T Cell Microvilli: Finger-Shaped External Structures Linked to the Fate of T Cells

The immunological synapse (IS) is a dynamic structure formed at the contact site between T cells and Ag-presenting cells (APCs) or target cells (1, 2, 3, 4, 5, 6, 7). The IS is a spatiotemporal signaling platform through which T cells are activated to initiate adaptive immunity. The first step in IS formation is the engagement of the TCR with specific Ag peptide-MHC molecules, leading to actin cytoskeletal rearrangement (8). Next, signaling molecules, including TCRs, form microscale clusters and migrate along retrograde actin flow toward central supra-activation clusters (c-SMACs), leading to the maturation of IS. lymphocyte function-associated Ag-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) interactions are critical for IS stabilization, and they are positioned at the peripheral SMAC surrounding the c-SMAC (5, 9). Until recently, when, how, and where TCRs microclusters on the T cell membrane are formed before and during IS formation has remained controversial.

A previous report suggested that the TCR is organized as pre-assembled nanoclusters in the plasma membrane (10). These TCR nanoclusters require cholesterol for stabilization as they are disrupted if cells are exposed to methyl-β-cyclodextrin (MβCD) (11, 12, 13, 14). TCR nanoclusters were visualized with high-speed photo-activated localization microscopy (hsPALM), which has a higher resolution than conventional fluorescence microscopy. The hsPALM analysis showed that the adapter protein linker for T cell activation also forms nanoclusters in resting T cells (15). Following T cell stimulation, TCR nanoclusters may allow cooperativity to develop microclusters. This cooperativity may explain T cells’ exquisite sensitivity and signal amplification in response to Ags (10). Like αβTCR nanoclusters, γδTCR nanoclusters also exist (16). Other multi-chains, such as FcεRIs and B cell receptors, are also found as preclusters in the plasma membrane of mast cells and B cells, respectively (17). However, whether TCR nanoclusters are located in a specific plasma membrane structure is unclear.

A study based on 2 complementary super-resolution microscopy methods demonstrated that TCRs are highly clustered at microvilli tips in resting T cells (18), highlighting these membrane projections as effective sensors for antigenic moieties on APCs or target cells. We corroborated that the TCR ζ chain is localized at microvilli tips (Fig. 1). Pre-clustering of TCRs on microvilli could add an important structural element to the kinetic segregation model of IS formation, which predicts that activated surface receptors segregate into distinct, concentric regions based on the relative sizes of their extracellular domains (19). Consistent with this model, we tracked the centripetal movement of both a microvilli-specific protein (V-set and transmembrane domain containing 5; Vstm5) and TCR clusters with total internal reflection fluorescence microscopy (20). The mechanism of how microvilli on T cells search the surface of opposing cells before and after Ag recognition has also been reported (19).

Figure 1
The TCR localizes to the microvilli tip. Jurkat T cells expressing Vstm5_GFP, a microvilli-specific protein, and TCRζ_tdTomato were imaged at the terminal stage of T cell activation. Reproduced from Kim et al. (20), licensed under Creative Commons (CC BY 4.0).

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Microvilli may serve as a structural scaffold for TCR pre-clustering and to deliver microclusters upon TCR engagement. This could lead to a major shift in the paradigm of IS research. Previously, microclusters were thought to arise from stochastic interactions between freely exchanged membrane proteins (10). Conversely, current evidence suggests that microcluster formation may be limited to areas of the membrane that initiate mutual microvillar contact between T cells and APCs. Moreover, microcluster size may be restricted to the physical dimensions of the microvilli tips. Additionally, this fact may mirror the selective localization of specific molecules involved in the sensing role of the ends of membrane protrusions in dendritic cells (DCs), B cells, and mast cells (21).

Typically, T cells express as many as 20,000 molecules on their membrane surface. [INSERT FIGURE 001]The sizes and dimensions of receptor molecules on the membrane surface can differ vastly. For example, one of the largest membrane glycoproteins is a CD45 with a rigid core of 15.2 nm and a variable mucin-like extracellular domain that extends the height to 10 (CD45R0), 20 (CD45RA), or 40 (CD45RABC) nm. On the contrary, TCR chains of the αβ or γδ type consist of a small extracellular domain. They create a ligand-binding heterodimer (TCRαβ or γδ) that senses or recognizes a peptide engaged with an MHC on the APC. However, how these molecules efficiently participate in sensing external signals to trigger TCR signaling quickly remains unclear. The organization of proteins into distinct membrane compartments such as lipid rafts may facilitate the initial signaling events (22, 23). The surface of T cells is densely covered with finger-like membrane protrusions called microvilli (20). Lipids and lipid domains are important for T cell microvillus morphogenesis (24). Although a comprehensive analysis of lipids in microvilli has not been performed, sphingolipids and phosphatidylinositol 4,5-bisphosphate molecules, which occasionally form dimpling domains in the membrane, may be essential for microvillus formation (25).

