Christopher Bricogne

PD-L1 co-stimulation contributes to ligand-induced T cell receptor down-modulation on CD8(+) T cells

T cell receptor (TCR) down‐modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down‐modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD‐L1) on dendritic cells (DCs) and programmed death 1 (PD‐1) on CD8 T cells contributes to ligand‐induced TCR down‐modulation. We provide evidence that this occurs via Casitas B‐lymphoma (Cbl)‐b E3 ubiquitin ligase up‐regulation in CD8 T cells. Interference with PD‐L1/PD‐1 signalling markedly inhibits TCR down‐modulation leading to hyper‐activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD‐L1 silencing accelerates anti‐tumour immune responses and strongly potentiates DC anti‐tumour capacities, when combined with mitogen‐activated kinase (MAPK) modulators that promote DC activation.

Retroviral and Lentiviral Vectors for the Induction of Immunological Tolerance

Retroviral and lentiviral vectors have proven to be particularly efficient systems to deliver genes of interest into target cells, either in vivo or in cell cultures. They have been used for some time for gene therapy and the development of gene vaccines. Recently retroviral and lentiviral vectors have been used to generate tolerogenic dendritic cells, key professional antigen presenting cells that regulate immune responses. Thus, three main approaches have been undertaken to induce immunological tolerance; delivery of potent immunosuppressive cytokines and other molecules, modification of intracellular signalling pathways in dendritic cells, and de-targeting transgene expression from dendritic cells using microRNA technology. In this review we briefly describe retroviral and lentiviral vector biology, and their application to induce immunological tolerance.

PD-L1 co-stimulation, ligand-induced TCR down-modulation and anti-tumor immunotherapy

PD-1 engagement on the surface of effector T cells strongly suppresses their cytotoxic function, which constitutes a major obstacle for T cell-mediated anti-tumor activities. Surprisingly, PD-1 is strongly upregulated in T cells, engaging its ligand PD-L1 during antigen presentation. However, our recent published data may provide an explanation for this apparent contradiction.

PD-L1/PD-1 Co-Stimulation, a Brake for T cell Activation and a T cell Differentiation Signal

For T cell activation, three signals have to be provided from the antigen presenting cell; Signal 1 (antigen recognition), signal 2 (co-stimulation) and signal 3 (cytokine priming). Blocking negative co-stimulation during antigen presentation to T cells is becoming a promising therapeutic strategy to enhance cancer immunotherapy. Here we will focus on interference with PD-1/PD-L1 negative co-stimulation during antigen presentation to T cells as a therapeutic approach. We will discuss the potential mechanisms and the therapeutic consequences by which interference/inhibition with this interaction results in anti-tumour immunity. Particularly, we will comment on whether blocking negative co-stimulation provides differentiation signals to T cells undergoing antigen presentation. A major dogma in immunology states that T cell differentiation signals are given by cytokines and chemokines (signal 3) rather than co-stimulation (signal 2). We will discuss whether this is the case when blocking PD-L1/PD-1 negative co-stimulation.

Immune modulation by genetic modification of dendritic cells with lentiviral vectors

Our work over the past eight years has focused on the use of HIV-1 lentiviral vectors (lentivectors) for the genetic modification of dendritic cells (DCs) to control their functions in immune modulation. DCs are key professional antigen presenting cells which regulate the activity of most effector immune cells, including T, B and NK cells. Their genetic modification provides the means for the development of targeted therapies towards cancer and autoimmune disease. We have been modulating with lentivectors the activity of intracellular signalling pathways and co-stimulation during antigen presentation to T cells, to fine-tune the type and strength of the immune response. In the course of our research, we have found unexpected results such as the surprising immunosuppressive role of anti-viral signalling pathways, and the close link between negative co-stimulation in the immunological synapse and T cell receptor trafficking. Here we review our major findings and put them into context with other published work.

Lentiviral Vectors in Immunotherapy

Genetic immunotherapy can be defined as a therapeutic approach in which therapeutic genes are introduced into defined target cell types to modulate immune responses. A major challenge for this therapeutic strategy is the delivery of these genes into target cells in an efficient, stable manner. Possibly one of the best systems to achieve this is the use of lentivi‐ ral vectors (lentivectors) as gene carriers, as they are capable of transducing both dividing and resting cells [1].

