Role of Extracellular Vesicles in Shaping Immune Responses in the Lymph Node Microenvironment

ABSTRACT

Extracellular vesicles (EV) secreted by living cells are important players in cell‐to‐cell communication, particularly in the diffuse immune system, where they contribute to local and systemic coordination. Indeed, EV are in the ideal size range for diffusion in interstitial and lymphatic fluids, which is the main route of tissue EV trafficking towards secondary lymphoid organs that orchestrate immune responses. In the lymph nodes, EV interact with multiple immune and stromal cell populations sensitive to antigens and co‐delivered tissue messages via degradative and non‐degradative pathways. Natural conveyers of bioactive compounds inherited from their mother cells and their surroundings, EV modulate innate and adaptive immune responses in health and disease. The aim of this scoping review is to aggregate information from the literature on cellular effectors and molecular pathways of EV‐mediated immune responses in the lymph node environment and to gain insight into practices using EV for immune intervention .

Functional alliances of stromal, innate and adaptive immune cells in EV immunity in the lymph node. EV reflect the phenotype of their mother cell and cluster material from their surroundings. In the lymph node, EV preserve valuable context information between multiple cellular effectors to coordinate immunity in adaption to change.

1. Extracellular Vesicles (EV) in Immune Surveillance

The new perception of the body as a holobiont, harbouring complex microbiota and viromes in addition to its own cells, has profoundly changed our understanding of the immune system. Beyond its defensive action against pathogens, the immune system continuously monitors the maintenance of a physiological balance, distinguishing between the ‘good’ and ‘bad’ agents that the body must tolerate or eliminate, respectively. Earlier models grounded on self/non‐self‐discrimination have been replaced by more evolutionary theories based on ‘danger’ or ‘discontinuity’, which can arise from within or outside the organism throughout its lifetime (Pradeu 2018). Congruently, signals from the tissue dictate immune decision‐making, rather than a specific antigen itself. By draining interstitial fluids, the lymph becomes enriched with compounds processed extracellularly. EV are membrane‐bound particles into the extracellular space released by living cells. The current nomenclature distinguishes three major subtypes of EV: (i) exosomes (50–150 nm), which are formed as intraluminal vesicles inside late endosomes and released upon fusion of the latter to the plasma membrane; (ii) ectosomes (50–1000 nm), which result from plasma membrane protrusion and scission; and (iii) apoptotic bodies (100–5,000 nm), which are generated by cell fragmentation during apoptosis. As the three subtypes are of overlapping size, and biogenesis pathways are difficult to establish, the generic term EV will be used throughout the manuscript in the application of the current Minimal Information for Studies of EV guidelines (van Niel et al. 2018; Welsh et al. 2024). EV offer unique properties as immune sentinels. As their composition reflects the phenotype of their mother cell and their surface clusters ligands and soluble material from their surroundings, EV continuously provide a flow of information on peripheral tissue function, metabolism, extracellular matrix degradation, stress, senescence, and infection (reviewed in Buzas 2023).

Adaptive immune responses result from the encounter of multiple actors in highly organised secondary lymphoid organs, that is the spleen and lymph nodes, which filter blood and lymph, respectively (Qi et al. 2014). Built to support high pressure, endothelial cells of the blood vascular tree form tight 10 nm junctions; in contrast, in lymphatic capillaries, loosely connected, overlapping flap‐like lymphatic endothelial cells (LEC), form large openings, suited for particle absorption (Schudel et al. 2019). In addition to paracellular transport, the transcellular transport of vesicles across the lymphatic endothelium under flow conditions has been described (Triacca et al. 2017). Similar to viruses, with whom they share overlapping biogenesis pathways, physical structures, and functions, their reduced size allows EV to easily enter and travel through lymphatic vessels and access secondary lymphoid organs within minutes, against 8–12 h required by their cellular counterparts (Figure 1) (Byersdorfer and Chaplin 2001; Mempel et al. 2004; Oussoren et al. 1997; Srinivasan et al. 2016). In vivo tracking of exogenous and endogenous EV suggests that lymphatics, rather than blood vessels, are the main route of dissemination of EV secreted into the interstitial space (Broggi et al. 2019; Ekström et al. 2022; Pucci et al. 2016; Sakurai et al. 2023; Wang et al. 2025; Zeng et al. 2021). For example, intramuscular DNA vaccination triggers local EV secretion and lymph node drainage within 4 h, priming subsequent adaptive immune responses (Wang et al. 2025).

FIGURE 1.

Draining and retention of EV in the lymph node. EV recombinant for the green fluorescence protein (GFP) (6 × 10⁹ part/mouse, EV‐GFP, SIGMA) were administered by intradermal injection to C57BL/6 mice. Two hours later, draining and distant lymph nodes were sampled, transparised, and stained with VioG570 anti‐CD31 (lymphatic and blood vessels) and VioR667 anti‐GFP antibodies. 3D images were acquired by light sheet microscopy (UltraMicroscope Blaze, Miltenyi Biotec) and analysed using Imaris software (version 10.1, Oxford Instruments). Scale bar: 200 µm. GFP‐positive spots automatically detected in the lymph node using the Imaris Spot Detection module are represented by magenta spheres of arbitrary size, based on their size and intensity.

In the steady state, the lymph node constitutes a home of peripheral tolerance (Mueller 2010). Migratory dendritic cells (DC), lymph node resident DC, and stromal cells actively promote tolerance to self or innocuous antigens by clonal deletion or anergy of T lymphocytes and induction of regulatory T lymphocytes (Treg) (Bonifaz et al. 2002; Cohen et al. 2010; Fletcher et al. 2010; Lee et al. 2007; Wilson et al. 2003). EV have been shown to interact with various populations of immune and stromal cells in the lymph node through surface and intracellular signalling via the endosomal pathway (García‐Silva et al. 2021; Hood et al. 2011; Leary et al. 2022; Nambiar et al. 2024; Pucci et al. 2016; Saunderson et al. 2014; Sun et al. 2019). Besides the well‐documented roles of stem and immune regulatory cell‐derived EV and the efferocytosis of EV from dying cells in immune tolerance, knowledge on the contributions of small EV from tissue cells remains in its infancy (Ge et al. 2024; Poon et al. 2019; Prieto‐Vila et al. 2024). The purpose of this review is to summarise the current evidence on pathways exploited by EV in the lymph node microenvironment in health and disease and to provide an outlook on the use of EV for systemic immune intervention.

