The transcription factor forkhead box P3 (FOXP3) is the best known Treg biomarker and is recognized as the master regulator of Treg cell generation and function. As a transcription factor, FOXP3 induces the expression of Treg-associated genes, including IL-2, CD25, CTLA-4, and miR-155 (28, 29). More than a recognitional marker, FOXP3 possesses the ability to control the switch between Treg cells and effector T cells. Ectopic FOXP3 expression transforms T cells into suppressor cells, while the failure of constant FOXP3 expression impairs the potency to inhibit effector cells (30–32). Based on FOXP3 expression, Treg cells can be dissected into three subpopulations: CD45RA(+)FOXP3(lo) resting Treg cells (rTreg cells), CD45RA(-)FOXP3(hi) activated Treg cells (aTreg cells) and cytokine-secreting CD45RA(-)FOXP3(lo) nonsuppressive T cells. Both rTreg cells and aTreg cells are effective suppressor cells in vitro. aTreg cells die rapidly but rTreg cells proliferate and convert into aTreg cells (24).

Due to FOXP3’s indispensable role in Treg biology, the regulation of FOXP3 expression has drawn considerable attention. Several transcription factors have been reported to induce FOXP3 transcription, including Forkhead transcription factor of the O class (FOXO)1, FOXO3, c-Rel, Smad2, and Smad3 (33–39). Interestingly, the glycolytic enzyme enolase (ENO)-1 inhibits FOXP3 transcription through binding to the promoter region (40), suggesting a major role in the direct metabolic control of Treg cell function. At the post-transcriptional stage, several microRNAs are predicted to directly bind to the FOXP3 3’-UTR. Specifically, changes in the abundance of miR-31 and miR-15a/16 have been associated with significant modulation of FOXP3 expression (41–44). FOXP3 can also be regulated through post-translational modifications. High expression of the deubiquitinase (DUB) ubiquitin specific-processing protease (USP)7 in Treg cells is required for sustained FOXP3 expression (45). Vice versa, the E3 ubiquitin ligase Stub1 leads to FOXP3 ubiquitination and degradation (46, 47). Uncontrolled FOXP3 ubiquitination has been proposed as a relevant mechanism in autoimmune diseases. In patients with psoriasis, the (C-C motif) ligand (CCL)3 and the protein kinase B (PKB)a/Akt1 pathway induce polyubiquitination of FOXP3, which is highly associated with defective Treg function (48). Besides ubiquitination, FOXP3 is also regulated by other modifications, including acetylation (by SIRT1 and TIP60) and phosphorylation (by CDK2 and NLK) (49–54). In the autoimmune disease rheumatoid arthritis (RA), instability of FOXP3 expression and the subsequent failure of Treg-dependent immunosuppression are due to the insufficient expression of the histone acetyltransferase TIP60 (55).

Although CD25⁺FOXP3⁺ T cells amongst CD4⁺ T cells are often considered to be classical Treg cells, the compartment of CD8⁺ T cells also contains a regulatory subset. Like CD4⁺ Treg cells, CD8⁺ Treg cells express FOXP3, but at a lower level (56, 57). Recent studies have emphasized that it is not accurate to identify CD8⁺ Treg cells based only on CD25 expression (58, 59). Instead, additional cell surface markers, such as CD39⁺ and CD26^- are now considered useful markers of CD8⁺ Treg cells (60, 61). The subpopulation of CD8⁺CD39⁺CD26^- T cells represents a highly purified Treg cell subset, which possesses strong inhibitory effects in T cell activation assays (12).

Similar to CD4⁺ Treg cells, CD8⁺ Treg cells may be reduced in number or quality in patients with autoimmune disease. Specifically, lowered numbers of CD8⁺ Treg cells have been reported in patients with systemic lupus erythematosus (SLE) and recovery of CD8⁺FOXP3⁺ Treg cells after transplantation of autologous hematopoietic progenitor cells has been associated with good control of disease activity (62). In patients with giant cell arteritis (GCA), frequencies of circulating CD8⁺ Treg cells are largely maintained, but an altered gene expression program results in impaired suppressive capacity and unopposed inflammatory activity of pathogenic CD4⁺ T cells. Experiments designed to repair the expression of relevant gene products in patient-derived CD8⁺ Tregs have been sufficient to restore fully functional Tregs in vitro and in vivo, which prevented the invasion of the vessel wall by inflammatory cells (13).