Conversely, these outer membrane layers may allow for a more complex 3-dimensional (3D) receptor organization than a flat membrane. A recent super-resolution microscopy study of microvilli revealed that T cell activation receptors, such as TCR, CD2, CD4, and CD28, accumulate exclusively at the microvilli tips (18, 26). Thus, the non-random 3D distribution of receptors is likely important to optimize signaling and cellular responses (24, 26, 27).

Microvilli are composed of cross-linked actin filaments laterally connected by bundling proteins. The most well-characterized are the microvilli in intestinal epithelial cells, which have a constant length and are specialized for nutrient absorption (Fig. 2A) (25, 28). Although T cell microvilli have characteristics of epithelial microvilli, they are also similar to filopodia (27, 29). Filopodia are dynamic structures that vary in length (up to 100 µm) (Fig. 2B) (30, 31, 32). Filopodia play pivotal roles in cellular processes such as cell adhesion to the extracellular matrix, cell migration toward chemokine gradients, wound healing, development, and cell signaling. Key proteins involved in cell adhesion or probing environmental changes localize at the tips of filopodia, mediating their functions (30). These activities of filopodia are similar to those of microvilli in T cells but not in epithelial cells. Microvilli in epithelial cells are strikingly uniform in length, exhibiting only around 5% variation in mean length (up to 1.1 μm) in the mouse small intestine (30).

Figure 2
Morphological comparison of microvilli between epithelial cells and T cells and characteristics of T cell microvilli. (A) Epithelial cell microvilli are constant in length and specialized for nutrient absorption; however, T cell microvilli are irregular and more like filopodia, which extend and shrink intermittently via the alternate assembly and disassembly of their actin filaments. (B) T cells use microvilli to sense Ags on the surface of APCs. Filopodia are actin-rich surface projections specialized for sensing the external environment and mediate cell adhesion and migration.

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T cell microvilli are important structures that determine the external morphology of T cells, but their importance has only recently been recognized. Two lymphocyte receptors, L-selectin and α4β7 integrin, are segregated on the microvillus tip and play critical roles in lymphocyte attachment and rolling under physiologic flow (33, 34), suggesting an important role for extravasation. Nevertheless, the structural and biophysical features of these abundant outer layers imply important functional capabilities. For instance, microvilli tips are a structural scaffold for TCR and functionally important T cell protein clustering, enabling highly effective, rapid, and spatially defined signal processing (35). Moreover, EVs are released through microvillus fragmentation in activated T cells (Fig. 3A), suggesting that microvilli are a source of Evs associated with activated T cells (20, 27). Protrusive membrane architectures can provide sites for producing microvesicles from the cell surface. Primary and motile cilia/flagella, which function as cellular antennae, can produce microvesicles from their tips (36). Unlike the cross-linked F-actin bundles in microvilli, the internal cilium structure is mainly composed of a microtubule-based core architecture, implying that membrane protrusions provide sites for vesicle shedding (27, 36, 37, 38). Furthermore, cilia formation is similar to IS formation, and ciliary microvesicles contain the endosomal sorting complex required for transport (ESCRT) (6).

Figure 3
Generation of TMPs from T cells necessarily requires TCR signaling and adhesion on APCs via integrin activation. (A) Fragmentation of TMPs during and after disconnection from T cells in the kinapse on a lipid bilayer presenting cognate peptide-MHC and ICAM-1 (yellow arrows = fragmented sites; blue arrows = sites yet to be fragmented). Reproduced from Kim et al. (20), licensed under Creative Commons (CC BY 4.0). (B) Schematic illustration of the proposed mechanism of TMP generation. When T cells recognize a cognate Ag presented on APCs via elongated microvilli, T-cell-receptor-induced inside-out activation of integrins, such as LFA-1 and VLA-4, leads to enhanced cell adhesion and spreading on dendritic cells. When the immune synapse terminates and switches back to the kinapse phase, the T cells leave abundant TMPs on the surface of the APCs and surface – anchored large microvilli particles are further fragmented into nano-sized microparticles of approximately 50–100 nm by plasma membrane budding complexes, such as Arrdc1, TSG101, and Vps4.