2. Nanoparticle Retention in the Lymph Node

In the lymph node (Figure 2), the afferent lymph flows through the subcapsular and transverse sinuses before merging into the medullary sinus and efferent lymphatic vessel. Blood cells enter the lymph node through the high endothelial venules that vascularise the organ. Between these borders, the lymphoid pulp is composed of a mesh of stromal cells that supports the lymph node architecture and creates niches filled with B and T lymphocytes in the cortex and paracortex, respectively (Gray and Cyster 2012). This intriguing spatial organisation functions as a molecular sieve (Roozendaal et al. 2009). Small solutes (≤ 70 kDa; hydrodynamic radius ≤ 4 nm), like cytokine/chemokine signalling molecules, can enter directly the tubular conduit network formed by fibroblastic reticular cells (FRC) for rapid delivery towards perivenular regions where they regulate blood cell attraction (Roozendaal et al. 2008, Roozendaal et al. 2009; Y.‐N. Zhang et al. 2019). This allows for the instant diffusion of alarm signals to mount an immune response. In addition, conduit‐associated macrophages and DC may capture and process small soluble antigens (Sixt et al. 2005). In contrast, the sampling of larger nano‐ and microparticles relies on specialised macrophages and LEC that line the sinuses and lymphatic vessels. These cells transfer particles to lymph node‐resident DC or marginal zone B (MZB) cells for the subsequent presentation of antigens to B and T lymphocytes gathering in germinal follicles and T cell zones, respectively (Chen et al. 2025). The use of synthetic lipid formulations and gold/magnetic nanoparticles showed prolonged lymph node retention for particles in the size range of EV and of non‐vesicular material like exomeres and supermeres (Hu et al. 2015; Zhang et al. 2018; Zhang et al. 2021), whilst small particles (≤ 15 nm) are not retained (Rohner and Thomas 2016; Y.‐N. Zhang et al. 2019). Collectively, this evidence aligns with the assumption that the lymph node structure supports distinct and probably complementary actions of soluble and particulate mediators.

FIGURE 2.

Functional alliances in EV‐mediated immune responses in the lymph node microenvironment. Lymph node anatomy and enlarged views of (1) EV in the afferent lymph with cargo and elements of the surface corona, and (2–6) interaction with cellular effectors. (2) In the subcapsular sinus, EV can be captured by (2a,b) SCS macrophages through sialylated surface glycans that bind to CD169 macrophage receptors, enabling relay to MZB cells in the lymph node cortex. If present, DAMP/PAMP co‐delivered with EV may bind to innate immune receptors, inducing macrophage activation. In addition, (2c) LEC lining lymph node sinuses expose VCAM‐1 and CD151 cell adhesion molecules that interact with extravesicular integrins, leading to EV uptake and processing. LEC present EV antigens as well as inhibitory immune signals, impairing effector T cell responses. (3) EV draining through the medullary sinus can be scavenged by MS macrophages bearing TIM‐4 surface receptors that bind to PtdSer. EV internalisation by MS macrophages may trigger IL‐33 cytokine release, promoting immune tolerance in tumour settings. (4) In the lymph node cortex, FRC form tubular conduits (in dark blue) tunnelling small solutes (< 70 kDa) for rapid diffusion. In response to EV, distinct populations of FRC in the T and B cell zones (in brown) increase the expression of anti‐inflammatory mediators, for example, PD‐L1 and TGF‐β blocking T cell responses. (5) In lymph node germinal centres, non‐degradative EV retention and surface exposure on FDC contributes to high affinity BCR selection during precursor B cell differentiation into antibody‐secreting plasma and memory B cells. (6) Depending on their cargo and co‐delivered tissue cues, EV may either promote inflammatory B and T effector cells responses or the development of immune regulatory cells in parallel to the inhibition of inflammatory cells. Data on the precise contribution of intermediate stromal or innate immune effectors is still lacking. Abbreviations: BCR, B cell receptor; Breg, regulatory B cell; DAMP, danger‐associated molecular pattern; FDC, follicular dendritic cell; FRC, fibroblastic reticular cell; HEV, high endothelial venules; ITGA/ITGB, integrins α/β; LEC, lymphatic endothelial cell; MHC, major histocompatibility complex; MS, medullary sinus; MZB, marginal zone B cell; PAMP, pathogen‐associated molecular pattern; PD‐1, programmed death 1; PD‐L1, programmed death ligand 1; PGE2, prostaglandin E2; PtdSer, phosphatidylserine; SCS, subcapsular sinus; TCR, T cell receptor; TGF‐β, transforming growth factor β; Th17, T helper 17; Tim‐4, T‐cell immunoglobulin and mucin domain‐containing protein 4; TLR, Toll‐like receptor; tolDC, tolerogenic dendritic cell; Treg, regulatory T cell; VCAM‐1, vascular cell adhesion molecule. Image created with https://BioRender.com.

Findings on tumour EV have demonstrated that the natural homing of EV to the lymph nodes is lost upon proteinase K pre‐treatment, suggesting that surface protein expression enhances the accumulation of EV in the lymph nodes (Charoenviriyakul et al. 2018; Sakurai et al. 2023). In addition, depending on the nature, status, and environment of the cell of origin, biologically active molecules—such as cytokines, immunoglobulins, integrins, proteoglycans, proteases, glycosidases, and angiogenic proteins—travel attached to the surface or encapsulated inside EV (Figure 2‐1), further expanding their biodistribution, stability, and signalling capacity (Cvjetkovic et al. 2016; Fitzgerald et al. 2018; Hakulinen et al. 2008; Ko et al. 2019; Purushothaman et al. 2016; Skog et al. 2008; Tóth et al. 2021; Wolf et al. 2022). EV exposure to biofluids further conditions the composition of the protein corona, the removal of which may impair EV activity, as shown by the abrogation of the angiogenic effects induced by placental stem cell EV (Wolf et al. 2022).

3. Partners of EV Activity in the Lymph Node Environment

Within the lymph nodes, EV from afferent lymph may first engage with cells at the interface of the subcapsular and medullary sinuses (Figure 2‐2–6). Consistent with their well‐documented roles in viral clearance, several studies have identified macrophages in the subcapsular and medullary sinuses of the lymph node as the primary targets of EV. Subsequent EV relay to secondary effectors, namely B lymphocytes and FRC in the lymph node pulp, contributes to shaping innate and adaptive immune responses. Four studies further described strong EV interactions with LEC, supporting the survival and/or recruitment of resident and migratory immune cells. However, the contributions of other cells may be masked by the technically challenging isolation of rare lymph node‐resident stromal and immune cell populations required for study (Broggi et al. 2019; García‐Silva et al. 2021; Leary et al. 2022; Lin et al. 2024). In the following sections, we describe the direct and indirect encounters of EV with specific cell populations in the lymph node, as well as the EV components involved and the signalling pathways activated in downstream target cells (Figure 2) (Table 1).

TABLE 1.

Cellular and molecular effectors of EV‐driven immune responses in the lymph node. Data from the literature on EV components and molecular pathways activated in target cells. For human SCS and MS macrophages, distinction relies on anatomical rather than marker‐based immune detection.