Determination of the mechanisms of how Treg cells function is critical to understanding the role of these specialized T cells in protective and pathogenic immunity. Ever since Treg cells were initially described, attention has been directed at uncovering, both on the cellular and molecular level, how these cells can inhibit signaling to affect the survival of their target cells. Here, we are going to summarize the spectrum of mechanisms used by Treg cells to exert their immune-regulatory role ( Figure 1 ).

Giant Cell Arteritis (GCA) is the most common autoimmune vasculitis, with incidence and prevalence rates growing as the population ages. Patients with GCA suffer from aggressive wall inflammation in medium and large arteries, including the aorta, subclavian arteries, axillary arteries, extra-cranial branches of the carotid arteries, and vertebral arteries. Infrequently, GCA is diagnosed in lower extremity arteries. Clinically, the most feared disease manifestations are ischemic stroke of the optic nerve and the posterior brain. Potentially fatal consequences such as aneurysm formation, dissection and rupture are related to the destruction of the aortic wall. On a cellular level, the disease is characterized by the formation of granulomas within inflamed arteries, assembled from CD4⁺ T cells, macrophages and multinucleated giant cells (102, 103).

Numeric and qualitative deficiencies in Treg cells are well recognized in GCA. Terrier and colleagues reported decreased frequencies of CD4⁺FOXP3⁺ Treg cells in the peripheral blood of GCA patients and FOXP3 was suspiciously absent in T cells infiltrating vasculitic lesions (104).

Recent cellular and molecular studies have shifted attention from CD4⁺ Treg cells to the CD8⁺ Treg cell subset. CD8⁺ Treg cells contain a functionally specialized subpopulation that imposes immunosuppression by secreting NOX2 containing exosomes that are absorbed by nearby CD4⁺ effector T cells to suppress their activation and proliferative expansion (12). In patients with GCA, the frequency of CD8⁺ NOX2⁺ Treg cells is diminished, and, in addition, their function is essentially paralyzed. Molecular mechanisms underlying the loss of functional fitness in patient-derived CD8⁺ Treg cells have been uncovered and are closely related to a defect in exosome production (13) ( Figure 2 ). Both, the loading of NOX2 into the exosomes and the generation of exosomes were attenuated due to the rewiring of the endosomal system. The endosomal machinery is critically involved in processes of protein quality control and is responsible for the trafficking of intracellular proteins between different cellular compartments and the release of proteins through exosomes. New insights into the different vesicular trafficking pathways have emerged, and the endosomal sorting complex required for transport (ESCRT) pathway is now recognized as the pathway for the formation of intraluminal vesicles and multivesicular bodies (MVB) (105). Exosomes are born by intraluminal budding of the MVB (106). The multiprotein complex of the ESCRT machinery regulates the invagination of vesicles into the MVB while recognizing, capturing, and sorting ubiquitinated protein cargo. Sequestering of ubiquitinated membrane proteins can occur at the endosomal membrane as well as the plasma membrane. When the MVB fuses with the plasma membrane, exosomes can be secreted into the extracellular space. Much has yet to be learned about the trafficking, docking and membrane integration of exosome carrying MVB, but several Rab GTPases within the endolysosomal trafficking machinery, including Rab27a and Rab27b, are associated with exosome loading and secretion (107). In essence, effector proteins recruited by Rab GTPases ultimately determine the collection of cargo, the movement of vesicles throughout the subcellular compartments, the docking of MVBs to the target membrane and the delivery of exosomes. In the case of CD8⁺ Treg cells derived from GCA patients, hyperactivation of NOTCH4 signaling disrupts the exosomal secretion of NOX2 through transcriptional control of Rab GTPases. Precisely, NOTCH4^hiCD8⁺ Treg cells upregulate the expression of RAB5A and RAB11A but repress RAB7A, accumulating NOX2 in the intracellular compartment, including the early and recycling endosomes. GCA CD8⁺ Treg cells fail to translocate NOX2 to MVBs and the cell surface, disrupting the exosomal release of immunosuppressive NOX2 ( Figure 2 ). Ultimately, dysfunctional CD8⁺ Treg cells are unable to control the expansion of pro-inflammatory CD4⁺ T cells, paving the way for the invasion of an immune privileged tissue site (13). These data will allow the development of new strategies to modulate the balance between pro and anti-inflammatory T cells in GCA. Inhibition of NOTCH signaling repaired CD8⁺ Treg function in vitro and in vivo and ameliorated vascular inflammation in NSG mice carrying human arteries (13) provide a rationale for drug targeting of this vaso-inflammatory pathway.

