CD81 Controls Sustained T Cell Activation Signaling and Defines the Maturation Stages of Cognate Immunological Synapses

Abstract

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin-2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activation. The interaction between T lymphocytes and antigen-presenting cells (APCs) is essential for the initiation of the immune response. The dynamic structure formed at cell-to-cell contacts between T cells and APCs, called the immune synapse (IS), is characterized by controlled recruitment of membrane receptors to specific subcellular sites (1). Upon activation by an APC, T cell molecules involved in the IS redistribute in highly organized structures at the T cell-APC contact (2). The T cell receptor (TCR) and associated molecules concatenate into the central area (central supramolecular activation cluster [cSMAC]), whereas adhesion receptors rearrange in a surrounding external ring called the peripheral supramolecular activation cluster (pSMAC) (3). During IS formation, preclustered TCR "protein islands" converge into larger aggregates that translocate toward the cSMAC (4, 5), from where they are internalized and degraded (6). The balance between the generation and degradation of TCR microclusters is critical for sustained T cell activation (5, 7) and is modulated by ligand mobility (8). However, the mechanisms regulating protein receptor movement and the basis for IS molecular segregation are still poorly understood.A plethora of molecules are translocated to the IS during T cell activation (9). These include the tetraspanins CD81 (10) and CD82 (11), which are known to associate with several IS components such as major histocompatibility complex class II (MHCII) molecules, CD4, and LF...

Tetraspanins CD9 and CD151 at the immune synapse support T‐cell integrin signaling

“…At all times analyzed, colocalization of CD9 or CD151 with CD3 (Supporting Information Fig. 1A-B) was lower than previously observed for CD81 [5], suggesting that, unlike CD81, CD9, and CD151 might not regulate the reorganization of CD3 at the IS. …”

“…2014. 44: 1967-1975 The role of CD9 and CD151 during T-cell activation might be TEM-mediated, contrasting with CD81, which regulates synapse formation and subsequent T-cell activation through TCR signaling events [5], and with CD82, which regulates the T cell actin cytoskeleton [7]. Adhesion of lymphocytes to the extracellular matrix and to other cells is mediated by integrins [8], and well characterized interactions between integrins and tetraspanins are important for the regulation of cell adhesion and migration [4,9].…”

“…The increased IS stability enhances T-cell sensitivity, stimulates cytoskeletal processes, induces downstream signaling, and drives effective T-cell differentiation [2,3]. How integrins regulate these processes is not fully understood.Several membrane receptors and integrins concentrated at the IS -CD3, CD4, ICAM-1, lymphocyte function-associated antigen 1 (LFA-1), and very late antigen 4 (VLA-4) -are associated with tetraspanins [4,5] 1968 Rocha-Perugini Vera et al Eur. J. Immunol.…”

“…T and B cells were mixed (1:1) and incubated at 37°C for the indicated periods. Measurements of IL-2 secretion and immunoblotting were performed as described [5]. …”

“…Primary SEE T lymphoblasts were transfected by electroporation with siRNA 4, 5 days after isolation using the same protocol. Lentiviral silencing of endogenous CD9 or CD151 was performed as described [5]. …”