Cell type	Human markers	Mouse markers	EV‐related function	EV components	Pathways	References
Macrophages
SCS macrophages	CD169+ CD163+ CD204+	CD169+ F4/80−	EV relay, DAMP/PAMP signalling	α2,3‐Linked sialic acid glycoconjugates	Sialoadhesin binding Innate immune surface receptors	(Black et al. 2016; Card et al. 2014; Pucci et al. 2016; Saunderson et al. 2014; Srinivasan et al. 2017)
MS macrophages	CD169+ CD163+ CD204+	CD169+ F4/80+	EV scavenging	Phosphatidyl‐serine	TIM‐4/IL33 signalling, Treg activation Degradative uptake?	(Lamorte et al. 2023) (Leary et al. 2022)
LEC	Prox1+ CD31+ Lyve1+/−	CD31+ PDPN+ Lyve1+ Prox1+ PD‐L1+	EV uptake/cross‐dressing Antigen‐presentation CD8+ T‐cell deletion/exhaustion DAMP/PAMP signalling	ITGA4/ITGB1 and ITGA6/ITGB4 Antigens MicroRNA‐142, microRNA‐1468	E‐selectin ↑ Msr‐1↑ bst2 ↑ Jak2/Stat3 activation (IDO↑ PD‐L1↑ TGFβ ↑) Innate immune receptors	(Hirosue et al. 2014; Leary et al. 2022; Lin et al. 2024; Zhou et al. 2021; Zhou, Zhang, et al. 2021)
FRC	CD45− CD31− CD90+ PD‐L1+	PDPN+ CD31− ER‐TR7+	Treg induction T effector inhibition	IFNGR1‐P	PD‐L1↑ Jak/Stat1 TGFβ↑ signalling MHC II antigen presentation	(Chen et al. 2025; Denzer et al. 2000; Han et al. 2023; Karasu et al. 2018; Pasztoi et al. 2017)
FDC	CR1+ CR2+ VCAM‐1+ MFGE8+ CXCL‐13+	CR1+ CR2+ VCAM‐1+ MFGE8+ CXCL‐13+ −	(Non) degradative EV recycling, MHC II antigen presentation, B cell activation/maturation Effector T‐cell inhibition	Integrins Complement Immunoglobulins
B cells	CD19+ CD20+ (panB)	CD19+ CD20+ (panB)				(Chen et al. 2025; Karasu et al. 2018; Kumar et al. 2021)
MZB			Extracellular vesicle shuttling	IgM opsonisation Antigen Integrins	Complement‐dependent/independent capture, cell adhesion
Germinal centre B cells			B cell activation & maturation
Breg	CD19+ IL10+	CD19+ IL10+	Breg ↑ Th2 shift			(Cosenza et al. 2018)
T cells	CD3 (panT marker)	CD3 (panT marker)
CD8+ T cells	CD8+	CD8+	CD8+ T cell inhibition	CD155, CD276	Negative co‐stimulation	(Guetter et al. 2025)
Treg	CD25+ Foxp3+	CD25+ Foxp3+	Treg induction		PgE2↑ CDK8↓ FoxP3↑ Th17 inhibition TGFβ	(Liu et al. 2025) (Li et al. 2025) (Ou et al. 2025) (Burlingham et al. 2023)
T and B memory cells			Antigen restimulation	Antigen

Abbreviations: Breg, regulatory B cell; Bst‐2, bone marrow stromal antigen 2; CDK8, cyclin‐dependent kinase 8; CR1/ CR2, complement receptors 1/2; CXCL‐13, C‐X‐C motif chemokine ligand 13; DAMP, danger‐associated molecular pattern; FDC, follicular dendritic cell; FoxP3, forkhead‐box P3; FRC, fibroblastic reticular cell; IDO, indoleamine 2,3‐dioxygenase; IFNGR1‐P, interferon gamma receptor 1 phosphorylated; IgM, immunoglobulin M; IL‐33; interleukin 33; ITGA/ITGB, integrins α/β; JAK, Janus kinase; LEC, lymphatic endothelial cell; LYVE‐1, lymphatic vessel endothelial receptor 1; MFGE8, milk fat globule‐EGF and factor V/VIII domain‐containing protein 8; MHC, major histocompatibility complex; MS, medullary sinus; MSR1, macrophage scavenger receptor 1 (CD204); MZB, marginal zone B cell; PAMP, pathogen‐associated molecular pattern; PD‐L1, programmed death ligand 1; PDPN, podoplanin; PGE2, prostaglandin E2; PROX1, prospero homeobox protein 1; SCS, subcapsular sinus; STAT, signal transducer and activator of transcription; TGF‐β, transforming growth factor β; Th2, T helper 2; Th17, T helper 17; Tim‐4, T‐cell immunoglobulin and mucin domain‐containing protein 4; Treg, regulatory T cell; VCAM‐1, vascular cell adhesion molecule.

3.1. Subcapsular Sinus (SCS) Macrophages

Lymph node‐resident macrophages located on top, in between, and below sinus‐lining cells have been shown to pass lymph‐borne viral particles to antigen‐presenting cells, including B lymphocytes, without leaving their position (Junt et al. 2007; Phan et al. 2009; Winkelmann et al. 2014). This unidirectional ‘flypaper’ function relies on macrophage processes that protrude into the lymph node sinus, preventing systemic pathogen spread (Figure 2‐2a). Similarly, over the past decade, strong evidence has emerged indicating that EV are neither retained nor internalised by CD169⁺ F4/80⁻ SCS macrophages, which are endowed with low phagocytic and degradative capacities (Phan et al. 2009; reviewed in Gray and Cyster 2012). Instead, SCS macrophages have been described to transfer endogenous melanoma‐derived tumour EV to B cells or follicular dendritic cells (FDC). This barrier function is supported by experiments using clodronate liposomes for transient macrophage depletion, which led to the spread of EV into the lymph node cortex (Pucci et al. 2016). The adhesion receptor CD169 (also known as sialoadhesin, SIGLEC1) on the macrophage surface assists in this function by binding to sialylated molecules on syngeneic B cell EV membrane surfaces (Saunderson et al. 2014). Likewise, a study on small EV from syngeneic mouse EL4 lymphoma apoptotic cells reported co‐localisation with CD169⁺ macrophages in the marginal zone of the spleen, suggesting that CD169 is a generic EV receptor in secondary lymphoid organs (Black et al. 2016) (Figure 2‐2b).

