Single-Cell Transcriptomics of Human TET2 Knockout CD4 T-Cells and Their Clonal Evolution

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.22-23
• Main Authors: Li, Yuping, Liu, Xiaoqian, Liu, Xuxiang, Wu, Xiwei, Bouska, Alyssa, Lone, Waseem G., Lio, Chan-Wang J., Herek, Tyler, Lee, Logan, Dong, Gehong, Wang, Jinhui, Lou, Chih-Hong, Qi, Wei, Shetty, Kunal, McKeithan, Timothy W., Iqbal, Javeed, Chan, Wing (John) C.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-143113

Angioimmunoblastic T-cell lymphoma (AITL), the most frequent subtype of peripheral T-cell lymphoma (PTCL), is a neoplasm with characteristics of mature T follicular helper (TFH) cells. We and others have identified frequent (~75%) inactivating mutations in the TET2 (Ten-Eleven Translocation-2) gene in AITL. TET2 belongs to a 3 member family of TET dioxygenases that catalyze DNA demethylation by oxidation of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and further oxidative cytosine products. Thus, loss of function (LOF) of TET2 will cause aberrant genome hypermethylation and reduction in 5-hmC. Studies of the variant allele fraction (VAF) of TET2 mutants suggest that this mutation is a founding abnormality in AITL. However, how TET2 loss promotes the development of AITL is still unclear. To study LOF of TET2 in CD4 T-cell lymphomagenesis without the noise generated by other mutations in an established lymphoma, we generated a human TET2 knock-out (KO) CD4 T-cell model using CRISPR/Cas9 technology, which allows us to perform functional genomic studies by directly editing genes at their genomic loci. Whole transcriptome sequencing and single-cell transcriptome sequencing were used to study the cell evolution after KO. We generated multiple TET2 KO primary CD4 T-cell models using two different CRISPR/Cas9 methods. The first approach used the plasmid PX458-a, which expresses green fluorescent protein (GFP) fused Cas9 and guide RNA-a targeting TET2 exon 6, to electroporate CD4 T-cell from healthy donor F25. The second approach used homologous DNA repair (HDR) mediated knock-in (KI) of tandem GFP gene and a SV40 transcription stop signal to terminate TET2 expression at exon 3. Cas9/sgRNA-e RNP complex, along with a long DNA template (about 1.6 kb), was electroporated into CD4 T-cells from two healthy donors, F25 and M40. GFP-positive cells were sorted by FACS after electroporation and were considered to be edited cells. Edited CD4 T-cells were cultured in vitro with 50 U/ml IL-2, and stimulated regularly (every 7~10 days) with 1:1 ratio of anti-CD3/CD28 T activator beads. TET2 KO in these cells was confirmed by qRT-PCR, Sanger sequencing and Western blotting. Compared with wild-type (WT) CD4 T-cells under the same culture conditions, a lower level of 5-hmC in TET2 KO cells was observed, indicating successful editing of TET2. Compared to WT cells, KO cells had a higher growth rate, due to a lower apoptosis rate and a higher proliferation rate, by