P150
Role of ZEB2 in the function of antigen-specific human CD8+ T cells
SASIDHARAN NAIR V. 1, YU Z. 1, BONIFACIUS A. 2, BECKSTETTE M. 3, NIEMZ J. 1, PIETZSCH B. 1, BUSCH D. 4,5, CICIN-SAIN L. 6,7, MULLER F. 8, EIZ-VESPER B. 2,7, FLOESS S. 1, HUEHN J. 1
1 Department Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany; 2 Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School , Hannover, Germany; 3 Bielefeld Institute for Bioinformatics Infrastructure, Bielefeld University, Bielefeld, Germany; 4 Institute of Microbiology – Clinical Microbiology, Immunology and Hygiene, University Hospital Erlangen, Friedrich-Alexander-University (FAU) Erlangen-Nurnberg, Erlangen, Germany; 5 German Center for Infection Research (DZIF), Partner site Munich, Munich, Germany; 6 Department of Viral Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany; 7 German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Braunschweig, Germany; 8 Integrative Cellular Biology and Bioinformatics, Saarland University, Saarbrucken, Germany
Title:
Role of ZEB2 in the function of antigen-specific human CD8+ T cells
Objectives:
ZEB1 and ZEB2 are zinc finger E homeobox binding proteins known to be modulated during T cell differentiation. However, the precise regulatory role of ZEB2 in cytotoxic T cells remains obscure. The purpose of this study is to determine the functional characteristics of ZEB2 on CD8+ T cells.
Methods:
We performed whole-genome bisulfite sequencing on cytomegalovirus (CMV)-specific human CD8+ T cells to identify differentially methylated regions (DMRs) in the ZEB2 locus. The identified DMRs were further validated by pyrosequencing on different T cell subsets from CMV-seropositive donors. Furthermore, ZEB2 expression levels were determined through quantitative RT-PCR. Finally, the impact of ZEB2 on the cytotoxic potential of virus-specific CD8+ T cells was studied by CRISPR-Cas9-mediated deletion of ZEB2 on CD8+ TEMRA cells followed by a cytotoxic killing assay.
Results:
We have identified four DMRs within the ZEB2 locus that were significantly hypomethylated in CMV-specific CD8+ memory T cells when compared to conventional memory and naive CD8+ T cells analyzed as controls. ZEB2 expression is significantly upregulated in effector memory CD8+ T cells when compared to central memory and naive CD8+ T cells isolated from the peripheral blood of CMV-seropositive donors. Moreover, the demethylation of ZEB2 DMRs in CD8+ TEMRA cells correlated significantly with ZEB2 expression and was stable upon long-term in vitro culture. Intriguingly, ZEB2 gene disruption in CD8+ effector T cells significantly reduced their cytotoxicity and also affected the viability of the T cells.
Conclusion:
Altogether, our data reveal that ZEB2 expression not only contributes to effector CD8+ T cell differentiation, but also intricately regulates the cytotoxicity of CD8+ T cells via multiple mechanisms.