Unlike T cells, Natural Killer (NK) cells are negative for T cell receptors (TCRs). The functional TCR complex protein consists of TCRαβ chains and downstream signaling subunits of CD3δ, CD3γ, CD3ε and CD3ζ chains. T cells are positive for all, while NK cells lack all these polypeptides except CD3ζ. NK92 cells were transduced by viral sequential transduction to have T cell phenotypes by inserting TCRs. Ectopic TCR complex proteins positive NK92 cells (TCR CD3+, TCR CD8 CD3+ and NY ESO1 1G4 CD8 CD3+ NK92 cells) were successfully generated as a basic to create TCR positive NK biobank. Furthermore, synthesis of TCR polypeptides and the percentage of TCR complex protein positive NK92 cells elevated by keeping transduced NK92 cells in culture for a longer period. Moreover, flow cytometry data revealed that TCRαβ or CD3 elements, when expressed alone, were detected only in the cytoplasm but not on the NK92 cell surface.
Regarding the functionality of generated TCR CD3+, TCR CD8 CD3+ and NY ESO1 1G4 CD8 CD3+ NK92 cells, they have revealed the capacity to degranulate to release cytotoxic granules and produce interferon gamma (IFNγ) in response to engagement of the cognate peptide/major histocompatibility complex (MHC) ligand on Epstein-Barr virus (EBV) B target cells. In addition, In Vitro cytotoxic studies have shown that generated TCR complex protein positive NK92 cells could recognize and kill relevant peptide/MHC ligand on EBV B target cells.
Concerning the isolated peripheral blood (PB) NK cells, transduced PB NK cells showed well growth and viability under the inverted microscope. However, only intracellular expression of the TCR complex proteins observed and even keeping transduced cells in culture for long period did not help translocation of TCR complex into the plasma membrane.
Another part of this study performed to make of single chain gene constructs (encoding for chimeric receptors) with either tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) or 2B4 upstream (extracellular part of chimeric receptor) of the different nucleotide chains (transmembrane and cytoplasmic domains) by running of overlapping polymerase chain reaction (PCR). Gel images for PCR products showed clear correct bands of all generated single chain gene constructs. Then, produced single chain gene constructs were amplified by One Shot™ Stbl3™ chemically competent cells with respect of conserving the sequence of nucleotides based on gel electrophoresis images and deoxyribonucleic acid (DNA) sequencing results.
In conclusions, different TCR complex protein positive NK cells produced, especially NK92 cells. The produced TCR complex protein positive NK92 cells were functional and cytotoxic upon coculturing with relevant pulsed peptide EBV B target cells. In addition, all different designed chimeric receptor single chain gene constructs made successfully by running of overlapping PCR and their sequence of nucleotides conserved upon multiplication of competent cells based on gel images and DNA sequencing results.