Abstract 612: Methylation changes in DNA of CD8 T cells following CD137 costimulation

Abstract Introduction: CD137 ligation imprints long term changes in the behavior of costimulated T-cells (1). There is not a satisfactory mechanistic explanation yet. Experimental procedures: to determine the specific DNA methylation changes occurring upon CD137 costimulation, purified human CD8+ T cells from three healthy donors were activated in vitro for 5 days with anti-CD3 monoclonal antibody and either with an anti-CD137 monoclonal antibody or its corresponding isotype (huIgG4). Activated lymphocytes were left 5 days in culture with huIL-7 without further stimulation. Such back-to-resting CD8+ lymphocytes were reestimulated with anti-CD3 for 12, 24 and 36h to validate the expression of the genes differentially methylated upon primary stimulation at mRNA and protein levels. DNA methylation profiles of both activated and resting cell subsets were characterized with Infinium 450K DNA methylation array (Illumina). To further confirm our observations, identical experimental procedure was performed with a different anti-CD137 agonist antibody (mIgG1 isotype) in a CD8+ T-cell donor. Differentially methylated genes between anti-CD3+anti-CD137 versus their corresponding control counterparts were validated by pyrosequencing on activated and resting CD8+ from independent group of healthy donors (n=8 for hIgG anti-CD137 and n= 11 for mIgG anti-CD137). Expression changes were confirmed by qRT-PCR and flow cytometry in activated, rested and reestimulated CD8+ lymphocytes. Results: 853 genes were differentially methylated in hIgG4 anti-CD137-treated CD8+ T cells as compared with their controls, 52 of which were shared with mIgG1 anti-CD137-costimulated CD8+ T lymphocytes. A number of differentially methylated genes are involved in i) T cell migration, ii) T cell activation, survival and homeostasis and iii) regulation of gene expression including key T-cell transcription factors. Conclusions: CD137 costimulation induces CD8+ T lymphocytes that are poised to respond more effectively to a second antigen exposure. These acquired functions are imprinted in the genomic DNA of the CD8+ T cells by DNA methylation changes upon CD137 co-stimulation, and involve key genes for CD8+ T cells. References: 1. Hendriks, J., Y. Xiao, J.W. Rossen, K.F. van der Sluijs, K. Sugamura, N. Ishii, and J. Borst, During viral infection of the respiratory tract, CD27, 4-1BB, and OX40 collectively determine formation of CD8+ memory T cells and their capacity for secondary expansion. J Immunol 2005, 175: p. 1665-76. Citation Format: M. Angela Aznar, Sara Labiano, Angel Diaz-Lagares, Manel Esteller, Juan Sandoval, Ignacio Melero. Methylation changes in DNA of CD8 T cells following CD137 costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 612. doi:10.1158/1538-7445.AM2017-612