Takayasu’s arteritis (TAK) is an inflammatory vasculopathy that primarily affects young women and leads to aortitis and vasculitis of the primary aortic branch vessels (108). While the histopathology of vascular involvement and the consequences of vascular inflammation have similarities in GCA and TAK, there are also differences in the cellular and molecular immunopathology of the two vasculitides (103). The percentage of circulating activated Treg has been reported to be lower in TAK patients than in healthy age-matched controls, while resting Tregs are similar (109, 110) suggesting the possibility of abnormal Treg cell maturation. On account of their plasticity, Treg cells can acquire new effector functions, e.g. differentiating into Th1, Th2, or Th17-like cells (20). But this transformation of Treg cells can possibly strengthen the inflammatory processes and further diminish physiological immunosuppression. Another possibility based on recent studies proposes that Tregs derived from TAK patients insufficiently differentiate into Th2-like cells, thereby detracting from IL-4 and IL-13 production and contributing to excess inflammation (110).

Polyarteritis nodosa, formerly known as periarteritis nodosa, is a systemic necrotizing vasculitis affecting medium and small-sized vessels (111). Vascular damage occurs preferentially in the gastrointestinal tract, skin and peripheral nervous system. Unlike GCA and TAK, PAN patients have been reported to have increased Treg cells in their blood (112). However, in co-culture experiments with effector T cells the suppressive abilities of Tregs from PAN patients are significantly depressed, and this loss of function appears to be associated with lower expression of CTLA-4. Studies comparing Treg cell competence in patients treated with prednisolone or prednisolone plus cyclophosphamide have found normalization of Treg cell frequencies (112).

Kawasaki disease (KD) is a vasculitis of early childhood targeting medium and small-sized vessels. A classical complication of KD is the formation of coronary artery aneurysms. In the acute phase of KD, Treg cells frequency is lowered by 50% compared to age-matched healthy controls, regardless of whether the patients had coronary artery lesions (CAL) or not (113–115). The gold standard of therapy for KD is now the infusion of immunoglobulins (IVIG), which modulates abnormal immunity through the antibody-dependent pathways and increases the number of circulating Treg cells (116).

Fc-specific Treg clones have been generated from sub-acute KD patients without arterial complications following IVIG therapy. These Treg clones display an unusual phenotype, secreting IL-10 and IL-4 but not TGF-β. However, KD patients with CAL even despite IVIG treatment seem to be unable to expand these Fc-specific Treg populations (117). Similar Treg fine specificities have been isolated from IVIG-treated KD patients and healthy controls, suggesting that the absence of Fc-specific Treg cells in acutely ill KD patients may be an inflammation-imposed abnormality (118). Patients with a history of KD in their childhood did not respond to Fc protein in vitro, suggesting that the IVIG-induced Treg response in KD patients is short-lived. Infliximab, a chimeric monoclonal antibody targeting TNF-α, can increase Treg cell frequencies during acute KD, while Infliximab-resistant patients lack an adaptation of Treg cell frequencies (119). All of these observations support the hypothesis that an inflammatory state can alter the frequencies of CD4+ FOXP3+ cells in the circulation and can possibly strip these cells of their suppressive capabilities.

In KD, FOXP3 mRNA levels appear to be regulated by the miR-155/SOCS1 and the miR-31 signaling pathways. In patients with acute KD, decreased CD4⁺FOXP3⁺ Treg cells might be associated with decreased expression of miR-155, leading to aberrant SOCS1/STAT5 signaling and overexpression of miR-31. This abnormality may also represent an inflammation-imposed deviation as it can be corrected by IVIG (114).

Vasculitis associated with Cryoglobulinemia is an immune complex disease that unfolds primarily in small-sized vessels, such as the capillaries of renal glomeruli. All patients with cryoglobulinemic vasculitis require evaluation for chronic hepatitis C virus (HCV) infection (120). Early studies showed that patients with symptomatic HCV-associated cryoglobulinemic vasculitis have both reduced numbers and diminished function of Treg cells (121), consistent with the hypothesis that systemic inflammatory states are characterized by redistribution and functional impairment of Treg cells.

