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A single-cell approach to engineer CD8+ T cells targeting cytomegalovirus

Cellular & Molecular Immunology volume 18, pages 1326–1328 (2021)Cite this article

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T lymphocytes are crucial for antiviral responses and provide a promising repertoire for potential therapies of viral diseases such as cytomegalovirus (CMV) infection1 and the ongoing COVID-19 pandemic caused by SARS-CoV-2.2 CMV-related diseases occur once the host immune system is impaired or lacks a protective repertoire of virus-specific T lymphocytes.3 Adoptive transfer of T-cell receptor (TCR)-engineered T cells (TCR-Ts) provides an encouraging alternative treatment option for patients with CMV reactivation.4 However, generating TCR-Ts requires the identification of epitope-specific and functional TCR pairs. Modern single-cell sequencing techniques open up the ability to unravel TCR repertoires,5 which offers a potential opportunity to screen functional TCR pairs for TCR-T therapy. Here, we report an efficient approach that combines ex vivo CD8+ T-cell stimulation with single-cell RNA and TCR V(D)J sequencing to identify CMV-specific TCRs for generating TCR-Ts.

To identify sequences of NLV-specific TCRs, NLV-MHC tetramer+ and CD8+T cells were sorted by flow cytometry and analyzed with single-cell RNA and TCR V(D)J sequencing. A total of 781 single cells with high sequencing quality were captured by both single-cell RNA and TCR sequencing. T cells with identical TCR sequences were defined as one TCR clonotype and ranked according to frequencies (Fig. 1e). Unsupervised clustering showed that clonotypes 1, 2, and 4 were located in one cluster (cluster 1), while clonotype 3 and other low-frequency clonotypes had a distinct expression profile (cluster 2) (Fig. 1f). Differential gene expression (DGE) analysis revealed that cytotoxic effector molecules (GZMH and NKG7), the proinflammatory cytokine IFNG, and the exhaustion marker TIGIT were highly expressed in cluster 1, suggesting that cluster 1 cells had been activated and had differentiated. In contrast, naïve cell-related markers (CD27 and LEF1) were highly expressed in cluster 2, suggesting naïve populations (Fig. 1g). Furthermore, DGE analysis with a T-cell-specific functional gene set6 showed high expression of 40 functional genes (p value < 0.01, Wilcoxon test, and fold change ≥ 2) in cluster 1 (Fig. 1h). Gene ontology analysis and KEGG pathway enrichment analysis showed that these 40 genes are functionally involved in cytokine receptor–ligand interactions and lymphocyte activation (Fig. 1i, j). These results suggest that cluster 1 represents potentially functional NLV-specific T cells, whereas cluster 2 represents naïve CD8+ T cells.

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Acknowledgements

This project is supported by the Science, Technology and Innovation Commission of Shenzhen Municipality under grant No. JCYJ20170303151334808 and grant No. JSGG20180508152912700. The first author would like to acknowledge financial support from the China Scholarship Council (CSC) (grant No. 201904910476).

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  1. These authors contributed equally: Fei Wang, Qumiao Xu, Zhenkun Zhuang

Authors and Affiliations

  1. BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China

    Fei Wang, Zhenkun Zhuang & Xiuqing Zhang

  2. BGI-Shenzhen, Shenzhen, 518083, China

    Fei Wang, Qumiao Xu, Zhenkun Zhuang, Ziyi Li, Qianqian Gao, Yaling Huang, Yonglun Luo, Xiuqing Zhang, Linnan Zhu & Cheng-chi Chao

  3. Department of Biomedicine, Aarhus University, 8000, Aarhus, Denmark

    Fei Wang & Yonglun Luo

  4. China National GeneBank, BGI-Shenzhen, Shenzhen, 518120, China

    Qumiao Xu, Qianqian Gao, Yaling Huang & Xiuqing Zhang

  5. Shenzhen Key Laboratory of Genomics, Beishan Industrial Zone, Shenzhen, 518083, China

    Qumiao Xu, Xiuqing Zhang, Linnan Zhu & Cheng-chi Chao

  6. School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China

    Zhenkun Zhuang

  7. Northwest A & F University, Yangling, 712100, Shaanxi, China

    Ziyi Li

  8. Guangdong Enterprise Key Laboratory of Human Disease Genomics, Beishan Industrial Zone, Shenzhen, 518083, China

    Xiuqing Zhang, Linnan Zhu & Cheng-chi Chao

  9. AbVision, Inc, Milpitas, CA, USA

    Cheng-chi Chao

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  1. Fei Wang
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Correspondence to Xiuqing Zhang, Linnan Zhu or Cheng-chi Chao.

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Wang, F., Xu, Q., Zhuang, Z. et al. A single-cell approach to engineer CD8+ T cells targeting cytomegalovirus. Cell Mol Immunol 18, 1326–1328 (2021). https://doi.org/10.1038/s41423-020-0466-z

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