Phenotype and regulatory mechanisms of DOT cells. DOT cells are in vitro–expanded γδ T cells that mostly (>70%) express a Vδ1⁺ TCR, which controls their activation, proliferation, and differentiation during the 2–3-wk protocol (Almeida et al., 2016). The cytokines IL-2 and IL-4 provide important signals for DOT cell proliferation, whereas IL-15 drives their cytotoxic effector phenotype. During in vitro expansion, strong TCR stimulation in the presence of IL-15 upregulates a series of NKRs that are critical for tumor cell targeting: NKp30 (binding to B7-H6 on tumor cells), DNAM-1 (binding to PVR and Nectin-2), and NKG2D (binding to MICA/B and ULBP1–6 ligands). Conversely, DOT cell activity is negatively regulated by TIGIT and PD-1, which are induced in the TME. The DOT cell protocol also upregulates chemokine receptors, like CXCR3 and CXCR4, that control their migration and infiltration into tumor lesions, and several molecules associated with tissue retention and residency, namely CD69 or CD103, alike tissue-resident memory T cells. Finally, DOT cells are very amenable to genetic engineering, and transduction with CARs specific for tumor-associated antigens (TAA) enhances their potency in vitro and in vivo.