Background Gamma-delta (γδ) T cells are depleted during cancer progression resulting in the progressive loss of anti-cancer activity. Elevated numbers of γδ T cells are associated with greater survival outcomes in both hematopoietic and solid malignancies. Induced pluripotent stem cell (iPSC) derived γδ T cells could address the therapeutic challenges of multiple allogeneic γδ T cell infusions as iPSCs possess nearly unlimited self-renewal and multi-lineage differentiation potential. These can be genetically modified, selected, and propagated to provide a source of potentially ‘off-the-shelf’ immune cells.
Methods Precursor cells obtained from healthy volunteer donors were reprogrammed into iPSCs using non-integrating Yamanaka factors. A feeder-free multi-step strategy was used to differentiate iPSCs, leading to the generation of Vδ1+ γδ T cells. Characterization of the Vδ1+ T cell product included multiplex genomic PCR assays and Sanger sequencing to examine the rearrangement of the TCRγ and TCRδ gene loci, and G-band karyotype analysis. Pluripotent markers (Tra-1–60, OCT3/4 & SSEA4), HPC markers (CD34, CD43), γδ T cell surface markers (CD3, γδ TCR, CD4, CD8, CD16, CD56), effector memory markers (CD45RA, CD27), natural cytotoxicity receptors (NKG2D) were identified using multiparameter flow cytometry. T cell function was determined by flow cytometric cytotoxicity assays against K562, OLM13, U87MG, OVSAHO, OVCAR-3, KURAMUCHI targets at increasing Effector to Target (E:T) ratios. Th1/2/17 cytokine release was determined following PMA/ionomycin stimulation and LEGENDplex™ bead-based immunoassays.
Results We generated Vδ1T-iPSC lines (iVδ1T) identified as Vγ5-to-Jγ1/2 and Vδ1-to-Jδ1 recombination. One iPSC line showed normal karyotype with 99% cells expressing OCT3/4 & SSEA4. The differentiation process generated 70+ million iVδ1T cells from 3 million iPSCs expressing γδ T cell markers CD45, CD3, Vδ1-TCR, CD16, CD56, NKG2D, CD45RA, and CD27. Cytokine release following PMA/ionomycin stimulation showed increases of at least 50x for Granzyme A, 300x for IFN-γ, 1400x for TNF-α, ~10 to 20x for Granzyme B, ~5 to 10x for Perforin, ~6x for Granulysin. IL-6 was not detected either before or after stimulation, and IL17A was at low concentration. At a 16:1 E:T ratio, preliminary data shows that Vδ1+ γδ T cells killed K562 (CML) 95.7%; MOLM13 (AML) 60.3%; U87MG (glioblastoma) 70.3%; and ovarian cancer lines OVSAHO 57.1%, OVCAR-3 69.6%, and KURAMUCHI 55.1%.
Conclusions We generated Vδ1+ iPSC derived γδ T cells with effector cytokine phenotype and low risk for cytokine release syndrome. Robust cytotoxic activity was seen across a variety of cancer cell lines, potentially providing an off-the-shelf platform for allogeneic cell therapy.
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