Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis.

Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain partially elusive. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who beared biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein-protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4+ CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity and provides the molecular pathomechanism behind the identified here and previously unreported genetically determined form of HLH.

Granulysin- and granzyme-dependent elimination of myeloid cells by therapeutic ova-specific type 1 regulatory T cells.

The intrinsic immunosuppressive properties of regulatory T (Treg) cells can be harnessed for therapeutic approaches aiming at down-modulating harmful immune reactions. In this context, expanded type 1 Treg cells (Tr1 cells) specific for ovalbumin (ova-Tr1 cells) have been tested for clinical efficacy in the treatment of autoimmune disorders such as refractory Crohn's disease (CD). The clinical use of these therapeutic products warrants exploration of their mechanism of action. Here, we identified a relationship between the CD activity index and the expression of lytic molecules by the ova-Tr1 cells administered in the previously reported First-in-Man study [Crohn's And Treg cells Study 1 (CATS1) study]. Accordingly, ova-Tr1 cells were found to carry granules containing high levels of lytic molecules, including multiple granzymes and granulysin. These cells displayed a T-cell receptor (TCR)-independent cytotoxic activity, which was preferentially directed toward myeloid cell lines and monocyte-derived dendritic cells. Upon contact with myeloid cells, ova-Tr1 cells induced their apoptosis via a perforin-independent and a granulysin/granzyme-dependent mechanism. As compared to CD8+ cytotoxic T cells, ova-Tr1 cells required more time to lyse target cells and displayed a more gradual lytic activity over time. Notably, this activity was sustained over days resulting in the control of myeloid cell populations at a relatively low ratio. Our study reveals that ova-Tr1 cells are endowed with a sustained cytotoxic activity that relies on a unique combination of granulysin and granzymes and that preferentially eliminates myeloid target cells in a TCR-independent manner.

Switching between individual and collective motility in B lymphocytes is controlled by cell-matrix adhesion and inter-cellular interactions.

Lymphocytes alternate between phases of individual migration across tissues and phases of clustering during activation and function. The range of lymphocyte motility behaviors and the identity of the factors that govern them remain elusive. To explore this point, we here collected unprecedented statistics pertaining to cell displacements, cell:matrix and cell:cell interactions using a model B cell line as well as primary human B lymphocytes. At low cell density, individual B lymphocytes displayed a high heterogeneity in their speed and diffusivity. Beyond this intrinsic variability, B lymphocytes adapted their motility to the composition of extra-cellular matrix, adopting slow persistent walks over collagen IV and quick Brownian walks over fibronectin. At high cell density, collagen IV favored the self-assembly of B lymphocytes into clusters endowed with collective coordination, while fibronectin stimulated individual motility. We show that this behavioral plasticity is controlled by acto-myosin dependent adhesive and Arp2/3-dependent protrusive actin pools, respectively. Our study reveals the adaptive nature of B lymphocyte motility and group dynamics, which are shaped by an interplay between and cell:matrix and cell:cell interactions.