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Review
. 2024 Jan 3:14:1324557.
doi: 10.3389/fimmu.2023.1324557. eCollection 2023.

Extracellular vesicles and microvilli in the immune synapse

Javier Ruiz-Navarro  1 , Víctor Calvo  2 , Manuel Izquierdo  1
Affiliations
Review

Extracellular vesicles and microvilli in the immune synapse

Javier Ruiz-Navarro et al. Front Immunol. .

Abstract

T cell receptor (TCR) binding to cognate antigen on the plasma membrane of an antigen-presenting cell (APC) triggers the immune synapse (IS) formation. The IS constitutes a dedicated contact region between different cells that comprises a signaling platform where several cues evoked by TCR and accessory molecules are integrated, ultimately leading to an effective TCR signal transmission that guarantees intercellular message communication. This eventually leads to T lymphocyte activation and the efficient execution of different T lymphocyte effector tasks, including cytotoxicity and subsequent target cell death. Recent evidence demonstrates that the transmission of information between immune cells forming synapses is produced, to a significant extent, by the generation and secretion of distinct extracellular vesicles (EV) from both the effector T lymphocyte and the APC. These EV carry biologically active molecules that transfer cues among immune cells leading to a broad range of biological responses in the recipient cells. Included among these bioactive molecules are regulatory miRNAs, pro-apoptotic molecules implicated in target cell apoptosis, or molecules triggering cell activation. In this study we deal with the different EV classes detected at the IS, placing emphasis on the most recent findings on microvilli/lamellipodium-produced EV. The signals leading to polarized secretion of EV at the synaptic cleft will be discussed, showing that the IS architecture fulfills a fundamental task during this route.

