Cutting Edge: Bispecific gd T Cell Engager Containing Heterodimeric BTN2A1 and BTN3A1 Promotes Targeted Activation of Vg9Vd2+T Cells in the Presence of Costimulation by CD28 or NKG2D

Vg9Vd2+ T cell-targeted immunotherapy is of interest to harness its MHC-independent cytotoxic potential against a variety of cancers. Recent studies have identified heterodimeric butyrophilin (BTN) 2A1 and BTN3A1 as the molecular entity providing "signal 1" to the Vg9Vd2 TCR, but "signal 2" costimulatory requirements remain unclear. Using a tumor cell-free assay, we demonstrated that a BTN2A1/3A1 heterodimeric fusion protein acti-vated human Vg9Vd2+ T cells, but only in the presence of costimulatory signal via CD28 or NK group 2 member D. Nonetheless, addition of a bispecific gd T cell engager BTN2A1/3A1-Fc-CD19scFv alone enhanced granzyme B-mediated killing of human CD19+ lymphoma cells when cocultured with Vg9Vd2+ T cells, suggesting expression of costimulatory ligand(s) on tumor cells is sufficient to satisfy the "signal 2" requirement. These results highlight the parallels of signal 1 and signal 2 requirements in ab and gd T cell activation and demonstrate the utility of heterodimeric BTNs to pro -mote targeted activation of gd T cells.