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EVs are small, nano-sized vesicles surrounded by a lipid membrane that are released from cells into the extracellular space (39). They have been found in the blood, cerebrospinal fluid, bronchial fluid, amniotic fluid, ascites fluid, urine, semen, saliva, tears, bile, gastric acid, milk, and most laboratory cell lines (39, 40, 41, 42). There are 2 types of EVs according to their subcellular origins: exosomes and microvesicles. Exosomes are derived from endosomal compartments. The cell plasma membrane is internalized into the cell in the initial step of the endosomal pathway and is called an early endosome. During this process, intraluminal vesicles are formed by inward budding of the membrane in late endosomes to develop multivesicular bodies (MVBs). These are transformed into lysosomes or auto-phagosomes under specific conditions or are secreted into the extracellular space by exocytosis. Secreted intraluminal vesicles are defined as exosomes. Exosomes range from 30 to 150 nm in size (43, 44, 45). Unlike exosomes, microvesicles are directly derived from the external plasma membrane (40). Microvesicles are initially formed by various factors (physical and enzymatic pathways) and are known as microparticles, microvesicles, ectosomes, and shedding vesicles (46). Compared with exosomes, microvesicle size is heterogeneous, ranging from 30 to 1,000 nm (40, 43, 44, 45, 46).

The molecules in exosomes and microvesicles may be different as they originate from different cellular processes. Tetraspanin (CD63) modulates the formation of exosome and intraluminal vesicles (43, 45, 47, 48, 49). Studies have shown that molecules such as cholesterol, lysobisphosphatidic acid, and epidermal growth factor can determine the fate of MVBs into liposomes or exosomes (40, 45, 50). In contrast to exosome generation, the molecules involved in the generation and release of microvesicles are unclear. Wilson et al. reported that activation of purinergic receptors with ATP stimulates microvesicle release (51). Kuo et al. demonstrated that arrestin domain containing 1 (ARRDC1) is a mediator of microvesicle budding (52). Several reports indicated that the ESCRT machinery regulates microvesicle and exosome secretion (52, 53, 54). Thus, although exosomes and microvesicles are derived from different origins, they share secretion mechanisms in cellular systems.

T cells generate EVs (9, 55, 56, 57, 58, 59). Choudhuri et al. reported that TCR-enriched microvesicles are released in the IS formed between T and B cells (9). Saliba et al. demonstrated synaptic ectosomes enriched with tetraspanins, TCR signaling, and ESCRT proteins (55). Blanchard et al. (59) showed that TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/ζ complex. However, no reports have demonstrated that T cells generate EVs by shedding the outer layer membrane, particularly of microvilli, during T cell interactions with APCs. Moreover, whether TCR activation itself is enough to produce exosomes or microvesicles is unclear. Our recent work suggests that T cell microvilli, which serve as a structural platform to segregate TCR and TCR signaling complexes, are the most easily separable structures during T cell adhesion to APCs. Additionally, since microvilli are enriched with adhesion molecules at their tips, microvilli on the surface of T cells allow them to form multiple bridges with APCs (20, 27). Activation of TCR signaling at the microvilli tips leads to the activation of adjacent adhesion molecules, such as LFA-1 and very late Ag-4 (VLA-4), which may separate microvilli due to increased receptor affinity and avidity (Fig. 3B) (60, 61). Thus, microparticle or microvesicle generation from T cells requires T cell adhesion to the APC matrix. Additional experiments are necessary to examine whether TCR stimulation using soluble CD3/28 Abs can also release EVs.

However, since TMPs are derived from the cellular outer layers, overlapping origins with TCR-enriched microvesicles (9), synaptic ectosomes (55), and exosomes (59) cannot be completely excluded. Therefore, we termed all membrane particles released from activated T cells on adhesion substrates TISs.

