T cells represent a key component of the immune system that can recognize foreign molecules (antigens) via cognate cell surface receptors termed T cell receptors (TCRs). The two main families of T cells, known as αβ and γδ T cells, are defined by the different gene loci that they use to generate their respective TCRs. αβ T cells typically recognize antigens displayed on the surface of target cells in association with antigen-presenting molecules known as major histocompatibility complex (MHC) molecules. Much less is known about how γδ T cells recognize antigen, although it is clear they are also essential to protective immunity. In humans, many γδ T cells (classified as Vγ9Vδ2 T cells) respond to small phosphorylated nonpeptide antigens, called phosphoantigens (pAgs), which are produced by cellular pathogens and cancers. In turn, γδ T cells become activated, proliferate, rapidly produce proinflammatory cytokines such as IFN-γ, and exert cytotoxic activity. pAg recognition appears to involve a cell surface molecule, butyrophilin 3A1 (BTN3A1), which plays a necessary, but not sufficient, role in this process. Therefore, the molecular basis that underpins pAg recognition by Vγ9Vδ2 T cells remains unclear and represents a long-standing conundrum, which has impeded the study of these important immune cells.

In contrast to αβ T cells, Vγ9Vδ2 T cells are not MHC-restricted and can recognize pAg expressed by multiple cancers and infectious diseases. Thus, they represent an attractive target for the development of new immunotherapy treatments. A much clearer understanding of the molecular basis for pAg recognition is required to optimally harness these cells for immunotherapy. We undertook a multipronged approach to investigate which molecules are necessary for pAg detection by γδ T cells. We used a genome-wide screen to identify molecules that mediate pAg-driven γδ T cell activation. Furthermore, we asked if these molecules directly bind to the Vγ9Vδ2 TCR and how they work in conjunction with BTN3A1.

The top candidate molecule identified in our genome-wide screen was butyrophilin 2A1 (BTN2A1), a molecule distinct from, but related to, BTN3A1. We show that without BTN2A1, Vγ9Vδ2 T cells cannot be activated by either bacterial or mammalian pAgs, and that BTN2A1 expression was required for Vγ9Vδ2 T cell–mediated tumor cell killing. Neither BTN3A1 nor the other butyrophilin family members tested can compensate for loss of BTN2A1. BTN2A1 can bind directly to the Vγ9Vδ2 TCR and associates closely with BTN3A1 on the surface of target cells. We also identify an important role for the transmembrane and/or intracellular domain of BTN2A1. Furthermore, pAg-mediated activation of γδ T cells requires coexpression of both BTN2A1 and BTN3A1, which together appear to convey pAg recognition and responsiveness by Vγ9Vδ2 T cells. Lastly, we show that BTN2A1 binds to the side of the Vγ9 domain of the TCR, and also reveal the existence of a critical putative second ligand-binding domain on a separate region of the TCR that incorporates Vδ2. Disruption of either of these binding sites abrogated the ability of Vγ9Vδ2 T cells to respond to pAg.

Our findings suggest that γδ T cells recognize pAg in an entirely different way to how any other immune cell recognizes antigen. We propose a model whereby BTN2A1 and BTN3A1 co-bind the Vγ9Vδ2 TCR in response to pAg. This pAg likely modifies the BTN2A1–BTN3A1 complex to make it stimulatory, which may occur through BTN molecule remodeling and/or conformational changes. Targeting these molecules will create new …