Intradermal or subcutaneous administration of fluorescently labelled EV engenders strong co‐staining of EV with macrophage markers at early time points (2–3 h), followed by delayed co‐staining with CD19⁺ B lymphocytes (Sakurai et al. 2023; Saunderson et al. 2014; Shimoda et al. 2017; Srinivasan et al. 2016). Elegant studies using intravital two‐photon microscopy on endogenous EV produced by tumours, skin, or cardiac allografts revealed early detection of EV antigens at SCS macrophage locations and subsequent trans‐display to cognate and non‐cognate B cells in the underlying B cell follicles, initiating their maturation (Pucci et al. 2016; Zeng et al. 2021). Non‐cognate mechanisms are less proficient in EV B cell association, though membrane α2,6‐sialoconjugates may also interact with CD22 on B cells, dampening B cell activation (Lanoue et al. 2002). Observations made on allogeneic and xenogeneic transplant‐derived vesicles suggest that MZB cells capture the EV in an immunoglobulin M and complement receptor‐dependent manner (CR1/2) during non‐cognate transfer (Chen et al. 2025; Kumar et al. 2021). These findings were further corroborated by the enhanced spleen and lymph node B cell targeting of DC EV coated with a complement receptor 2 binding domain following intravenous injection (Offens et al. 2025). Although SCS macrophage/B cell synapses are the most documented mechanism of EV relay to the lymph node cortex, a recent study showed that SCS macrophages also shed CD169⁺ EV that associate with particulate antigens and accumulate on light‐zone FDC, where high‐affinity B lymphocyte selection occurs (Chen et al. 2022).

In the absence of uptake, the extent to which lymph node SCS macrophages remain neutral during EV transfer or instruction of recipient immune cells remains an open question. CD169 ligation does not trigger canonical outside‐in signalling cascades in macrophages. However, the capture of pathogen‐ and/or danger‐associated molecular patterns stimulates surface innate and co‐stimulatory receptor‐driven cytokine secretion by SCS‐macrophages, particularly interferon type I secretion, which is conducive to the downstream priming of both innate and adaptive immune cells. Likewise, MZB cells express Toll‐like receptor 4, allowing the rapid release of interleukin 6 (IL‐6) while awaiting germinal centre B cell maturation (Palm et al. 2016). Intradermal administration of EV derived from Toll‐like receptor‐3/4 ligand‐stimulated Hey human ovarian cancer cells enhances nodal retention and primes the activation of IL‐12 and nitric oxide synthase 2 inflammatory macrophage responses (Srinivasan et al. 2017). The use of CD169‐deficient mice has led to conflicting evidence of either boosted or inhibited adaptive immune responses following SCS macrophage capture of tumour or graft‐derived EV in vivo (Black et al. 2016; Gonzalez‐Nolasco et al. 2025; Saunderson et al. 2014; Zeng et al. 2021). Growth and hypoxia‐inducible factors induced by syngeneic mouse B16‐F10 tumour EV cargo have been suggested to promote lymph node macrophage polarisation from pro‐inflammatory to anti‐inflammatory states during tumour development (Hood et al. 2011). In transplantation settings, tissue injury engenders extracellular ATP release, a potent danger signal that directs donor EV release and composition, as well as allo‐responses (Gonzalez‐Nolasco et al. 2025).

In summary, strong evidence supports a role of SCS macrophages in EV capture and relay to B cell zones. Co‐delivery of complement and danger‐ or pathogen‐associated molecular patterns with EV further initiates immune responses. In‐depth exploration of phenotypic changes in SCS macrophages induced by EV and EV‐associated components derived under steady and inflammatory conditions is required to elucidate their contributions to the outcome of EV‐mediated immune responses.

3.2. LEC

3.2.1. LEC Function Relevant to Immune Regulation

LEC are flat cells lining all lymph node sinuses and ensheathing the meshwork of macrophages and vessels running through the cortex and paracortex at the interface with the lymph node pulp (Figure 2‐2a, 2c). LEC can be identified by the expression of platelet endothelial cell adhesion molecule‐1 (CD31), podoplanin (38 kDa glycoprotein), vascular endothelial growth factor receptor‐3, and prospero homeobox transcription factor 1 (Wilting et al. 2002). LEC function as stationary immune cells, with extensive roles in immune cell trafficking, survival, and regulation. Notably, they guide immune cell transmigration through the expression of lectin‐like adhesion molecules and the constitutive secretion of chemoattractants, such as chemokine C–C motif ligand (CCL) ‐21 and CCL‐19, and the pleiotropic cytokine IL‐7 (Byrum et al. 2026; Xiang et al. 2020). CCL‐21 binds to C–C chemokine receptor 7 on migratory DC, macrophage subpopulations, and B and T lymphocytes (Förster et al. 2008; Gunn et al. 1999). CCL‐21 enhances the binding of lymphocyte function‐associated antigen 1 to intracellular adhesion molecule 1 (ICAM1), which is expressed on LEC and blood endothelial cells, thereby promoting leukocyte transmigration (Eich et al. 2011; Gunn et al. 1998). Conversely, LEC produce sphingosine‐1‐phosphate, regulating lymph node egress (Pham et al. 2010). LEC are also the main source of colony‐stimulating factor 1 and express the receptor activator of nuclear factor kappa‐B, which supports SCS macrophage survival and positioning in a functional alliance (Camara et al. 2019; Mondor et al. 2019). Antigen expression in LEC has been shown to promote CD4⁺ regulatory T cell expansion and cognate CD8⁺ T cell deletion with the involvement of programmed death ligand 1 (PD‐L1) and lymphocyte‐activation gene 3 immune checkpoint pathways. In addition, LEC may transfer endogenous cytosolic or membrane antigens to DC, thereby mediating subsequent CD4⁺ T cell anergy (Gkountidi et al. 2021; Rouhani et al. 2015).

3.2.2. Evidence for EV Binding, Uptake, and Transcytosis by LEC

Murine tumour EV cross lymphatic endothelial barriers via transcellular and paracellular routes (Sakurai et al. 2023; Srinivasan et al. 2016) and dynamin‐dependent vesicular transcytosis of large biomolecules allows rapid delivery to cells in the lymph node pulp (reviewed in (Takeda et al. 2023)). In contrast to SCS macrophages, LEC efficiently internalise tumour integrin (ITG) α positive EV (Leary et al. 2022; Lin et al. 2024). Experiments using LEC lineage‐specific knockout models or neutralising antibodies demonstrated that this uptake involves the binding of integrins ITGA4/ITGB1 and ITGA6/ITGB4 in EV membranes to vascular cell adhesion protein 1 or CD151, respectively, both of which are constitutively expressed by LEC. Melanoma‐derived EV administration upregulates macrophage scavenger receptor 1 and bone marrow stromal antigen 2 in LEC, which belong to the functional family of scavenging and tethering proteins that may participate in the sensing of EV particles in the lymph node. Intradermal administration of EV from melanoma cell lines upregulates the LEC expression of ICAM 1, extracellular signal‐regulated kinases, and nuclear factor κb, which are responsible for cell recruitment and lymphangiogenesis in the pre‐metastatic niche (García‐Silva et al. 2021).

