RhoB Mediates Phosphoantigen Recognition by Vγ9Vδ2T Cell Receptor.

Zsolt Sebestyen,Cassie-Marie Peigné,Jürgen Kuball,Anna Vyborova,Daniel Olive,Gerrit Jan Schuurhuis,Siyi Gu,Rimke Oostvogels,Zuzana Rychnavska,Coraline Chéneau,Emmanuel Scotet,Martje Van Noorden,Astrid Cleven,Erin J Adams,Tuna Mutis,Wouter Scheper,Marleen Schiffler,Robert Jan Lebbink

doi:10.1016/j.celrep.2016.04.081

Abstract

Human Vγ9Vδ2 Tcells respond to tumor cells by sensing elevated levels of phosphorylated intermediates of the dysregulated mevalonate pathway, which is translated into activating signals by the ubiquitously expressed butyrophilin A1 (BTN3A1) through yet unknown mechanisms. Here, we developed an unbiased, genome-wide screening method that identified RhoB as a critical mediator of Vγ9Vδ2 TCR activation in tumor cells. Our results show that Vγ9Vδ2 TCR activation is modulated by the GTPase activity of RhoB and its redistribution to BTN3A1. This is associated with cytoskeletal changes that directly stabilize BTN3A1 in the membrane, and the subsequent dissociation of RhoB from BTN3A1. Furthermore, phosphoantigen accumulation induces a conformational change in BTN3A1, rendering its extracellular domains recognizable by Vγ9Vδ2 TCRs. These complementary events provide further evidence for inside-out signaling as an essential step inthe recognition of tumor cells by a Vγ9Vδ2 TCR.

Highlights

GdT cells are unconventional T cells with strong reactivity toward a broad spectrum of tumors of diverse tissue origin. gdT cells combine potent anti-tumor effector functions with the recognition of broadly expressed tumor-associated molecules, and these features have put gdT cells in the spotlight for clinical application in cancer immunotherapy
CD4+ abT cells engineered to express one defined Vg9Vd2 TCR were utilized for the functional screening in order to eliminate fluctuations in recognition by a diverse gdTCR repertoire (Gru€nder et al, 2012) and varying expression of natural killer (NK) receptors (Scheper et al, 2013; Correia et al, 2013)
The hypothetical loci zygosities were correlated with available genotype information of SNPs within the study’s population (Spaapen et al, 2008), resulting in the identification of 17 SNPs whose genotypes correlated perfectly (100%) with predicted zygosities (Figure 1B). Since none of these 17 SNPs, nor their proxy SNPs within high linkage disequilibrium (r2 > 0.8), directly affected genes by causing changes in protein coding sequences, we speculated that rather than playing direct roles, the SNPs we identified could represent surrogate markers for genetic regions associated with susceptibility to Vg9Vd2

Summary

Introduction

GdT cells are unconventional T cells with strong reactivity toward a broad spectrum of tumors of diverse tissue origin. gdT cells combine potent anti-tumor effector functions with the recognition of broadly expressed tumor-associated molecules, and these features have put gdT cells in the spotlight for clinical application in cancer immunotherapy. Activation of gdT cells involves the sensing of metabolic changes in cancer cells that result in the expression of generic stress molecules. These molecules are upregulated upon transformation or distress (Bonneville et al, 2010; Vantourout and Hayday, 2013). Intracellular phosphoantigen (pAg) levels accumulate in tumor cells due to dysregulation of the mevalonate pathway or upon microbial infection, allowing the targeting of transformed or infected cells by Vg9Vd2 T cells. Intracellular phosphoantigen levels can be pharmaceutically increased by treating cells with mevalonate pathway inhibitors such as aminobisphosphonates (ABPs), thereby sensitizing cells toward recognition by Vg9Vd2 T cells. The involvement of the Vg9Vd2 TCR in detecting elevated phosphoantigen levels was demonstrated as early as the 1990s (Bukowski et al, 1998; Davodeau et al, 1993; Wang et al, 2010), the molecular determinants required for activation of Vg9Vd2 TCRs on target cells have long remained elusive

Results

Discussion

Conclusion