Joëlle Riond

Cutting Edge: CTLs Rapidly Capture Membrane Fragments from Target Cells in a TCR Signaling-Dependent Manner

Upon encounter of a CTL with a target cell carrying foreign Ags, the TCR internalizes with its ligand, the peptide-MHC class I complex. However, it is unclear how this can happen mechanistically because MHC molecules are anchored to the target cell’s surface via a transmembrane domain. By using antigenic peptides and lipids that were fluorescently labeled, we found that CTLs promptly capture target cell membranes together with the antigenic peptide as well as various other surface proteins. This efficient and specific capture process requires sustained TCR signaling. Our observations indicate that this process allows efficient acquisition of the Ag by CTL, which may in turn regulate lymphocyte activation or elimination.

Melanoma Chemotherapy Leads to the Selection of ABCB5-Expressing Cells

Metastatic melanoma is the most aggressive skin cancer. Recently, phenotypically distinct subpopulations of tumor cells were identified. Among them, ABCB5-expressing cells were proposed to display an enhanced tumorigenicity with stem cell-like properties. In addition, ABCB5+ cells are thought to participate to chemoresistance through a potential efflux function of ABCB5. Nevertheless, the fate of these cells upon drugs that are used in melanoma chemotherapy remains to be clarified. Here we explored the effect of anti-melanoma treatments on the ABCB5-expressing cells. Using a melanoma xenograft model (WM266-4), we observed in vivo that ABCB5-expressing cells are enriched after a temozolomide treatment that induces a significant tumor regression. These results were further confirmed in a preliminary study conducted on clinical samples from patients that received dacarbazine. In vitro, we showed that ABCB5-expressing cells selectively survive when exposed to dacarbazine, the reference treatment of metastatic melanoma, but also to vemurafenib, a new inhibitor of the mutated kinase V600E BRAF and other various chemotherapeutic drugs. Our results show that anti-melanoma chemotherapy might participate to the chemoresistance acquisition by selecting tumor cell subpopulations expressing ABCB5. This is of particular importance in understanding the relapses observed after anti-melanoma treatments and reinforces the interest of ABCB5 and ABCB5-expressing cells as potential therapeutic targets in melanoma.

T cell activation correlates with an increased proportion of antigen among the materials acquired from target cells

We have investigated the density of peptides required to elicit different biological responses in cytotoxic T lymphocytes (CTL), including trogocytosis (i.e., the phenomenon whereby the lymphocytes actively capture fragments of plasma membrane from those cells with which they establish an immune synapse). We have used two separate mouse models of CTL recognising defined peptides presented by MHC class I molecules. In both systems, triggering of cytotoxicity and capture of membrane components reached saturation with low densities of ligand. On the other hand, down‐modulation of cell‐surface levels of TCR, induction of IFN‐γ production and detection of peptide captured required much higher ligand densities. Interestingly, fratricide (i.e., killing between CTL sharing the same specificity), a mechanism proposed to account for CTL exhaustion, was detected only at antigen concentrations still well above that second threshold leading to full blown activation. Taken together, our results show that the different thresholds that govern the elicitation of different CTL functions correlate with different proportions of antigen among the target cell components being captured via trogocytosis.

Agonist‐induced signaling and trafficking of the μ‐opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C‐terminal domain

The human μ‐opioid receptor and a mutant form, μS/T[i3+Cter]A, in which all Ser and Thr residues from the third cytoplasmic loop and C‐terminal domain were changed to Ala, were studied after expression in CHO‐K1 cells. Although the mutant receptors had similar affinities for agonists and EC50 values for inhibition of adenylyl cyclase as compared to wild‐type receptors, the E max were almost 2‐fold decreased, suggesting a role of the mutated residues in G‐protein coupling. After chronic morphine or etorphine, the EC50 values of the agonists were about 5‐fold increased at both receptors but the E max values were not altered; upon agonist withdrawal forskolin‐stimulated cAMP levels were increased to almost 200% of control levels. Sequestration and rapid down‐regulation of the μ‐opioid receptor were induced by DAGO and etorphine but not morphine. In contrast, the μS/T[i3+Cter]A receptor was not sequestered and was up‐regulated (150–380%) after treatment with agonists. The results indicate that the Ser and Thr residues in the third cytoplasmic loop and C‐terminus of the μ‐opioid receptor are not involved in the limited desensitization or in the adenylyl cyclase superactivation promoted by agonists but that their integrity and/or their phosphorylation is required in the intricate and coordinately regulated pathways involved in receptor signaling and trafficking.

