Cytotoxicity and IFN-γ production by human γδ T cells underlie their potent antitumor functions. However, it remains unclear where and how human γδ T cells acquire these key effector properties. Given the recent disclosure of a major contribution of the thymus to murine γδ T cell functional differentiation, in this study we have analyzed a series of human pediatric thymuses. We found that ex vivo–isolated γδ thymocytes produced negligible IFN-γ and lacked cytolytic activity against leukemia cells. However, these properties were selectively acquired upon stimulation with IL-2 or IL-15, but not IL-4 or IL-7. Unexpectedly, TCR activation was dispensable for these stages of functional differentiation. The effects of IL-2/IL-15 depended on MAPK/ERK signaling and induced de novo expression of the transcription factors T-bet and eomesodermin, as well as the cytolytic enzyme perforin, required for the cytotoxic type 1 program. These findings have implications for the manipulation of γδ T cells in cancer immunotherapy.

The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy.

The thymus is the major site for normal and leukemic T-cell development. The dissection of the molecular determinants of T-cell survival and differentiation is paramount for the manipulation of healthy or transformed T cells in cancer (immuno)therapy. Casein Kinase 2 (CK2) is a serine-threonine protein kinase whose anti-apoptotic functions have been described in various hematological and solid tumors. Here we disclose an unanticipated role of CK2 in healthy human thymocytes that is selective to the γδ T-cell lineage. γδ thymocytes display higher (and TCR-inducible) CK2 activity than their αβ counterparts, and are strikingly sensitive to death upon CK2 inhibition. Mechanistically, we show that CK2 regulates the pro-survival AKT signaling pathway in γδ thymocytes and, importantly, also in γδ T-ALL cells. When compared to healthy thymocytes or leukemic αβ T-cells, γδ T-ALL cells show upregulated CK2 activity, potentiated by CD27 costimulation, and enhanced apoptosis upon CK2 blockade using the chemical inhibitor CX-4945. Critically, this results in inhibition of tumor growth in a xenograft model of human γδ T-ALL. These data identify CK2 as a novel survival determinant of both healthy and leukemic γδ T-cells, and may thus greatly impact their therapeutic manipulation.

Proteasome inhibitors, such as bortezomib, are first-line therapy against multiple myeloma (MM). Unfortunately, patients frequently become refractory to this treatment. The transcription factor NRF1 has been proposed to initiate an adaptation program that regulates proteasome levels. In the context of proteasome inhibition, the cytosolic protease DDI2 cleaves NRF1 to release an active fragment that translocates to the nucleus to promote the transcription of new proteasome subunits. However, the contribution of the DDI2-NRF1 pathway to bortezomib resistance is poorly understood. Here we show that upon prolonged bortezomib treatment, MM cells become resistant to proteasome inhibition by increasing the expression of DDI2 and consequently activation of NRF1. Furthermore, we found that many MM cells became more sensitive to proteasome impairment in the context of DDI2 deficiency. Mechanistically, we demonstrate that both the protease and the HDD domains of DDI2 are required to activate NRF1. Finally, we show that partial inhibition of the DDI2-protease domain with the antiviral drug nelfinavir increased bortezomib susceptibility in treated MM cells. Altogether, these findings define the DDI2-NRF1 pathway as an essential program contributing to proteasome inhibition responses and identifying DDI2 domains that could be targets of interest in bortezomib-treated MM patients.

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