Torralba-Raga, L. | Search bioRxiv

bioRxiv2025

Toggling of NKG2A expression drives functional specialization of iPSC-derived CAR NK cells

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. NK cell function is dynamically regulated through education driven by inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). However, the acquisition of inhibitory receptors in iNK cells and their role during differentiation and education remains poorly defined. In this study, we monitored receptor repertoires, transcriptional states, and functional responses in a range of genetically engineered iNK cell lines. Transcriptional reference mapping placed iNK cells close to cytokine-activated NKG2A+ CD56dim peripheral blood (PB) NK cells. Despite their early differentiation stage, iNK cells displayed a well-developed cytotoxic effector program, which was also reflected in high protein expression of Eomes, granzyme B, and activating receptors DNAM-1 and NKG2D. Acquisition of NKG2A by iNK cells was associated with a more differentiated transcriptional state and superior functional responses against a broad range of targets, including those expressing low to moderate levels of HLA-E, suggesting attenuated inhibitory signaling through NKG2A in iNKs. CRISPR knockout of {beta}2-microglobulin (B2) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of educating interactions with HLA-E in cis or trans. Finally, CRISPR-mediated ablation of NKG2A led to a spontaneous compensatory surface expression of CD94/NKG2C heterodimers, associated with enhanced IFN-{gamma} production and cytotoxic activity against target cells with forced high expression of single-chain {beta}2m-HLA-E-peptide trimers. Our results indicate an education-independent functional maturation of iNK cells, characterized by potent effector programs coupled with a favorable early-stage transcriptional profile.

immunology↗

bioRxiv2023

Targeting HLA-E Positive Cancers with a Novel NKG2A/C Switch Receptor

HLA-E is overexpressed by approximately 80% of solid tumors, including malignant glioblastoma, and is emerging as a major checkpoint for NKG2A+ CD8+ T cells and NK cells in the tumor microenvironment and circulation. This axis operates side-by-side with PD-L1 to shut down effector responses by T and NK cells. Here, we engineered a novel chimeric A/C switch receptor, combining the strong HLA-E binding affinity of the NKG2A receptor ectodomain with the activating signaling of the NKG2C receptor endodomain. We found that A/C Switch-transduced NK and T cells displayed superior and specific cytotoxic function when challenged with tumor cells exhibiting medium to high HLA-E expression. Furthermore, A/C Switch-expressing human T cells demonstrated enhanced anti-tumor function in a xenograft model of glioblastoma. Importantly, the activity of the modified T cells was governed by an equilibrium between A/C Switch transduction level and HLA-E expression, creating a therapeutic window to safeguard against on-target off-tumor toxicities. Indeed, normal cells remained insensitive to A/C Switch engineered T cells even after pre-treatment with IFN-{gamma} to induce HLA-E expression. We propose that this novel A/C switch receptor may operate alone to control tumor cells expressing high levels of HLA-E or in combination with other engineered specificities to overcome the suppressive NKG2A/HLA-E checkpoint.

immunology↗