Precision Targeting of human iNKT Cells for Cancer Immunotherapy

Type 1 natural killer T cells (NKTs) are a unique T lymphocyte population expressing semi-invariant T cell receptors (TCR) that recognise the lipid presenting CD1d protein. Formerly we have shown that the human NKT repertoire in healthy individuals consists of clones that bear NKT-TCRs with varied affinities for CD1d (low and high), which directly translated into NKT subsets with diverse functions. However, the differential anti-tumour role of these NKT subsets is unknown. Cancer patients exhibit reduced NKT numbers and those that are present are functionally inept. In mouse models, these defects can be reversed in vivo through activation with Alpha-galactosylceramide (AlphaGalCer); however, translation into cancer patients has not been inefficacious. Likely due to reduced NKT cell numbers in cancer patients and functionally impaired repertoire of the NKT cells. We propose that particular subsets low or high will exert anti-tumour responses when adoptively transferred and can be engineered with chimeric antigen receptors to enhance their cytotoxicity. Therefore, we aim to (1) isolate high and low affinity NKTs utilising in-house produced CD1d tetramers, (2) characterise NKT subsets both phenotypically and functionally, and 3) investigate their anti-tumour properties in relevant pre-clinical models. To this end, NKT cells were isolated from healthy donors, expanded with AlphaGalCer and characterised. Low and high-affinity NKT clones were co-cultured with CD1d+ve T2 lymphoblasts and tumour cell killing was quantified by flow cytometry. Preliminary data show that the in vitro cytotoxicity of high-affinity NKT cells is greater than low-affinity NKTs. However this may be attributable to CD4 expression status. Moreover, AlphaGalCer pulsing enhanced the cytotoxic effect; however, the high-affinity NKTs still outperformed low-affinity NKTs in some cases. These observations suggest that precision targeting of NKT cell subsets may yield a better therapeutic anti-tumour response in patients. Although the NKT affinity alone does not appear confer enhanced cytotoxicity in all cases it is likely a contributing factor that needs to be considered in context of other cell surface markers, such as NKG2D