T cell-derived EVs, including microvesicles, exosomes, or synaptic ectosomes, interact with other cells to form EV networks and modulate target cell functions (62, 63). Previously, we showed that TISs contain proteins, cytokines, miRNA, lipids, and other functional substances. Indeed, treatment of TISs from CD4⁺ T cells in vitro immediately triggered calcium influx into DCs and induced their maturation and activation by upregulating costimulatory molecules such as CD40, MHCII, CD80, and CD86 (20). Cytokine arrays provided clues to explain the underlying mechanisms of how TISs directly regulate DC functions. We found that various functional cytokines and chemokines, such as IL-33, TNF-α, and CCL5, which regulate DC activation or maturation, are highly enriched in TISs compared to the cell lysate. In particular, IL-33 activates myeloid DCs to produce IL-6, IL-1β, TNF-α, and CCL17 and to express high levels of CD40, CD80, OX40L, and CCR7 (64). Importantly, IL-33-activated DCs prime naive lymphocytes to produce the Th2 cytokines IL-5 and IL-13 (64). Additionally, TNF-α is critical for activating the adaptive immune response to viral infection by the maturation of local DCs (65). CCL5 is a potent chemoattractant for eosinophils, basophils, monocytes, effector memory T cells (CD4⁺/CD45RO⁺), B cells, natural killer cells, and immature DCs (66).

Evidence suggests that the “licensing” of DCs by CD4⁺ T cells is required for cytotoxic T cell responses (67). In particular, IFN-γ produced by CD4⁺ T cells is necessary for classical DC licensing by upregulation of costimulatory molecules such as CD80 and CD86, subsequently inducing the cross-priming of cytotoxic T cells. Interestingly, TISs contain a high amount of IFN-γ and may facilitate DC licensing by sustaining IFN-γ mediated signaling by membrane fusion on the DC surface.

How are TISs transferred and fused to other cells? Proteomic analysis revealed that molecules involved in regulating TCR signaling, including TCR subunits, CD2, or CD28, and in proximal signaling cascades, including Lck, zap70, or Syk, are exclusively enriched in TISs. DC activation by TISs is inhibited by treatment with blocking Abs against CD2, suggesting that TISs can be transferred and fused to the membrane on target cells via CD2–CD58, broadly expressed on APCs including DCs. Indeed, the interaction of T lymphocytes with APCs and target cells is critically dependent on CD2–CD58-mediated adhesion (68).

Many immunotherapeutic treatments have unpredictable or uncontrollable side effects (69, 70, 71, 72, 73, 74, 75, 76, 77). Since TIS is a product naturally produced by activated T cells, unlike current artificial therapeutics, the application of TIS to immunotherapy may be safe with fewer side effects. In a previous report, we demonstrated that TISs directly activate DCs independent of Ags (20), suggesting that TIS could be useful as an immune adjuvant to induce DC recruitment and activation. An important adjuvant mechanism is to induce DC maturation and prolong Ag exposure (78). Various adjuvants have been studied in association with aluminum-based nanoparticles, saponin-based adjuvants, and TLR ligands or pathogen-associated molecular patterns (78). However, most adjuvants strongly trigger adaptive immunity by inducing DC activation but cause side effects such as redness, edema, pain, fever, and chills (46, 78, 79, 80, 81). Since TISs are naturally generated in the early stages of adaptive immunity, TIS could be utilized in cancer vaccinations based on tumor Ag-selective high immunogenicity. Although TISs derived from a patient’s T cells may be the safest immune adjuvant, TISs from allogeneic healthy individuals will not induce immune rejection because exosomes do not induce a severe immune reaction, as shown in mice subjected to repeated administration of allogenic mouse or human cell-derived EVs (82). Despite the potential benefits of TIS application, however, there may still be potential unintended immune responses to auto-Ags carried by TISs. Therefore, it will be important to systematically investigate and understand the various aspects possibly that can occur in the body after TIS treatment.

Among TISs, TMPs are mainly derived from microvilli, so molecules can be engineered to be localized and expressed on the microvilli tips. For example, Vstm5 is specifically distributed throughout the entire stem region of microvilli with little localization in the intracellular region (20). Therefore, engineering fms-like tyrosine kinase 3 ligand (FLT3L) or GM-CSF, known to induce DC proliferation and development, to localize at the microvilli using the Vstm5 transmembrane domain, we can generate FLT3L or GM-CSF-enriched TISs. TISs generated in this way will induce a more robust immune response.

Although the functions of TISs on APCs are somewhat understood, what happens to T cells themselves is largely unknown. This section will address the physiological implications of TIS release in T cells. TCR is thought to be downregulated from the cell surface due to increased TCR internalization, decreased recycling, and increased degradation (83, 84, 85, 86). If T cells are activated by cross-linking TCR by soluble anti-CD3/28 Abs, surface TCR and TCR complex are significantly internalized (86). However, we identified that a large portion of TCR⁺ particles is released from T cells if the cells are stimulated on plate-bound (immobilized) anti-CD3/28 Abs or on planar lipid bilayers presenting cognate peptide-MHC and the recombinant ICAM-1 in a density-dependent manner (87). These findings demonstrate that T cells lose surface TCRs during T cell-APC interactions, but not by internalization.

