Indirect Stimulation of Human Vγ2Vδ2 T Cells through Alterations in Isoprenoid Metabolism

Abstract

Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-D-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/ApppI that directly stimulate. In this study, we further characterize stimulation by these compounds, and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates. However, the continuous presence of aminobisphosphonates was toxic for T cells, and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known antigen presenting molecules, and resistant to fixation. New classes of stimulatory compounds–mevalonate, the alcohol of HMBPP, and alkenyl phosphonates–likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct antigens. Transfection of APC with siRNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells, and increased cellular IPP. siRNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy.

Zoledronate can induce colorectal cancer microenvironment expressing BTN3A1 to stimulate effector γδ T cells with antitumor activity

“…There is general agreement on the role of gd T lymphocytesinfiltrating solid tumors, including CRC, in the anticancer surveillance, [3][4][5]9,11 so that treatments focused on gd T cell-mediated immune responses are now considered as an attractive and promising therapeutic approach in oncology. [6][7][8]39,40 Stimulation of gd T lymphocytes with PAg, 3,6-11 through the engagement of the BTN3A1 molecule, [25][26][27][28][29][30][31] leads to the generation of an efficient antitumor immune response.…”

“…[1][2][3] Vd2 T lymphocytes recognize unprocessed nonpeptide molecules, including phosphoantigens (PAg) derived from the mevalonate pathway in mammalian cells, and via the 1-deoxy-D-xylulose-5-phosphate pathway, in bacterial cells. [1][2][3][4][5] gd T cells also recognize NKG2D ligands (MICA, MICB and ULBPs), overexpressed at the cells surface by viral infections or tumor transformation. [1][2][3] Another activation signal can be delivered via FcgRIIIa/CD16 that, upon interaction with the Fc of IgG, initiates the antibody-dependent cellular cytotoxicity to destroy opsonized cells or microorganisms.…”

“…1,2 For this unconventional antigen recognition and multiple activation pathways, gd T lymphocytes are antitumor effector cells in several cancer types, including colorectal cancer (CRC), and potential suitable tools for anticancer therapy. [3][4][5][6][7][8] Different drugs can be utilized to improve the mechanisms of gd T cell activation: 2,3,8 in particular, synthetic pyrophosphate-containing compounds have been proposed for cancer immunotherapy on the basis of their ability to stimulate gdT cells. [9][10][11][12] Moreover, aminobisphosphonates (N-BPs), in addition to their effect of inhibiting osteoclastic bone resorption, 13 lead to gd T cell activation and proliferation, with a consequent increased number of gd T cells in peripheral blood, displaying antitumor activity [14][15][16][17][18] Indeed, N-BPs, such as zoledronate (Zol), are chemically stable analogs of inorganic pyrophosphate (IPP) that inhibit the mevalonate pathway and upregulate IPP accumulation, promoting antitumor Vg9Vd2 T cells in vitro and in vivo.…”

Essential Requirements of Zoledronate-Induced Cytokine and γδ T Cell Proliferative Responses

“…1), which translate into either stimulatory or inhibitory effects. Zoledronate-induced upstream accumulation of the cognate gd T cell Ag IPP, which is recognized by the Vg9Vd2 TCR (16,17), initiates the immediate response within hours. Early upregulation of IL-2Ra (CD25) on gd T cells increases their sensitivity to concomitant stimulation with IL-2.…”

“…In an attempt to explain this clinical observation, N-BPs initially were suggested to be cognate phosphoantigens that can be directly recognized by gd T cells (direct pathway). Isopentenyl pyrophosphate (IPP), which accumulates as a consequence of N-BP-induced inhibition of FPP synthase, was later identified as the true cognate Ag that is recognized by the Vg9Vd2 TCR (indirect pathway) (16)(17)(18). However, the context of IPP recognition by the Vg9Vd2 TCR remains elusive.…”