Precision targeting of human iNKT cells for cancer immunotherapy

Abstract

Invariant natural killer T (iNKT) cells are a distinct subset of T lymphocytes that recognise lipid antigens presented by CD1d. iNKT cells have been classified into subsets based on both their TCR affinity for CD1d and coreceptor expression, such as CD4⁺ and double-negative (DN) populations, each with distinct functional properties. However, their differential roles in anti-tumour immunity remain unclear. To address this, we employed single-cell RNA sequencing to compare the gene expression and transcription factor profiles of low and high affinity iNKT cells, revealing distinct molecular signatures. TCR sequencing using Oxford Nanopore technology uncovered substantial variability,particularly within the complementarity-determining region 3 (CDR3) of both the α and βchains. Functional assays demonstrated that high affinity iNKT clones exhibit superior cytotoxicity against CD1d-expressing target cells. Additionally, we found that DN iNKT cells display greater cytotoxic potential than CD4⁺ iNKT cells, correlating with their strong expression of the activating receptor NKG2D. Blocking experiments confirmed that NKG2D mediates their enhanced tumour cell lysis. Given the importance of CD1d in iNKT cell activation, we explored strategies to enhance its expression on cancer cells. We optimised an in vitro treatment regimen combining Enhancer of Zeste Homolog 2 (EZH2) inhibitors and all-trans-retinoic acid (ATRA),demonstrating that this approach significantly increases CD1d surface expression and enhances iNKT cell-mediated lysis of acute myeloid leukaemia (AML) cells. Collectively, our findings underscore the functional and phenotypic heterogeneity of iNKT cells and their significance in cancer immunotherapy. Selectively targeting specific iNKT subsets may enhance therapeutic efficacy, while tumour micro environment modulation to upregulate CD1d could further improve direct iNKT-mediated tumour lysis. Future studies should optimise these strategies for clinical application in cancer immunotherapy.

More information

Identifiers

ORCID for Salah Mansour: orcid.org/0000-0002-5982-734X

ORCID for Ali Roghanian: orcid.org/0000-0003-1316-4218

ORCID for Diana Garay Baquero: orcid.org/0000-0002-9450-8504

Catalogue record

Export record