A French team of investigators has evaluated the effectiveness of different therapies on the Treg cell population in 3 prospective trials. PEGylated interferon alfa-2b plus ribavirin treatment induced a significant and stable increase of Treg cell frequencies compared with baseline in patients with clinical and viral remission (122). In contrast, Treg cell frequencies did not differ after treatment for non-responders or partial responders. The frequency of Treg cells was positively correlated with plasma complement levels and inversely correlated with cryoglobulin levels, again indicating that circulating CD4⁺ FOXP3⁺ T cells are a sensitive biomarker of systemic inflammation. Since Treg cells are dependent on IL-2 as a growth and survival factor, therapeutic trials have tested the potential benefit of supplementing low-dose IL-2 (aldesleukin) in patients refractory to conventional antiviral therapy and/or B cell depletion therapy. Monitoring of peripheral Treg cells following treatment with exogenous IL-2 has shown numeric and functional improvements (123). Beneficial effects and reversal of Treg deficiency have also been reported for combination antiviral therapy with sofosbuvir plus daclatasvir (124).

Henoch-Schoenlein purpura (HSP), also known as IgA vasculitis, is the most common small vessel vasculitis in children (125). Target organs include skin, kidneys, gastrointestinal tract, and joints. Like most systemic vasculitides, HSP patients have lower numbers and weak suppressive function of Treg cells (126–129). As a reflection of a shift in the balance between pro-inflammatory effector T cells and suppressive Treg cells, the Th17/Treg ratio has been positively correlated with the erythrocyte sedimentation rate, kidney lesions, and multiorgan involvement (126). Treg cells that secrete IL-10 and TGF-β accumulate in the vasculitic tissue lesions in the kidneys (129). FOXP3 staining is preferentially localized in renal interstitial areas but does not correlate with proteinuria, serum albumin levels, and the histological classification of vasculitic involvement (130). In a recent study, miR-1-3p, miR-19b-1-5p, and miR-29b-1-5p were found to be up-regulated in peripheral blood mononuclear cells (PBMCs) of HSP patients, while miR-483-5p and miR-1246 were down-regulated. This shift was correlated with the Th17/Treg ratio (131). Again, numeric and functional alterations in peripheral blood Treg cells may simply reflect the high inflammatory status.

Behçet’s disease (BD) is an auto-immune/auto-inflammatory syndrome that can lead to vasculitis. The risk for BD is highest amongst individuals living along the Silk Road and the prototypic lesions are mucosal and genital ulcerations. BD stands out amongst the vasculitides by its often concurrent venulitis (132). Like other vasculitides, Treg cells are decreased in BD and correlate with disease activity (133–138). Imbalances in miRNA expression and excessive IL-21 have been considered to be key underlying abnormalities leading to Treg cell dysfunction (135, 138). Infliximab, but not colchicine or cyclosporine, increases the percentage of FOXP3⁺ cells in BD (134). Observation studies have reported that treated BD patients with low circulating Treg populations experience more ocular inflammation than those higher numbers (134). T cells exposed to infliximab had increased expression of FOXP3 and TGF-β and suppressed the activation of bystander T cells in in vitro experiments (134).

AAV is a necrotizing vasculitis affecting mostly small-sized vessels. Typically, few or no immune complex deposits are seen in affected tissues. The term AVV encompasses three disease entities: granulomatosis with polyangiitis (GPA, Wegener’s granulomatosis), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome) (139, 140). These three vasculitides are associated with specific autoantibodies, antineutrophil cytoplasmic antibodies (ANCA) that target either proteinase 3 (PR3) or myeloperoxidase (MPO). Each vasculitic entity has a specific phenotype with a particular pattern of organ damage. GPA is more often associated with anti-PR3 ANCAs and manifests with necrotizing granulomatous inflammation involving the upper and lower respiratory tract and necrotizing glomerulonephritis. MPA is typically associated with anti-MPO antibodies. Granulomatous inflammation is not a feature of MPA which presents with necrotizing glomerulonephritis and pulmonary capillaritis. Patients with EGPA have asthma, eosinophilia, and eosinophil-rich, necrotizing granulomatous inflammation often involving the respiratory tract. While the respiratory tract and the kidneys are targeted in most AAV patients, manifestations in the skin and the peripheral nerves are not unusual (141, 142).