Keywords: FMNL1β; T lymphocytes; actin cytoskeleton; extracellular vesicles; immune synapse; microvilli; multivesicular bodies; protein kinase C δ.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Immune synapse, microvilli and F-actin depletion at the cIS. C3 (control) and P5 (PKCδ-interfered) Jurkat clones were challenged with Cell tracker blue (CMAC)-labelled Raji cells (blue) pulsed with Staphylococcal enterotoxin (SEE) to induce IS formation. After 1 h of conjugate formation, fixed cells were labelled with phalloidin (green) and anti-CD63 (magenta) to label F-actin and MVB, respectively. Upper panels: Maximal Intensity Projections (MIP) of merged CMAC, anti-CD63 and phalloidin channels. Lower panels: enlarged XZ plane area of the IS interface area (1.5x and 2.5x zoom for C3 and P5, respectively, from the white rectangles shown in the upper planes) of the phalloidin channel, generated as shown in Supplementary Video 2 . White arrows indicate the direction to visualize the IS interface views enclosed by the regions of interest (ROIs, white rectangles). Yellow arrows label some microvilli emanating from Jurkat T lymphocytes. The area of the F-actin-low region at the cIS (Fact-low cIS area) (yellow line) and the IS (IS area) (white line) were defined and measured as indicated in (4), and the relative area of the F-actin-low region at cIS (Fact-low cIS area/IS area) was calculated and represented. As seen in the figure, PKCδ is involved in the clearing of F-actin at the cIS, since its depletion impedes the creation of a cortical actin hypodense region at the cIS, which facilitates MVB polarization and secretion (4). This figure is related to Supplementary Video 2 .
Figure 2
Figure 2
T lymphocyte cortical actin cytoskeleton reorganization and centrosome polarization. After the initial scanning contact of TCR with pMHC on the APC ( Supplementary Video 1 ), both naive and effector T lymphocytes form mature IS with APC. Th IS lasts many hours during which de novo cytokine (i.e. IL-2, IFN-γ) production and secretion occur, that require continuous TCR signaling. Primed effector CTL establish more transient, mature IS after scanning the target cells (i.e. a virus-infected cell), and secrete cyto lytic granules (CG) within a few minutes. Secretory vesicles (CG in CTL, cytokine-containing vesicles in Th cells, and MVB in both T lymphocyte classes) are rapidly transported (several minutes for effector Th cells and very few minutes or seconds for effector CTL) towards the centrosome (in the minus “-” direction) and, almost simultaneously, the centrosome polarizes towards the cSMAC of the IS, a F-actin poor area that constitutes the secretory domain at the cIS (13). Centrosome translocation to the IS appears to be dependent on DAG production at the IS (14) and DAG-activated PKCθ controlling dynein anchored to adhesion and degranulation adapter protein (ADAP) (–17) at the pSMAC, that pulls centrosome in the minus direction. In addition, it has been shown that DAG-activated PKCδ controls FMNL1β phosphorylation (18), F-actin clearing at cSMAC and subsequent MVB and centrosome polarization (4). In synapses made by both effector CTL and Th, initial F-actin positive reorganization in the cell-to-cell contact area, and later F-actin decrease at the cSMAC (19) and F-actin accumulation at the dSMAC (20, 21) appear to be involved in centrosome and vesicle polarization and secretion.
Figure 3
Figure 3
Immune synapse, microvilli and EV. (A) Schematic picture illustrating the secretion of different EV, SMAPs and cytotoxic molecules by an effector CTL at the synaptic cleft. A mature IS formed by a CTL and an APC is represented. The different EV (blue characters) and some relevant molecules that can be found at the synaptic cleft are depicted. In addition, canonical CG or single core granules involved in the secretion of soluble perforin and granzyme B at the IS (11, 55, 56) are represented. Depletion of F-actin at the cIS and accumulation at the dSMAC occur, that are concomitant to MVB/centrosome polarization towards the IS and subsequent MVB and CG degranulation, which ultimately leads to exosome (4, 18, 57, 58), perforin/granzyme (11) and SMAP (56, 59) focused secretion. More recently, a new role of centrosomal F-actin on centrosome polarization has been established both in Jurkat cells and primary, effector CD4+ T lymphocytes forming synapses with superantigen-loaded Raji cells (18, 36). In addition, ectocytosis of shedding vesicles (ectosomes), via TSG101 and Vps4 (60, 61), and generation of TMPs from microvilli may occur (62, 63). (??) symbol means that is unclear whether TCR on endocytosed TMP may trigger APC activation via pMHC stimulation (62). All these secretion events are represented together to save space in the figure, although is not clear whether these events may simultaneously occur at the same IS. (B) Schematic representation of the trogocytosis mechanism and TMP production carried out by an APC acting upon a T lymphocyte. The figure represents three sequential stages of TMP formation via trogocytosis at the IS, namely initiation, processing and scission, as well as the distribution of some relevant T lymphocyte signaling molecules along microvilli (3, 62, 64). TCR, and proteins involved in early T lymphocyte signaling (i.e. Lck, LAT), accumulate in microvilli tips (53, 64, 65) prior to IS formation (66) whereas phosphoprotein phosphatase CD45 is excluded from microvilli tips (27). Molecules relevant for trogocytic TMP scission (Vps4, Arrdc1) remain accumulated into TMPs (64).
Figure 4
Figure 4
Role of FMNL1β in secretory polarized traffic and EV release at the IS. Schematic diagram depicting different steps in the polarization of MVB and exosome secretion in the Jurkat/SEE-Raji IS model. 1) IS formation is marked by an initial increase in cortical actin at the IS, where FMNL1β translocates and accumulates through a PKCδ-independent process. 2) FMNL1β undergoes a PKCδ-mediated phosphorylation at S1086, which reverses FMNL1β autoinhibition mediated by interaction of N-terminal Diaphanous inhibitory domain (DID) (yellow) with the C-terminal Diaphanous autoinhibitory domain (DAD) (green) and this may activate this formin at the IS. 3) Once phosphorylated at S1086, FMNL1β appears to govern the cortical actin reorganization process, creating a hypodense region at the cIS and the subsequent centrosome/MVB polarization to the IS. 4) These coordinated processes are crucial to enable a functional exosome release by T lymphocytes at the synaptic cleft (4, 18, 79).
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants from the Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (Grant PID2020-114148RB-I00 from the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033) and grant P2022/BMD-7225 funded by Consortia in Biomedicine of Comunidad de Madrid to MI.

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