Inhibitory Effects of Cytotoxicity of Cytokine Activated and Expanded Human CD8+ T Cells by a Human NKG2D Alternative Splice Variant.
Authors: Karimi M.A. , Cao T.M. , Soares L. , Negrin R.S. 
Published: October 2019

Abstract
NKG2D is an NK cell activating receptor that is also expressed on CD8+ T cells and γδ T cells where it up-regulates activation through TCR co-stimulation. NKG2D is triggered by ligands that are structurally related to MHC1 and expressed either by normal tissues early in ontogeny or by stress, infection or malignant transformation. The function of NKG2D on NK cells and CD8+ T cells is mediated by two distinct pathways of signaling through its association with two adapter proteins, DAP10 and DAP12. Two NKG2D isoforms that preferentially bind to either DAP 10 or DAP12 have been described in murine species. In evaluating human NKG2D gene expression by RT-PCR in cytokine activated CD8+T cells derived from 7 different donors, we consistently observed two distinct PCR bands differing by 218bp. Cloning and sequencing of both PCR amplicons revealed a previously described alternative spliced NKG2D variant resulting from inclusion of intron 6. The intron 6 inclusion introduces a stop codon almost immediately after the start of the intron 6 sequences resulting in a predicted protein product truncated without an extracellular domain. To determine the functional consequences of truncated NKG2D expression we isolated and cloned both full length NKG2D and truncated NKG2D into pRJ3 retroviral vector for expression as NKG2D full length with dsRed and NKG2D truncated EGFP fusion proteins. Activated human CD8+ T cells were trandused and FACS-sorted for dsRed or GFP+ cells and used against human cell line targets in Cr51 release cytotoxicity assays. We found that expression of the full length NKG2D dsRed resulted in ~15% increase in cytotoxicity while expression of the truncated NKG2D GFP resulted in ~70% (p.0009) reduction in cytotoxicity. Results were controlled for cytotoxicity obtained against the same tumor targets using non treated CD8+ T-cells as well as CD8+ T-cells transduced with an empty vector.
To further elucidate the role of each NKG2D isoform, we co-expressed both full length NKG2D and truncated NKG2D-EGFP with either DAP-10 or DAP-12. A similar cloning strategy was employed to generate a retroviral expression vector for DAP10-dsRed, DAP10-gfp, DAP12-dsRed and DAP12-gfp fusion proteins. Co expression of NKG2D with dsRed with either DAP10-gfp or DAP12-gfp resulted in comparable 20–25% increase in cytotoxicity (p=.0001) verses NKG2D alone. Co expression of the full length and truncated form also resulted in significant reduction in cytotoxicity of up to 65% (p.0001). Coexpression of the truncated NKG2D-gfp isoform with DAP10-dsRed or DAP12 dsRed did not alter the reduction in cytotoxicity observed with truncated NKG2D alone. We also performed similar experiments using murine CD8+ T cells with human cell line targets RPMI and U266. erexpression of full length NKG2D resulted in >32% increase in cytotoxicity while co-expression of full length NKG2D and DAP10 resulted in 42% increase in cytotoxicity against these human target cell lines. Coexpressing truncated NKG2D and DAP12 resulted in increased cytotoxicity slightly above the background and always less than full length NKG2D. Addition of DAP 10 and DAP 12 to the truncated NKG2D had a minimal impact on cytotoxicity. These results show that the NKG2D isform lacking the extracellular domain inhibits cytotoxicity of activated human CD8+ T-cells. Studies are ongoing to distinguish whether cytotoxocity inhibition is caused by hetrodimerization of the full length and truncated NKG2D incapable of ligand binding, or constitutive and competitive binding by truncated NKG2D of adaptor signaling.