Abstract B052: Exploring human iNKT cell functionality to enhance adoptive cell therapy against non-small cell lung cancer

Abstract Invariant Natural Killer T (iNKT) cells are unconventional T cells with semi-invariant T cell receptors that recognize glycolipid antigens presented by the MHC-I-like molecule, CD1d. iNKT cells are promising candidates for adoptive cell therapy (ACT), as they exhibit potent antitumor responses and are suitable for allogeneic transfer due to the non-polymorphic nature of CD1d. However, a limited understanding of human iNKT cell functionality, heterogeneity, and anti-tumor activity may hinder the efficacy of these cells in ACT applications. Spectral flow cytometry and Phenograph clustering analysis on peripheral blood mononuclear cells (PBMCs) from 14 healthy donors (7 male, 7 female) revealed distinct human iNKT cell subsets. Of these, one subpopulation demonstrated enhanced expression of CD57 along with cytotoxic molecules Granzyme B and Granulysin. This subset was also found to exhibit activated or exhausted phenotypes within human non-small cell lung cancer (NSCLC) tumors, suggesting anti-tumor activity. The primary objectives of this study were to effectively expand human iNKT cells from PBMCs and to assess their cytotoxic capacity against non-small cell lung cancer (NSCLC) in vitro. To further investigate human iNKT functionality, we generated a protocol for expanding large numbers of iNKT cells in vitro. Briefly, iNKT cells were enriched from PBMCs using magnetic activated cell sorting and stimulation with CD1d-expressing artificial antigen presenting cells loaded with iNKT ligand, α-galactosylceramide. Cells were subsequently purified using fluorescence-activated cell sorting, maintained in culture with IL-2 and IL-21, and stimulated every 3 weeks with anti-CD3. Expanded iNKT cells were functional, as they produced cytokines and cytotoxic effector molecules such as Granzyme B, TNFα, and IFNγ following anti-CD3/28 stimulation. Furthermore, iNKT cells were cytotoxic against NSCLC cell lines NCI-H460, and to a lesser extent, A549 in vitro, as assessed through IncuCyte live-cell imaging and AnnexinV staining. Interestingly, co-culture with iNKT cells induced upregulation of CD1d on NCI-H460 cells, which may result in enhanced cytotoxicity. Collectively, these findings reinforce the potential for human iNKT cells as candidates for ACT against NSCLC and establish the experimental framework to facilitate future investigations into subset-specific contributions to antitumor immunity. Furthermore, understanding the mechanisms that promote CD1d expression on NSCLC cells may result in therapeutic benefits that can enhance tumor susceptibility to iNKT-based ACT. Citation Format: Milea DiPonzio, Carolina de Amat Herbozo, Thierry Mallevaey. Exploring human iNKT cell functionality to enhance adoptive cell therapy against non-small cell lung cancer [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B052.