3.2.3. Consequences for Antigen Presentation, PD‐L1, IDO, TGF‐β, or T Cell Deletion/Exhaustion

LEC have been shown to inhibit T‐cell proliferation independently of antigen presentation via a nitric oxide synthase 2‐based mechanism (Dubrot et al. 2014; Lukacs‐Kornek et al. 2011). They also express Major histocompatibility complex (MHC) class I and II molecules. In line with their well‐documented roles in self‐ and non‐self‐antigen archiving and immunological memory, LEC cross‐present melanoma EV ovalbumin to T lymphocytes on MHC class I, promoting antigen‐specific CD8⁺ T‐cell deletion in the pre‐metastatic niche (Hirosue et al. 2014; Leary et al. 2022). This experimental evidence suggests an EV‐associated antigen retention by LEC, although bona fide antigen‐archiving signatures following EV treatment have not yet been published (Sheridan et al. 2025). Alternatively, murine class 2 transactivator (CIITA) knockout models have highlighted the ability of LEC to acquire MHC II as well as antigenic peptide‐loaded MHC II complexes from DC, either through cell contact or EV transfer (Dubrot et al. 2014). Devoid of co‐stimulatory molecules, LEC display the highest level of the immune checkpoint inhibitor PD‐L1 in the lymph node and secrete the anti‐inflammatory cytokine TGF‐ β (Cohen et al. 2010; Dubrot et al. 2014; Hirosue et al. 2014; Piao et al. 2022; Tamburini et al. 2014; Tewalt et al. 2012). The uptake of the cervical cancer extravesicular microRNAs miR‐142 and miR‐1468 has been shown to modulate indoleamine 2,3‐dioxygenase and PD‐L1 expression in LEC isolated from dermal lymphatic vessels, leading to Janus kinase 2 and signal transducers and activators of transcription pathway activation and subsequent CD8⁺ T cell exhaustion (Zhou et al. 2021; Zhou, Zhang, et al. 2021). Similarly, Helicobacter pylori‐infected gastric cancer cells ferry miR‐1246 to LEC, promoting PD‐L1 expression and CD8⁺ T cell deletion (Lu et al. 2024). Despite their inherent tolerogenic machinery, LEC express a panel of cytokine‐ and innate immune receptors that make them sensitive to inflammatory or danger signals (reviewed in Card et al. 2014).

Briefly, LEC have been shown to internalise lymph‐borne EV and cross‐present EV‐shuttled antigens, triggering profound transcriptional shifts. LEC upregulate immune checkpoints and suppress local immune reactivity in response to environmental EV, such as tumour EV. However, the relevance of EV targeting of lymph node LEC in the maintenance of peripheral tolerance requires further research.

3.3. Medullary Sinus (MS) Macrophages

Following the passage of the lymph through the subcapsular sinus, the particles may encounter CD169⁺ F4/80⁺ MS macrophages. They express MHC class II molecules and process internalised materials for antigen presentation. However, many studies have indicated that SCS macrophages and DC are more efficient in initiating adaptive immune responses, whereas MS macrophages act as terminal scavengers and a backup source of antigens for professional antigen‐presenting cells. F4/80⁺ macrophages have been identified as cellular targets of adipose tissue stem cell EV in inflamed lymph nodes in mouse models of experimental autoimmune encephalomyelitis (Turano et al. 2024). Moreover, lymph node MS macrophages further express T cell immunoglobulin and mucin domain‐containing protein 4, a well‐known receptor for phosphatidylserine exposed on EV from dying cells, promoting tolerance (Albacker et al. 2013). Phagocytosis of tumour apoptotic bodies further mediates interleukin‐33 immunosuppressive signalling and macrophage polarisation towards an anti‐inflammatory phenotype (Lamorte et al. 2023) (Figure 2‐3). Peinado's team demonstrated the uptake of exogenous and endogenous tumour EV by MS macrophages in draining lymph nodes. However, RNA sequence analysis has revealed only moderate changes in gene expression in CD169⁺ F4/80⁺ flow cytometry‐sorted lymph node MS macrophages (García‐Silva et al. 2021; Leary et al. 2022). In comparison, the authors described profound alterations induced in immune‐related genes in LEC, suggesting that LEC may be key drivers of tumour EV‐mediated immune responses.

3.4. FRC and FDC

Distinct subsets of FRC compartmentalise the parenchyma of the lymph node into marginal, perivascular, and T‐ and B‐cell zones (Figure 2‐4). Sheltered from the lymph flow, they provide substratum, vascular and intercellular adhesion molecules (VCAM and ICAM, respectively), and chemokines to actively guide immune cell locomotion and increase the likelihood of their encounter with cognate antigen‐presenting cells, as evidenced by intravital two‐photon microscopy analysis (Bajénoff et al. 2006; Fletcher et al. 2010). T‐zone FRC secrete CCL‐19 and CCL‐21 to attract chemokine receptor 7‐positive T cells and DC, as well as IL‐7, which is essential for T lymphocyte survival. Similarly, primary and secondary B‐cell follicle FRC secrete CXC motif chemokine ligand 13 and the B‐cell activation factor of the tumour necrosis factor family, acting as attractant and survival factors for CXC motif chemokine receptor 5‐positive B cell, respectively. Transmission electron microscopy revealed antigen retention on the long processes of FRC, forming intricate interdigitations with B lymphocytes (Nossal et al. 1968). This led to their misleading but still frequently used denomination as FDC before genetic fate‐mapping tools undoubtedly ascertained the non‐haematopoietic origin of these radioresistant antigen‐presenting cells (Jarjour et al. 2014). In transgenic mouse models, ovalbumin‐expressing FRC promote antigen‐specific CD4⁺ regulatory T‐cell development and control autoreactive follicular T helper cell activation (Nadafi et al. 2020). Conversely, FRC express constant antibody fragment and complement receptors, and opsonised particles deposited on FDC surfaces, thereby fostering B cell activation by binding to CD19/CD21 complement receptors. Long‐term, non‐degradative antigen‐presentation by FDC further supports affinity maturation in the germinal centre and memory B cell maintenance (reviewed in Carrasco and Batista 2006a; Heesters et al. 2013).

