Butyrophilin 3A1 Plays an Essential Role in Prenyl Pyrophosphate Stimulation of Human Vg2Vd2 T Cells

Most human gd T cells express Vg2Vd2 TCRs and play important roles in microbial and tumor immunity. Vg2Vd2 T cells are stimulated by self-and foreign prenyl pyrophosphate intermediates in isoprenoid synthesis. However, little is known about the molecular basis for this stimulation. We find that a mAb specific for butyrophilin 3 (BTN3)/CD277 Ig superfamily proteins mimics prenyl pyrophosphates. The 20.1 mAb stimulated Vg2Vd2 T cell clones regardless of their functional phenotype or developmental origin and selectively expanded blood Vg2Vd2 T cells. The gd TCR mediates 20.1 mAb stimulation because IL-2 is released by b 2 Jurkat cells transfected with Vg2Vd2 TCRs. 20.1 stimulation was not due to isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. In addition, stimulation was not inhibited by statin treatment, which blocks IPP production. Importantly, small interfering RNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expression of BTN3A1 but not by BTN3A2 or BTN3A3. Rhesus monkey and baboon APC presented HMBPP and 20.1 to human Vg2Vd2 T cells despite amino acid differences in BTN3A1 that localize to its outer surface. This suggests that the conserved inner and/or top surfaces of BTN3A1 interact with its counterreceptor. Although no binding site exists on the BTN3A1 extracellular domains, a model of the intracellular B30.2 domain predicts a basic pocket on its binding surface. However, BTN3A1 did not preferentially bind a photoaffinity prenyl pyrophosphate. Thus, BTN3A1 is required for stimulation by prenyl pyrophos-phates but does not bind the intermediates with high affinity. U nconventional T cells, such as gd T cells, ab iNKT cells, and mucosal-associated invariant ab T cells, have distinct recognition properties. Conventional CD8 and CD4 T cells use diverse ab TCRs to recognize foreign and self-peptides presented by MHC class I and class II molecules. The peptides are derived from proteins and loaded into MHC molecules through distinct intracellular processing and loading pathways. The number of ab T cells recognizing a particular peptide– MHC complex is generally very low such that expansion of re-active cells is required to mount effective T cell immunity. In contrast, gd TCRs recognize nonpeptide compounds or self-cell surface molecules. Humans and mice have limited numbers of Vg and Vd gene segments, and many gd TCRs exhibit limited or no junctional diversity. Thus, although gd T cells constitute a minor subpopulation among human and murine T cells, the actual precursor frequencies for …