Patients with AVV follow the general rule that systemic inflammation is associated with low numbers and functional impairment of Treg cells (143–146). Some studies have shown increased numbers of Treg cells (147, 148). Treg cells from GPA patients are still able to suppress proliferation of T cells from ANCA-negative patients, which has led to the hypothesis that part of the Treg deficit derives from target cell resistance (149). In AAV patients, expression of a FOXP3 isoform lacking exon 2 has raised suspicion of Treg cell instability (143). Also, miR-142-3p is upregulated in memory Tregs in GPA (150). In vitro overexpression of miR-142-3p produces functionally impaired Treg cells characterized by decreased cAMP levels. Pharmacological induction of cAMP production restores suppressive capacity (150). In MPA, attention has focused on the potential impact of diminished serum tryptophan and elevation of the tryptophan metabolite, kynurenine. Inhibition of tryptophan degradation enhances immune responsiveness, with a tendency to more severe glomerulonephritis (151). Correlative studies associating improvement of CCR4⁺FOXP3⁺ Treg cell frequencies with drug-free remission have supported the concept that ultimately, Treg cell frequencies are sensitive markers of the inflammatory status (152). In a mouse model, blocking IL-6 activity ameliorated the disease and increased the migration of Tregs into the kidney and the regional lymph nodes (152). In both GPA and MPA, B cell depletion therapy and conventional immunosuppressants yielded similar CD4⁺ Treg cell numbers (153).

Patients diagnosed with Eosinophilic Granulomatosis and Polyangiitis (EGPA) typically present with the triad of asthma, eosinophilia, and necrotizing vasculitis (154). Whether eosinophils are causally involved in the damage of small blood vessels remains unresolved. Compared to asthma control patients, EGPA patients have lower frequencies of Treg cells (155, 156). Production of IL-10, TGF-β, and expression of CTLA-4 by Treg cells are correlated to the inflammatory activity of the disease (156, 157). Relapsing patients, compared to those in remission, have a lower proportion of inducible Treg cells (134) and IL-2 production is predictably low. There is some evidence that the expression of the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO) is diminished in EGPA. IDO is thought to cause immune suppression through the breakdown of tryptophan. In relapsing EGPA, Treg cells are positively correlated with the CD19⁺ B cell count and inversely related to CD80⁺CD19⁺ B cells (156). Percentages of Treg cells, Treg-derived IL-10 and TGF-β are positively correlated with the percentage of CD83⁺ dendritic cells and inversely correlated with CD206⁺ DCs (158). These data suggest that Treg cells might play a role in DC and B cell biology.

As autoimmune disorders, vasculitides share features with other autoimmune diseases raising the possibility that Treg cell dysfunction is critically involved in the loss of self-tolerance. We will briefly review the current state of knowledge about Treg cell biology in the two classical autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

CD4⁺FOXP3⁺ T cells have been extensively studied in RA patients, but the mechanistic implications continue to be debated. Much of the discussion has focused on the methodologies of Treg analysis. One meta-analysis arrived at the conclusion that numbers of Treg cells in peripheral blood are diminished but the cells redistribute and accumulate in synovial fluid (159). Similarly, functional studies have supported the idea that systemic inflammation results in reduced numbers and reduced function. Functional analyses have shown that inhibitory competence is partially impaired. Specifically, Treg cells isolated from the peripheral blood of RA patients block the proliferation of effector T cells but fail to limit pro-inflammatory cytokine production (160). This effect can be partially reversed by anti-TNFα therapy, which successfully restores the capacity of Treg cells to inhibit cytokine production, and in parallel, supports recovery of Treg cell numbers in peripheral circulation (160). Attempts at defining the underlying mechanism have implicated reduced expression of CTLA-4 and enhanced expression of IL-6 (161, 162). Work analyzing the stability of FOXP3 has yielded valuable insights into the post-translational modification of the transcription factor as a determining feature of Treg phenotype and fitness. Specifically, loss-of-function of the histone acetyltransferase TIP60 (KAT5) has been associated with the instability of FOXP3, impaired Treg cell differentiation and failed immunosuppression (55) ( Figure 3 ).