Although immune electron microscopy revealed MHC class II antigen presentation by endogenous EV adsorbed onto FDC as early as 2000 (Denzer et al. 2000), evidence of EV interactions with FRC remains scarce. Sentinel work from Morelli's team showed that, while EV are excluded from FRC conduits, MZB cells shuttle allograft‐derived EV from the lymph node sinuses to FDC. Subsequent antigen presentation is supported by intravital imaging showing diminished velocity and increased arrest for cognate compared to control B cells scanning FDC displaying EV‐antigens at the onset of antibody‐mediated graft rejection (Chen et al. 2025) (Figure 2‐5). Surprisingly, allogeneic but not syngeneic donor antigens were detected on FDC in graft‐draining lymph nodes, despite a similar inflammatory parental cell milieu due to surgery. Serum exposure appears to be a prerequisite, suggesting that distinct lymph node FRC targeting of opsonised EV. However, autologous EV from head and neck squamous cell carcinoma have been shown to reach sentinel lymph nodes and be taken up by FRC, where they deliver phosphorylated interferon gamma receptor 1 and activate Janus kinase 1 and the signal transducers and activators of transcription pathway, leading to increased PD‐L1 expression and impaired T effector cell responses (Han et al. 2023). Furthermore, FRC retention could be a plausible explanation for the persistence of antigens from DC‐derived EV, days after the disappearance of the parental cells from the lymph node (Luketic et al. 2007). Again, these seemingly contradictory observations suggest that the co‐delivery of environmental cues shapes the outcome of EV‐mediated immune responses (Prunevieille et al. 2021). FRC themselves release EV that carry bioactive cargo, such as TGF‑β, and these FRC‑derived vesicles can promote de novo induction of forkhead‐box P3 ‐positive regulatory T cells, especially in mesenteric lymph nodes (Pasztoi et al. 2017). Based on this evidence, it can be speculated that EV are part of a bidirectional communication axis: FRC respond to incoming EV (e.g., from cancer cells) and release their own EV to shape T‐cell differentiation and peripheral tolerance in the lymph node.

3.5. Roles of EV in the Regulation of Adaptive Immunity in the Lymph Node

Pioneering work in the mid‐1990s and early 2000s revealed that EV from B lymphocytes and DC bear functional MHC class I and II molecules capable of directly priming T‐cell responses (Raposo et al. 1996; Segura et al. 2005; Segura, Nicco, et al. 2005). In mouse models of mastocytoma and mammary carcinoma, DC‐derived EV cross‐priming of cytotoxic T cells proved to be an efficient means of immune response amplification and tumour rejection (Zitvogel et al. 1998), providing the foundation for the clinical evaluation of DC EV vaccines. The role of EV in antigen presentation, cross‐presentation, and adaptive cellular and humoral responses has been reviewed in detail elsewhere (Buzas 2023). However, most studies are performed in vitro or assess systemic outcomes (e.g. clinical scores and serum cytokines), so cell‑type–resolved analyses within specific lymph node compartments (FRCs, LECs, SCS macrophages, or B‐ and T‐cell zones) and antigen‐specific effects are largely missing. As discussed above, EV influence immune responses by interacting with a diversity of stromal and innate target cells. In addition to lymph node resident cells, diverse populations of lymphoid cells (such as T and B lymphocytes, natural killer cells, γδT cells, and natural killer T cells) and myeloid cells (such as DC and macrophages) located between the T‐ and B‐cell zones contribute to the orchestration of innate and adaptive immunity (Figure 2‐6).

3.5.1. B Cells

B cells appear to be important effectors of EV‐mediated immune responses, possibly because they recognise membrane‐bound antigens more efficiently than soluble antigens, through stabilised interactions between integrins and cell adhesion molecules (Carrasco and Batista 2006b; Heesters et al. 2016). In vitro studies on PBMC revealed preferential association of fluorescent human islet EV to CD14⁺ and CD19⁺ cells (Rutman et al. 2018) and that B cells promote DC‐derived EV antigen T cell stimulation in vivo (Qazi et al. 2009). As stated above, graft‐derived EV captured in the SCS of the lymph nodes and transported across SCS macrophages present donor MHC molecules and complement components recognised by alloreactive B cells, leading to donor‐specific antibody production (Zeng et al. 2021). However, the role of EV in the development of allergic sensitisation remains unclear. Mast cell EV disseminate immunoglobulin E complexes to the lymph nodes, which may have bystander effects on allergic sensitisation (Pfeiffer et al. 2026). Patient‐isolated B‐cell EV have been shown to present birch pollen antigens and activate T cells in vitro, but lymph node‐related evidence is still lacking (Admyre et al. 2007). EV antigen‐specific B‐cell immune modulation has also been documented in the development of autoimmunity. Following immunisation, antigen‐experienced B cells secrete antibody‐bearing EV, which are detectable in B‐cell follicles of the lymph node that subsequently enter the general circulation (Rival et al. 2024). In lupus‐prone mice, these EV express self‐reactive immunoglobulin G antibodies on their surface, hinting at a new route of antibody diffusion. In contrast, immune‐regulatory mesenchymal stem cell (MSC)‐derived EV dampen disease severity in parallel with a significant increase in the relative frequency of CD19⁺IL‐10⁺ regulatory B‐cell populations in the lymph nodes in collagen‐induced arthritis and delayed‐type hypersensitivity mouse models of inflammation (Cosenza et al. 2018). In vitro restimulation with type II collagen led to a shift towards anti‐inflammatory cytokine secretion (IL‐1b↓ IL‐10 ↑) from lymph node cells isolated from MSC‐EV‐treated mice, suggesting antigen‐specific B‐cell‐dependent immune regulation of T‐cell responses.

Previous and current research has highlighted the importance of EV in the inception of humoral immune responses. Biomolecules of the EV corona, such as host‐derived complement, dictate EV trafficking to B zones in the lymph node cortex, which is a point to consider in the use of allogenic EV for therapeutic purposes.

3.5.2. T Cells

Unlike B‐cell receptors that recognise native, intact antigens, T‐cell receptors exclusively bind to processed peptide fragments presented on MHC molecules (Pishesha et al. 2022). EV antigen presentation can occur either by (i) direct presentation on EV surface of functional peptide‐MHC complexes and co‐stimulatory molecules inherited from their parental cell; (ii) indirect presentation following EV internalisation and antigen processing and MHC class II loading by recipient antigen‐presenting cells; (iii) cross‐presentation, in which antigen‐presenting cells route exogenous EV antigens into the MHC class I pathway to prime naïve CD8+ T cells; or (iv) cross‐dressing, in which peptide‐MHC‐complex‐containing EV are attached to the surface of the antigen‐presenting cell (Buzas 2023).

