Christelle Dousset

Early Triggering of Exclusive IFN-γ Responses of Human Vγ9Vδ2 T Cells by TLR-Activated Myeloid and Plasmacytoid Dendritic Cells

γδ T cells, a major innate-like T cell subset, are thought to play in vivo an important role in innate and adaptive immune responses to various infection agents like parasites, bacteria, or viruses but the mechanisms contributing to this immune process remain ill defined. Owing to their ability to recognize a broad set of microbial molecular patterns, TLRs represent a major innate pathway through which pathogens induce dendritic cells (DC) maturation and acquisition of immunostimulatory functions. In this study, we studied the effects of various TLR ligands on the activation of human Vγ9Vδ2 T cells, a main human γδ PBL subset, which has been recently involved in the licensing of mycobacteria-infected DC. Both TLR3 and TLR4, but not TLR2 ligands, induced a rapid, strong, and exclusive IFN-γ production by Vγ9Vδ2 T cells. This γδ subset contributed to a large extent to the overall PBL IFN-γ response induced after short-term TLR stimulation of human PBMC. Importantly, this phenomenon primarily depended on type I IFN, but not IL-12, produced by monocytic DC upon TLR engagement. Vγ9Vδ2 T cells were similarly activated by plasmacytoid DC upon TLR8/9 activation or Yellow Fever virus infection. Moreover TLR-induced Vγ9Vδ2 IFN-γ noncytolytic response led to efficient DC polarization into IL-12p70-producing cells. Our results support an adjuvant role played by Vγ9Vδ2 T cells along microbial infections through a particular cross-talk with pathogen-associated molecular patterns-activated DC. Moreover they provide new insights into the mechanisms underlying functional activation of this unique peripheral innate-like T cell subset during viral infections.

Complex Interplay of Activating and Inhibitory Signals Received by Vγ9Vδ2 T Cells Revealed by Target Cell β2-Microglobulin Knockdown

Tumor cells often escape immunosurveillance by down-regulating MHC class I molecule expression. For human Vγ9Vδ2 T cells, a major peripheral blood T cell subset with broad antitumor reactivity, this down-regulation can affect signals transmitted by both the inhibitory and the activating MHC class I and Ib-specific NK receptors (NKRs) that these lymphocytes frequently express. To assess the overall impact of MHC down-regulation on Vγ9Vδ2 T cell activation, we used stable β2-microglobulin knockdown to generate tumor cells with a ∼10-fold down-modulation of all MHC class I molecules. This down-modulation had little effect on T cell proliferation or cytokine production, but modified tumor cell killing efficiency. Ab-blocking studies identified ILT2 as an important inhibitor of tumor cell killing by Vγ9Vδ2 T cells. Down-modulation of MHC class I and Ib molecules severely reduced ILT2 inhibitory signaling, but still allowed signaling by activating CD94-based receptors. It also unveiled a frequent enhancing effect of NKG2D on tumor killing by Vγ9Vδ2 T cells. Current models suggest that activating NKRs have less affinity for their MHC ligands than homologous inhibitory NKRs. Our results show that, despite this, activating NKRs recognizing MHC class I molecules play an important role in the increased killing by Vγ9Vδ2 T cells of tumor cells with down-regulated MHC class I molecule expression, and suggest that these T cells will best lyse tumor cells combining MHC class I molecule expression down-regulation with up-regulated NKG2D ligand expression.

BH3 profiling as a tool to identify acquired resistance to venetoclax in multiple myeloma

BH3 profiling as a tool to identify acquired resistance to venetoclax in multiple myeloma Venetoclax/ABT-199 is the first in the class of BCL2-specific BH3 mimetics and the most promising targeted therapy in oncology (Souers et al, 2013). Venetoclax is currently under investigation in multiple myeloma (MM), which is heterogeneous and includes either patients with a translocation on chromosome 14 with different chromosomes (4, 6, 11 or 16) or a hyperdiploidy. We demonstrated that venetoclax induces cell death in a subgroup harbouring the t(11;14) transloca-tion, expressing a high BCL2/MCL1 gene expression ratio, and that intrinsic venetoclax resistance is mediated by high MCL1 expression in MM cells (Touzeau et al, 2014). Preliminary results from an ongoing phase I clinical trial testing venetoclax in relapsed/refractory MM patients indicate that BCL2 inhibition has a tolerable safety profile and single agent activity mostly in t(11;14) patients (Kumar et al, 2015). The anticipated use of venetoclax in the treatment of MM lead us to explore the mechanisms of acquired veneto-clax resistance. We generated two venetoclax-resistant mye-loma cell lines using in vitro selection and derived resistant sublines (named-199R) from KMS12-PE and XG5 t(11;14) myeloma venetoclax-sensitive cell lines (Figs 1A, 2A) (Data S1). Both resistant sublines showed a strong reduction in V

A simple flow cytometry‐based barcode for routine authentication of multiple myeloma and mantle cell lymphoma cell lines

lines are widely used in laboratories for in vitro experi-ments, especially for investigating abnormal hallmarks in can-cer cells and identifying therapeutic targets. Human cell linesare typically derived in academic laboratories from a widerange of cancer samples. To achieve a representation of intra-cancer heterogeneity, several laboratories, including ours, haveestablished cell line collections. However, the establishmentand maintenance of such collections significantly increase therisk of cross-contaminations and misidentification of celllines, leading to the publication of false data/interpretation(1). In addition to the risk of cross-contamination, widelyused cell lines can be described in contrasting manners for aparticular feature (e.g., the JJN3 myeloma cell line appearseither TP53