Capture of membrane components via trogocytosis occurs in vivo during both dendritic cells and target cells encounter by CD8+ T cells

Cytotoxic T lymphocytes recently stimulated by antigen‐presenting cells (APC) display major histocompatibility class (MHC) I and II molecules inherited from APC. We have previously reported that, in vitro, transfer of MHC molecules and several other proteins occurs through trogocytosis, i.e. the active acquisition of target cell membrane fragments by T lymphocytes. Here, using the model of viral antigen LCMVgp33‐41 recognition in transgenic P14 mice, we show that CD8+ T cells perform trogocytosis in vivo, as detected by the capture of biotin‐ or fluorescence‐labeled components of the APC surface. Trogocytosis occurs during interactions of CD8+ T cells with at least two kinds of cells: target cells and dendritic cells (DC). In lymph nodes, CD8+ T cells having performed trogocytosis with DC express the CD69 activation marker indicating that trogocytosis detects recently activated cells. Taken together, our findings suggest that trogocytosis may be a new in vivo marker of the recent interaction between a CD8+ T cell and its cellular partners or targets.

Molecular and Functional Dissection of the H-2Db-Restricted Subdominant Cytotoxic T-Cell Response to Lymphocytic Choriomeningitis Virus

ABSTRACT Infection of H-2 b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.

Interactions between CD8αβ and the TCRαβ/CD3-receptor complex

CD8+ T cells recognize antigenic peptides bound to major histocompatibility complex (MHC) class I molecules on normal antigen‐presenting cells (APC), as well as on virus‐infected cells or tumour cells (pMHC). At least two receptor types participate in recognition of these complexes: T‐cell receptor (TCR) αβ heterodimers and CD8αβ molecules. The former molecules react with antigenic peptide and variable regions of MHC class I molecules, whereas the latter molecules react with constant α3 regions of MHC class I molecules. As the avidity of both receptor‐MHC interactions is low, it is believed that TCRαβ and CD8αβ heterodimers collaborate in T‐cell recognition. We have established a TCR/CD3–CD8 capture ELISA, which can measure the interaction of pMHC with CD8αβ molecules and with TCR/CD3 complexes. The major findings are: (1) TCR/CD3 complexes derived from in vitro activated T cells and captured by anti‐CD3 MoAb, do bind specific pMHC and (2) CD8+ T cells express at least three forms of CD8αβ molecules: single CD8αβ, CD3–CD8 and TCR/CD3–CD8 complexes. Only the latter complexes are associated with CD3ζ homodimers, and the quantity of TCR/CD3–CD8 complexes relative to total CD8αβ molecules appears to increase and to be selected into sucrose‐gradient microdomains as a function of TCRαβ‐mediated T‐cell activation.

Abstract 5524: 5-aza-2′-deoxycytidine, a DNA demethylating agent, inhibits metastatic melanoma invasiveness

Metastatic melanomas are the deadliest form of skin cancer and are very aggressive tumors showing highly invasive properties and a rapid chemoresistance to standard treatment (Dacarbazine) and to specific BRAF-V600E kinase inhibitors (Vemurafenib). Thus, targeting these tumors remains a major concern for novel therapeutic proposals. Abnormal patterns of DNA methylation, an epigenetic modification that cells use to control gene expression, have been described in these tumors. These epigenetic modifications participate in melanoma formation and maintenance. The aim of our project is to characterize the DNA methylation changes that occur in the most aggressive form of melanoma and to reverse these changes by using clinically active DNA methyltransferase inhibitors (5-aza-2′-deoxycytidine, 5-aza-dC). To date a limited number of datasets have been published depicting the effects of inhibitors of DNA methylation on invasive capacities of metastatic melanoma cells. The work presented here, focuses on the study of the effects of DNA methylation inhibition on metastatic melanoma invasiveness. We have set up an original method to quantify DNA methylation by FACS and shown that non cytotoxic nanomolar 5-aza-dC concentrations were able to demethylate DNA of WM-266-4 metastatic melanoma cells. Then using an in vitro 3D spheroids cell invasion assay and fluorescent microscopy to measure invasion capacities of metastatic cell lines, we showed that 5-aza-dC was able to inhibit invasion of WM-266-4 cells at these non-cytotoxic demethylating concentrations. Citation Format: Chantal Etievant, Cecile Desjobert, Arnaud Carrier, Audrey Delmas, Jorg Tost, Gilles Favre, Joelle Riond, Paola Arimondo. 5-aza-2′-deoxycytidine, a DNA demethylating agent, inhibits metastatic melanoma invasiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5524. doi:10.1158/1538-7445.AM2014-5524