Targeted Lentiviral Vectors: Current Applications and Future Potential

About two decades ago recombinant human immunodeficiency virus type 1 (HIV-1) wasproposed as a blueprint for the development of lentiviral vectors (LVs) (Naldini, Blomer etal. 1996). Lentiviral vectors exhibit several characteristics that make them favorable tools forgene therapy, including sustained gene delivery through vector integration, transduction ofboth dividing and non-dividing cells, applicability to different target cell types, absence ofexpression of viral proteins after transduction, delivery of complex genetic elements, lowgenotoxicity and the relative ease of vector manipulation and production (Cattoglio, Facchi‐ni et al. 2007; Bauer, Dao et al. 2008). This is reflected in the numerous applications such as:transgene (tg) overexpression (Lopez-Ornelas, Mejia-Castillo et al. 2011), persistent gene si‐lencing (Wang, Hu et al. 2012), immunization (Breckpot, Emeagi et al. 2008), generation oftransgenic animals (Baup, Fraga et al. 2010), in vivo imaging (Roet, Eggers et al. 2012), induc‐tion of pluripotent cells, stem cell modification (Sanchez-Danes, Consiglio et al. 2012), line‐age tracking and site-directed gene editing (Lombardo, Genovese et al. 2007) as well asmany applications targeting cancer cells (Petrigliano, Virk et al. 2009).Recombinant LVs can be derived from primate as well as non-primate lentiviruses such asHIV-1 and simian immunodeficiency virus (SIV) next to the equine infectious anemia virus,caprine arthritis-encephalitis virus, maedi-visna virus, feline immunodeficiency virus (FIV)and bovine immunodeficiency virus respectively (Escors and Breckpot 2010). They are allmembers of the Retroviridae family with ‘retro’ referring to their capacity to retro-transcribetheir diploid single stranded (ss) RNA genome into a double stranded (ds) DNA copy that is

TMEM16F activation by Ca2+ triggers plasmalemma expansion and directs PD-1 trafficking

TMEM16F, an ion channel gated by high cytoplasmic Ca2+, is required for cell surface phosphatidylserine exposure during platelet aggregation and T cell activation. Here we demonstrate in Jurkat T cells and HEK293 cells that TMEM16F activation triggers large-scale surface membrane expansion in parallel with lipid scrambling. Following TMEM16F mediated scrambling and surface expansion, cells undergo extensive membrane shedding. The membrane compartment that expands the cell surface does not involve endoplasmic reticulum or acidified lysosomes. Surprisingly, T cells lacking TMEM16F expression not only fail to expand surface membrane, but instead rapidly internalize membrane via massive endocytosis (MEND). The T cell co-receptor PD-1 is selectively shed when TMEM16F triggers membrane expansion, while it is selectively internalized in the absence of TMEM16F. Its participation in this trafficking is determined by its single transmembrane domain. Thus, we establish a fundamental role for TMEM16F as a regulator of Ca2+-activated membrane trafficking.

Massive surface membrane expansion without involvement of classical exocytic mechanisms.

Activation of TMEM16F, a Ca2+ -dependent ion channel and lipid scramblase, causes massive surface membrane expansion in multiple cell types by unresolved mechanisms. We describe here that membrane expansion reflects opening of deeply invaginating surface membrane compartments when anionic phospholipids are lost from the cytoplasmic membrane leaflet. Compartments that open contain vesicle-associated membrane proteins (VAMPs) and can open with as little as one micromolar free Cai2+. Cationic peptides that sequester anionic phospholipids open the compartments from the cytoplasmic side without Ca2+. Monovalent cations facilitate membrane expansion via coupled permeation with anionic phospholipids through TMEM16F. When monovalent cation concentrations are reduced, membrane expansion can be reversed by changing ion gradients and membrane voltage. Depolarization closes the compartments by generating inward cation gradients through TMEM16F that promote influx of anionic phospholipids. In summary, TMEM16F-mediated membrane expansion likely does not reflect exocytosis but rather the relaxation of constrictions that close surface membrane invaginations. Summary The surface membrane of diverse cell types can be remodeled by opening and closing surface invaginations that are held shut by proteins that bind negatively charged lipids and constrict the orifices of these compartments.