Early and Dynamic Polarization of T Cell Membrane Rafts and Constituents Prior to TCR Stop Signals

Komen, Jeffrey S. Van; Mishra, Sumeet; Byrum, Jennifer N.; Chichili, Gurunadh R.; Yaciuk, Jane C.; Farris, A. Darise; Rodgers, William

doi:10.4049/jimmunol.179.10.6845

Cited by 12 publications

(23 citation statements)

References 46 publications

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“…Accordingly, treatment of J77 T cells with the actin-depolymerizing toxin latrunculin A before incubation with Raji/SEE cells prevented IS formation, with the very few T cell-B cell contacts observed being devoid of CD81-mCherry or endogenous CD3 accumulation at the IS (data not shown), in agreement with previous works (5,58). Cholesterol is an essential component of TEMs (46), with an important role in the IS context (59)(60)(61)(62). T cell-B cell conjugation as well as CD81 and CD3 accumulation at the IS were also significantly reduced by the treatment of J77 T cells with methyl-␤-cyclo-dextrin, a drug that extracts membrane cholesterol, before incubation with Raji/SEE cells (data not shown).…”

Section: Discussionsupporting

confidence: 90%

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

Rocha-Perugini

Zamai

González-Granado

et al. 2013

Molecular and Cellular Biology

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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...

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Section: Discussionsupporting

confidence: 90%

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

Rocha-Perugini

Zamai

González-Granado

et al. 2013

Molecular and Cellular Biology

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“…Lck10-GFP has been used as a probe to monitor the distribution of specific compact membranes in T cells (23). Similar to Lck and LAT, Lck10-GFP accumulated at the cSMAC or pSMAC depending on the place of exogenous MAL accumulation (Fig.…”

Section: Mal Distribution Controls Sorting Of Lck and Lat To Specificmentioning

confidence: 97%

“…The N-terminal 10-aa sequence of Lck is sufficient to confer myristoylation and double palmitoylation on a GFP chimera (Lck10-GFP) and to incorporate it into DRMs (23,27). Lck10-GFP has been used as a probe to monitor the distribution of specific compact membranes in T cells (23).…”

Section: Mal Distribution Controls Sorting Of Lck and Lat To Specificmentioning

confidence: 99%

MAL Protein Controls Protein Sorting at the Supramolecular Activation Cluster of Human T Lymphocytes

Antón

Andrés-Delgado

Reglero-Real

et al. 2011

The Journal of Immunology

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T cell membrane receptors and signaling molecules assemble at the immunological synapse (IS) in a supramolecular activation cluster (SMAC), organized into two differentiated subdomains: the central SMAC (cSMAC), with the TCR, Lck, and linker for activation of T cells (LAT), and the peripheral SMAC (pSMAC), with adhesion molecules. The mechanism of protein sorting to the SMAC subdomains is still unknown. MAL forms part of the machinery for protein targeting to the plasma membrane by specialized mechanisms involving condensed membranes or rafts. In this article, we report our investigation of the dynamics of MAL during the formation of the IS and its role in SMAC assembly in the Jurkat T cell line and human primary T cells. We observed that under normal conditions, a pool of MAL rapidly accumulates at the cSMAC, where it colocalized with condensed membranes, as visualized with the membrane fluorescent probe Laurdan. Mislocalization of MAL to the pSMAC greatly reduced membrane condensation at the cSMAC and redistributed machinery involved in docking microtubules or transport vesicles from the cSMAC to the pSMAC. As a consequence of these alterations, the raft-associated molecules Lck and LAT, but not the TCR, were missorted to the pSMAC. MAL, therefore, regulates membrane order and the distribution of microtubule and transport vesicle docking machinery at the IS and, by doing so, ensures correct protein sorting of Lck and LAT to the cSMAC.

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“…Spatiotemporal studies of T cells during Ag-presentation show that TCR triggering measured by intracellular Ca 2+ signaling is preceded by formation of a membrane complex composed of the actin cytoskeleton, membrane rafts, TCR, and TCR proximal proteins (4, 10). The membrane complex occurs seconds following initial contact between the T cell and APC, and minutes before measurable Ca 2+ signaling from the TCR(10).…”

Section: Introductionmentioning

confidence: 99%

“…The membrane complex occurs seconds following initial contact between the T cell and APC, and minutes before measurable Ca 2+ signaling from the TCR(10). The pre-TCR complexes are both Ag- and MHC II-independent, as they occur in CD4 + T cells when conjugated to MHC II-deficient B cells (10), and they correlate temporally with an Ag-independent adhesion of the T cell to the APC that precedes TCR triggering (4). Given the compositional similarities of the Ag-independent complexes and ISs, these structures have been referred to as an Ag-independent synapse (AIS) (11).…”

Section: Introductionmentioning

confidence: 99%

CD28 Sensitizes TCR Ca2+ Signaling during Ag-Independent Polarization of Plasma Membrane Rafts

Byrum

Komen

Rodgers

2013

The Journal of Immunology

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T cells become polarized during initial interactions with an APC to form an antigen-independent synapse (AIS) composed of membrane rafts, TCR, and TCR-proximal signaling molecules. AISs occur temporally before TCR triggering, but their role in downstream TCR signaling is not understood. Using both human and murine model systems, we studied the signals that activate AIS formation, and the effect of these signals on TCR-dependent responses. We show that CD28 produces AISs detectable by spinning disc confocal microscopy seconds following initial interactions between the T cell and APC. AIS formation by CD28 coincided with co-stimulatory signaling, evidenced by a cholesterol-sensitive activation of the MAP kinase ERK that potentiated Ca2+ signaling in response to CD3 crosslinking. CD45 also enriched in AISs, but to modulate Src kinase activity, since localization of CD45 at the cell interface reduced the activation of proximal Lck. In summary, we show that signaling by CD28 during first encounters between the T cell and APC both sensitizes TCR Ca2+ signaling by an Erk-dependent mechanism, and drives formation of an AIS that modulates the early signaling until TCR triggering occurs. Thus, early Ag-independent encounters are an important window for optimizing T cell responses to antigen by CD28.

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Early and Dynamic Polarization of T Cell Membrane Rafts and Constituents Prior to TCR Stop Signals

Cited by 12 publications

References 46 publications

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

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

MAL Protein Controls Protein Sorting at the Supramolecular Activation Cluster of Human T Lymphocytes

CD28 Sensitizes TCR Ca2+ Signaling during Ag-Independent Polarization of Plasma Membrane Rafts

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