HMBPP Analog Prodrugs Bypass Energy-Dependent Uptake To Promote Efficient BTN3A1-Mediated Malignant Cell Lysis by Vγ9Vδ2 T Lymphocyte Effectors

Abstract

Vγ9Vδ2 T effectors lyse cells in response to phosphorus-containing small molecules, providing primates a unique route to remove infected or malignant cells. Yet, the triggering mechanisms remain ill-defined. We examined lysis mediated by human Vγ9Vδ2 T effectors in response to the naturally-occurring (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) or a synthetic cell-permeable prodrug, bis (pivaloyloxymethyl) (E)-4-hydroxy-3-methyl-but-2-enyl phosphonate (POM2-C-HMBP). CD27+/CD45RA− Th1-like effector cells killed K562 target cells though a mechanism that could be enhanced by either compound or TCR antibody and blocked by Src inhibition or BTN3A1 disruption. Pre-treatment at 4°C decreased HMBPP-induced lysis but did not reduce lysis induced by POM2-C-HMBP. Together, our results show that internalization of HMBPP into target cells is required for BTN3A1-dependent lysis by Vγ9Vδ2 T effectors. The enhanced activity of the prodrug analog is due to its ability to bypass the pathways required for entry of HMBPP. These findings support an inside-out model of T cell triggering driven by small-molecule induction of BTN3A1.

Prevention of Vγ9Vδ2 T Cell Activation by a Vγ9Vδ2 TCR Nanobody

“…However, although this could explain the observed inhibitory effects of VHH 5E7, it does not explain or take into account reports indicating additional proteins encoded on chromosome 6 supplementary to BTN3A1 to be required for full HMPBB-induced stimulation of Vg9Vd2 T cells (55,73). Possibly, our observed in silico interaction between BTN3A1 and the Vg9Vd2 TCR is of relatively low affinity, which would be in agreement with earlier reports (55) and suggestions (8) and would therefore require additional (costimulatory) interactions for complete TCR engagement and Vg9Vd2 T cell activation (74).…”

The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region

“…5). Plasmids containing the empty vector, the WT BTN3A1, or the ST 296 AA or T 304 A mutations were transfected into K562 cells that were deficient in functional BTN3A1 (31). The 3 constructs expressed at the expected molecular weight and at similar levels, as demonstrated by Western blot analysis and expressed at similar surface levels, albeit slightly lower than parental cells, as demonstrated by flow cytometry (Supplemental Fig.…”

“…We have reported the generation of BTN3A1-deficient K562 cells by CRISPR/Cas9 and a pcDNA3.1-BTN3A1 rescue construct (31). Point mutations harboring either ST 296 AA or T 304 A in pcDNA3.1-BTN3A1 were generated with the same primers as above.…”

BTN3A is a prognosis marker and a promising target for Vγ9Vδ2 T cells based-immunotherapy in pancreatic ductal adenocarcinoma (PDAC)

“…25 Of note, binding of phosphoAntigen by the B30.2 intracellular domain of BTN3A1 has been demonstrated to be critical in this process. 23,24,[35][36][37] As a receptor that is devoid of an intracellular domain but can negatively regulate NK cells, it has previously been suggested that BTN3A2 may function as a decoy receptor via the binding of a putative ligand. Hence, we could postulate that the higher level of expression of the BTN3A2 isoform compared to BTN3A1 may facilitate tumor immune escape mechanisms that lead to Vg9Vd2 T cells exhaustion 38 and/or fail to activate Vg9Vd2 T cells in the absence of PAg.…”