NK cells are essential to an effective anti-tumour response. Thus, many tumours have developed diverse strategies to evade NK-cell recognition and activation, including the upregulation of the non-classical HLA molecule HLA-G. HLA-G is recognised by the inhibitory NK cell receptors ILT2 and KIR2DL4, protecting tumour cells from lysis and infiltration.<br/>We were interested in understanding the effect on the cytotoxicity of NK cells if either HLA-G or its associated NK cell receptors were removed. Using CRISPR/Cas9 genome editing, we generated ILT2 and KIR2DL4-deficient primary NK cells, as well as HLA-G-deficient JEG3 cells, a human trophoblast cell line. Using cytotoxicity assays, we quantified the specific lysis of polyclonal crispred NK and wild-type control cells on JEG3, CAPAN2 and CAOV3 cell lines and NK-cell degranulation during coculture.<br/>We observed a tendency to increased degranulation in KIR2DL4 knockout cells, whereas no significant effects on the cytotoxicity of NK cells against cancer cell lines. Using an ILT2 blocking antibody, we observed significant increases in degranulation in the wild-type primary NK cells relative to an isotype control. However, these increases seemed to be independent of HLA-G expression on target cells, so more work must be done to understand the specific interactions leading to this effect. We generated single cell JEG3 and CAOV3 clones in which HLA-G had been knocked out. However, due to time limits, we were unable to identify clones to be used in assays.<br/>We are currently working on improvements of the CRISPR on primary NK cells to corroborate our preliminary data. We plan to use the HLA-G knock out cells to determine whether the effects of KIR2DL4 and ILT2 knock out in primary NK cells are due to specific interaction.<br/>Understanding HLA-G regulation and interaction with NK cell receptors ILT2 and KIR2DL4 would open the possibility of using them as targets in cancer therapies to improve natural cytotoxicity against tumor cells.

Establishing the effects of interaction between HLA-G and cognate natural killer (NK) cell receptors ILT2 and KIR2DL4 on cytotoxicity in cancer cell lines

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.