Systemic Lupus Erythematosus (SLE) is the prototypic multi-organ autoimmune disease that affects the skin, kidneys, lungs, joints, and the central and peripheral nervous systems. SLE patients follow the classical pattern and have lower frequencies of Treg cells in their blood (163). Excellent progress has been made in defining the molecular abnormalities that produce Treg cell deficiency. A prime defect of SLE Treg cells lies in the low availability of IL-2, which is required for the expansion and survival of these immunoinhibitory T cells ( Figure 3 ). In SLE patients, the cAMP responsive element modulator-α (CREMα), a transcriptional repressor of the IL-2 promoter, is hyperactive, resulting in silencing of IL-2 and reduced FOXP3 expression (164). An alternative mechanism has been found for IL-2 deficiency in SLE. Mutations in the transcriptional repressor, Ikaros (165), lead to elevated protein phosphatase 2A (PP2A) expression in SLE T cells, restraining IL-2 expression (166). PP2A knockdown increased the expression of phosphorylated cAMP response element-binding protein (pCREB), restoring IL-2 expression (167). However, specific ablation of PP2A in murine Treg cells caused the development of autoimmunity in an mTOC-dependent manner (168), demonstrating that PP2A mediates different signaling pathways in effector and regulatory T cells.

Exosomes are nanosized extracellular vesicles (EV) that originate in the endosomal system and are secreted to the extracellular space when multivesicular bodies fuse with the cell membrane. As lipid bilayer membrane-enclosed vesicles, exosomes are a heterogeneous population, able to transport and deliver a multitude of proteins and nucleic acids. Exosomes are taken up by recipient cells, thus imposing strong immunomodulatory effects. Release of exosomes is one of the major mechanisms through which Treg cells communicate with surrounding cells ( Figure 1 ). CD8 Treg cells function by secreting NOX2-containing exosomes that then suppress membrane-proximal TCR signaling in nearby CD4⁺ T cells. The loss of NOX2-rich exosomes defines the loss of Treg activity in GCA (12, 13). In recent years, the concept of replacing dysfunctional Treg cells by transferring exosomes has attracted attention, as it might be possible to harness these vesicles for the therapeutic delivery of RNAs, peptides, proteins, and synthetic drugs. As cell-free reagents, therapeutic exosomes have numerous advantages, but challenges remain in achieving proper targeting and delivery. Some progress has been made in realizing the idea of therapeutic exosomes. One example are exosomes derived from anti-tumor CAR-T cells. Such exosomes were capable of attacking cancer cells in a CAR-T cell-free manner (183).

Methods have been developed to produce exosomes or exosome-like nanoparticles with defined payloads and progress is being made in designing ways of delivering the microvesicles (MV) to specific cells, thus increasing local concentrations and minimizing systemic side effects. Given the potent immunosuppression imposed by NOX2-containing exosomes, NOX2-loaded MV or exosome-like nanoparticles may provide the means to mimic Treg function in vivo. This approach would offer numerous advantages: (1) Better access to inflamed tissue sites because of the much smaller size of exosomes compared to Treg cells; (2) Potentially higher suppressive capacity. Treg cells need to use most of their energy and biosynthetic molecules for cellular maintenance, but artificial exosomes containing only their payload can deposit high local concentrations of suppressive proteins; (3) Easier control of unintended systemic immunosuppression. In cell-based Treg therapy, it is impossible to completely remove transduced Treg cells when generalized immunosuppression becomes a problem. In contrast, exosomes are short-lived. (4) Tight management of suppressive ability. Both cytokine and Treg therapy rely on the response of the immune cells in the host to produce the ideal effect. In patients that are immunocompromised, this goal may be difficult to reach. However, exosomes can directly manipulate the targeted cell population, thereby avoiding the intermediate steps, bringing about a more controllable result; (5) Low risk of contamination and rapid turnaround for production. Ex vivo cell engineering is demanding, complicated by possible contamination with pathogens and is a very time-consuming process. Production of exosomes can be achieved under industrialized conditions, can provide therapeutic reagents in a short time, with a low badge to badge variability; (7) Finally, exosomes can be designed to be independent of HLA restriction and could thus be used in the majority of patients.