In molting or shedding in invertebrates, ecdysis occurs when an animal routinely casts off a portion of its outer layer at specific points in its lifecycle, typically to facilitate the growth of the organism. Molting involves releasing the skin, hair, feathers, or other external layers, including the entire exoskeleton in arthropods. However, it remains a mystery whether mammalian cells, including immune cells, also have a molting process related to their growth or proliferation. TIS release from the T cell surface may be similar to molting. Strikingly, this event, in contrast to TCR internalization, leads to the rapid upregulation of surface TCRs and metabolic reprogramming to meet the demands of clonal expansion that drives multiple rounds of division and cell survival (87).

How this program works in T cells can be understood by examining the component molecules in TISs. First, membrane-essential lipids, such as cholesterols, glycerolipids, and fatty acids, indispensable for T cell metabolism (activation and proliferation), are concentrated in TISs. According to our lipidomics analysis, a molting process induces a substantial shift in essential lipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin—4 major phospholipid species present in the membrane. Physiologically, the major loss of essential lipids through the molting process can trigger de novo synthesis of gene clusters for lipid metabolism. Indeed, microarray analysis revealed that gene expression related to fatty acid synthetic metabolism—a critical pathway for cell survival and proliferation—is dramatically increased during T cell surface molting.

Second, TISs contain significant amounts of mitochondrial proteins. Although mitochondrial proteins are considered preferentially localized in mitochondria, plasma membrane lipid rafts are also enriched for mitochondrial oxidative phosphorylation (OXPHOS) proteins, a major source of ATP production for cell survival (88). Thus, the loss of energy-related mitochondrial proteins may trigger de novo synthesis of genes related to the OXPHOS pathway to compensate for and control homeostasis. T cells may release molecules essential for Ag recognition (TCR and its complex), energy metabolism (glycolysis, the TCA cycle, and ATP-binding proteins), and membrane components (fatty acids and cholesterols), which paradoxically promote T cell proliferation for clonal expansion.

Microvilli are a current research hotspot. Studies in recent years have shown that microvilli on T cells act as sensory organelles to survey APCs (19). Our group recently reported that microvilli not only provide a structural platform for TCR clustering to initiate TCR-mediated signaling but are also a source of nano-sized EVs, called TISs, delivering cytosolic proteins, lipids, and nucleic acids, which are potentially functional, to neighboring cells (20). Furthermore, these drastically change T cells after breaking off large portions of the cell membrane. Two recent findings challenge the long-held paradigm in T cell biology. First, immunologists have believed that TCR internalization is a major mechanism of surface TCR disappearance after T cell activation. Therefore, many studies have examined the internalization process of TCR and the TCR complex. However, our data suggests that large numbers of TCRs and membrane proteins are released as TMPs or synaptic ectosomes. Indeed, sufficient data support that the major cause of surface TCR loss is TCR⁺ TIS release, not TCR internalization. Second, TCR⁺ TIS release may have an important impact on T cell proliferation. If the TCR signal is activated, T cells can generate TISs even when they are not explicitly adhered; since T cells already acquired specificity from the peptide MHC (pMHC)-TCR signal, we believe another specificity signal may not be required in the molting process. If the pMHC-TCR signal is correct, molting occurs naturally by the adhesion process, which is thought to play a decisive role in the proliferation of T cells.

The transfer of TISs from T cells to the DCs induces a robust immune response by priming interacting DCs, which recruit polyclonal CD8⁺ T cells, natural killer cells, and other immune cells. Thus, TISs are a good candidate for cancer and antiviral vaccines that naturally boost immune functions. Moreover, as a delivery system for exogenous substances, TISs have an advantage compared with artificial vesicles such as liposomes, microspheres, micro-emulsions, and other synthetic drug-delivering systems. Since TISs are mainly derived from cell membrane microvilli, TISs may be useful as a carrier capable of inducing a more robust immune response by microvilli-specific engineering. Thus, TIS could be a promising tool as a novel, biologically compatible nano-based platform. Hence, additional systematic in vivo studies will provide potential clues regarding the potency and broad application of TISs a step closer to clinical usage.