EV contribute to the regulation of T‐cell responses. T‐zone macrophages express Tyro‐3, Axl, Mer (TAM) receptors—a family of tyrosine kinase receptors involved in apoptotic vesicle clearing—in cooperation with bridging ligands such as growth arrest‐specific protein 6 or Protein S in the lymph to capture phosphatidylserine‐positive vesicles. Besides, T‐zone macrophages do not prime T cells, supporting an essentially immune‐silent process of T cell deletion (Baratin et al. 2017). EV as mediators of immune suppressive conditions in the lymph node further led to the ‘nano‐seed and soil’ hypothesis of cancer. Congruent with Paget's original tumour metastasis theory (Paget 1889), the dissemination of tumour EV is conducive to tumour progression and the implementation of immune‐permissive pre‐metastatic niches in distant organs (Costa‐Silva et al. 2015; Peinado et al. 2012). In the lymph node microenvironment, this process also involves the suppression of T‐cell anti‐tumour immunity and the promotion of EV‐related antigen‐specific immune suppressive responses: In mice, following tumour EV uptake by LEC and subsequent PD‐L1 upregulation, tumour antigens cross‐presented on MHC class I molecules induce apoptosis of antigen‐specific CD8⁺ T cells (Leary et al. 2022). To complement this toolbox, tumour EV markedly increase the expression of extracellular matrix factors, such as laminin 5, which remodels the lymph node stroma permissive to tumour cell invasion (Hood et al. 2011). In melanoma patients, metastatic founder cells produce EV carrying immunomodulatory poliovirus receptors (CD155) and B7‐H3 (CD276), which participate in CD8⁺ T cell inhibition in the lymph node (Guetter et al. 2025).

In inflammatory settings, non‐immune tissue EV, such as keratinocyte EV, promote prostaglandin E2 secretion from skin keratinocytes following UV exposure, mast cell lymph node migration, inhibition of DC maturation, and Treg increase, in parallel with symptomatic relief in models of inflammatory bowel disease and psoriasis (Liu et al. 2025). Very recently, tests using pluripotent stem cell‐derived EV have suggested a strong tolerogenic capacity of these EV, in comparison with that of MSC‐derived EV, through reprograming of lymph node CD4⁺ and CD8⁺ T cells towards a regulatory phenotype, accompanied by the repression of cyclin‐dependent kinase 8 and the up‐regulation of its downstream target forkhead‐box P3, a master regulator of Treg function. Therapeutic effects were observed in a murine model of antigen‐specific type 1 diabetes; however, antigen specificity was missing from the functional assessment of the model (preprint, Li et al. 2025).

Although a partial picture of EV‐mediated T‐cell immunity is emerging, the cascade of events involved in the delivery and presentation of EV components to T cells in the lymph node has not yet been precisely identified. Technically, the degradative antigen processing required for T cell presentation constitutes a challenge in tracking T‐cell responses to EV cargo.

3.5.3. Contributions of EV From Tolerogenic Immune Cells

EV derived from tolerogenic immune cells sustain adaptive tolerance. In a murine model of psoriasis, drug‐loaded Treg‐derived EV induced local and systemic immune tolerance, accompanied by the inhibition of T helper 17 cell activity and a concomitant increase in the frequencies of Treg and tolerogenic DC in the spleen and lymph nodes (Ou et al. 2025). Likewise, EV derived from drug‐induced tolerogenic DC stimulated indoleamine 2,3‐dioxygenase/Treg and FAS ligand‐mediated improvement of autoimmune myasthenia gravis in rats (Li et al. 2016). In a mouse model of autoimmune alopecia, myeloid‐derived suppressor cell‐derived EV preferentially targeted skin‐draining lymph nodes, resulting in a strong increase in Treg, reduced T helper cell proliferation, mitigated cytotoxic activity, lymphocyte apoptosis, and clinical improvement in treated mice (Zöller et al. 2018). Lymph node allospecific Treg lymphocytes isolated from tolerised graft recipient mice have recently been shown to produce EV coated with a TGF‐β:latent‐associated peptide complex, which becomes active in an in vivo antigen‐specific suppression assay. These EV can further spread tolerance to naïve bystander T cells in vivo in a mechanism of ‘infectious tolerance’ (Burlingham et al. 2023). Interestingly, not all the lymph nodes appear to be equal with respect to Treg induction. Owing to their essential role in the acceptance of dietary antigens, neonatally ‘imprinted’ mesenteric lymph node stromal cells form a stable, gut‑specific microenvironment that is resistant to later inflammation and optimised for peripheral Treg development (Pasztoi et al. 2017; Pezoldt et al. 2018). The highest Treg cell induction to gut antigens was observed in the mesenteric lymph nodes of the duodenal segment (Esterházy et al. 2019). Enrichment of the intestinal epithelial cell marker A33 in the mesenteric lymph nodes aligns with the assumption that EV secreted by the intestinal epithelium may be involved in the transfer between the gut and its associated lymphoid tissues (Van Niel et al. 2003). In mouse models, intravenously or intraperitoneally administered MSC‐EV cargo can be detected in secondary lymphoid organs, including the mesenteric lymph nodes, indicating that at least a fraction of the MSC‑derived material reaches these lymph nodes (Court et al. 2020). How the duodenal dietary route should be targeted for the optimal delivery of therapeutic EV awaits further investigation.

Many autoimmune pathologies would benefit from antigen‐specific immune cell‐derived EV therapies, through in vivo reprogramming of secondary lymphoid organs to allow the establishment of immune memory for the lasting production of regulatory EV. The astonishing capacity of EV to train the immune system in adaption to change is highly evident in the case of maternal tolerance, where the migration of foetal placental EV to draining lymph nodes engenders microchimerism. Notably, intact foetal antigens are transferred between immune cells by EV cross‐dressing, highlighting the role of EV handover in the orchestration of multiple immune effectors (Bracamonte‐Baran et al. 2017). At present, data on intermediate stromal and innate immune effectors are lacking.

Collectively, these findings provide insight into the multifaceted roles of EV in adaptive immunity. However, most studies have focused on a single EV type and/or a specific subpopulation of effector cells. In perspective, systematic scanning of cellular responses to EV in the lymph node in healthy and diseased conditions (such as allergy, infection, and autoimmunity) should help to understand the synergistic and coordinated functional activities of EV immunity.

4. Implications for EV‐Based Immune Therapy

The lymphatic system constitutes the body's second vascular tree, which is subject to the uptake of soluble, particulate, and cellular materials for body‐wide scanning of tissue status and pathogen invasion. Initially, EV‐based treatments were performed in comparison to cell therapy and mainly administered intravenously. The evidence presented here suggests that subcutaneous or intradermal administration routes allow the targeting of immune cells via the lymphatic circulation, while avoiding undesirable dilution and rapid clearance in the blood stream (García‐Silva et al. 2021; Hao et al. 2006; Hood et al. 2011; Leary et al. 2022; Nambiar et al. 2024; Sakurai et al. 2023; Saunderson et al. 2014; Srinivasan et al. 2016; Sun et al. 2019). Notably, both vascular systems are interconnected, and EV not retained in the lymph nodes eventually reach the bloodstream at the intersection of the two systems at the veinous angle. To date, a single clinical case report has described inflammatory adverse reactions following repeated intradermal administration of lyophilised MSC‐EV (Tawanwongsri and Vachiramon 2024), whereas allogeneic MSC‐EV delivered via the intradermal route proved to be safe in case reports of epidermolysis bullosa (Conget et al. 2010).

