Cell stress activates γ9δ2 T cells via endogenous phosphoantigens and butyrophilin complex dynamics

Phosphoantigens (pAgs) are phosphate-containing small molecules that elicit an immune response. The pAgs bind to the intracellular domain of butyrophilin 3 (BTN3), enabling interactions with other butyrophilins to form complexes that trigger the T cell receptor (TCR) of Vγ9Vδ2 (γ9δ2) T cells. Despite multiple reports on this process, the conditions that regulate pAg levels leading to their detection remain unclear. Here we reveal a novel stress detection pathway, a type of lymphoid stress-surveillance response, in which mild cold stress triggers endogenous pAgs to engage with BTN family proteins, leading to the activation of γ9δ2 T cells. This stress response is dependent upon endogenous pAgs, as inhibition of HMG-CoA reductase abrogates the effect. It is also dependent upon BTN proteins, as depletion of BTN3A1 reduces the response. The ability of BTN2A1/BTN3A1 to respond is enhanced by the presence of BTN3A2 or BTN3A3. Furthermore, the internal domains of BTN2A1, BTN3A1, and BTN3A3 display differing abilities to dimerize, with BTN2A1 a constitutive dimer, BTN3A1 a monomer, and BTN3A3 a concentration dependent dimer. Full length BTN2A1/3A1 hybrid proteins additionally reveal that appropriately spaced multimers of BTN2A1 and BTN3A1 are critical in engaging the γ9δ2 TCR. In summary, our study uncovers a novel γ9δ2 T cell activation pathway mediated by cell stress and mevalonate pathway intermediates and highlights the critical roles of the BTN family members and their spacing in this process.