The repertoire of antigen receptors is extremely large, with only a few hundred naïve T cells present in the steady state, making random encounters with cognate antigen‐presenting cells in body fluids illusive (reviewed in Qi et al. 2014). The coordinated positioning of immune cells in secondary lymphoid organs supports the scanning and activation of close to all antigen‐specific T cells during their 12–24 h transit through the lymph node. EV and their biomolecular corona cluster multiple complex immune signals, such as antigens, co‐stimulatory signals, cytokines, and danger or tolerogenic mediators. By maximising the probability of co‐encounter with innate and adaptive immune cells, EV constitute unique ‘mobile signalling synapses’ integrating complex tissue messages (Dubrot et al. 2014; Nambiar et al. 2024; Wolf et al. 2022). The above‐described relay of EV between SCS macrophages, B cells, and FRC, together with non‐degradative endocytosis for antigen archiving and re‐stimulation, highlights the importance of EV cross‐dressing in immune communication. This allows for the preservation of valuable contextual information about the noxious or innocuous nature of a given antigen until final dissociation by degradative endocytosis required for MHC class I and II processing in antigen‐presenting cells.

Understanding how EV modulate the immune response outcomes is obviously important for defining new treatment strategies. Technical caveats are mostly linked to the difficulty of (i) tracking nanosized particles in vivo and (ii) extracting low‐abundance and manipulation‐sensitive lymph node‐resident stromal and immune cell populations. High‐resolution imaging in translucent zebrafish larvae at day 3–5 post‐fertilisation has revolutionised our knowledge of EV displacements at the organism scale; however, secondary lymphoid organ function is restricted by the absence of adaptive immunity at this stage of development, although innate immune effectors and primitive lymphatic vessels exist (Jung et al. 2017; Verweij et al. 2019). On the other hand, xenotransplantation of human cells in various transgenic fluorescent reporter strains allows the monitoring of EV activity in macrophages and neutrophils in grafted target tissues, independently of their ability to reprogram the lymph node environment. Translation to multiscale high‐resolution microscopy in combination with spatial omics analyses is required to contextualise EV interactions with immune effectors compartmentalised within the microanatomy of lymph nodes in adult organisms. Much progress stems from in situ single/two‐photon and positron emission tomography tracking of nanosized EV in mammals (Chen et al. 2025; Khan et al. 2022). The use of EV‐painting approaches and ultrasensitive nanoluciferase reporter constructs further alleviates the need to bind a large number of EV for recipient cell detection (Nambiar et al. 2024).

In line with recommendations from the International Council of Harmonisation (ICH Q6B), potency testing of EV‐based therapeutics should rely on their mechanisms of action in vivo. Despite major advances in the development of lymph nodes on chips (Belanger et al. 2021; Goyal et al. 2022), to date, no satisfactory in vitro models have recapitulated the temporal and spatial changes in lymph node cells that occur during EV‐driven immune responses. As anatomy, structure, and even lymphatic flow control the immune response outcome, holistic in vivo approaches remain the safest option for testing the effects of EV, which may differ in function of the administration route. It can be speculated that popular human vascular endothelial cells (HUVEC) or mixed lymphocyte reactions, frequently used in surrogate assays to evaluate neoangiogenic and immune‐regulatory properties, respectively, might be relevant for testing blood‐derived or intravenously administered EV. In contrast, functional evaluation of tissue or intradermally injected EV may benefit from an understanding of the molecular pathways driven by EV in draining lymph nodes for rational assay development combining crucial stromal and immune cell effectors.

The immune functions of EV in the lymph node have been studied mainly in the context of transplantation and cancer. Although MSC‐EV are the most abundant source of EV in clinical trials (Lightner et al. 2024; Norooznezhad et al. 2022), little is known about the various populations of EV with anti‐inflammatory or immunomodulatory activities (such as EV from stem cells, platelets, regulatory DC, B and T cells) in the lymph node microenvironment. For autoimmune and allergic diseases, the presentation of relevant antigens in the presence of appropriate tolerogenic signals is required to reinstate durable tolerance and adaptive memory. Owing to the importance of cross‐dressing, surface rather than cargo modifications may be actionable targets for the development of EV therapeutics. Treatment strategies include (i) EV from antigen‐specific immune regulatory cells, that are difficult to produce, (ii) immune regulatory MSC‐EV engineered to express target antigens or mixed with antigen‐expressing tissue EV, and (iii) surface coating with tolerogenic ligands on antigen‐expressing tissue EV. Conversely, to elicit anti‐tumour immune responses, DC EV engineered to harbour surface ligands, such as selectin CD62L and OX40L, show improved lymph node homing, extravasation from high endothelial venules, subsequent activation of effector T cells, and inhibition of Treg development (Ji et al. 2021). More recently, the combined expression of immune activating (CD80, CD137L, CD40L, CD2, and CD32) or immune modulatory (CD80, OX40L and PD‐L1) proteins has been achieved in HEK293 EV to elicit or neutralise anti‐tumour or autoreactive immune responses, respectively. Functional assessment confirmed efficient positive and negative co‐stimulation of mouse and human immune cells in vitro, symptom relief in mouse models of cancer and autoimmune disease, and increased CD11b⁺ CD3e⁻ myeloid and lymphoid T‐cell proliferation and activation in tumour‐draining lymph nodes (Luo et al. 2025). Furthermore, EV have been successfully employed as vaccine adjuvants: formulations with Saccharomyces cerevisiae EV containing glucan, DNA and RNA were efficiently delivered to the B‐cell zone, where they stimulate immune activation (Morishita et al. 2023). Extending the functionalisation of EV to promote antigen‐specific tolerance is a future research avenue for allergic, inflammatory, autoimmune, and vaccination therapies. In line with the danger and discontinuity theories of immunity, repeated administration of small doses or continuous release of hydrogel‐embedded EV may be necessary for antigen‐specific immune therapy (ASIT) (Li et al. 2025). To achieve this goal, dynamic dose‐response studies are required to determine the optimal treatment regimen for EV‐based ASIT. Based on the above‐described observations of opsonised EV, but not syngeneic particles, in the B‐cell zone (Chen et al. 2025), the therapeutic potential of syngeneic and allogeneic MSC‐EV may differ, calling for comparative analyses to delineate EV‐based drug conception.

5. Concluding Remarks

Longtime considered a means to get rid of cellular waste, there is now considerable evidence of the complex contributions of EV to local and systemic immune orchestration. By bridging the gap between ultra‐fast diffusing soluble mediators, tissue context information, and slowly moving low‐frequency specialised immune cells, EV guide, accelerate, and amplify the inception, execution, resorption, and recall of immune responses. Amenable to functionalisation, EV constitute flexible nanoparticle platforms to co‐opt multiple cellular effectors in the complex lymph node microanatomy for therapeutic intervention.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.