Glucocorticoids are potent anti-inflammatory and immunosuppressive agents used for treatment of autoimmune and inflammatory conditions (15–17). They mediate their effects through engagement with the nuclear glucocorticoid receptor inducing transcriptional regulation or rapid non-genomic effects (15, 17). In immune cells, the synthetic glucocorticoid dexamethasone suppresses production of most cytokines while increasing production of IL-10, inhibits lymphocyte activation and promotes lymphocyte apoptosis (15). Glucocorticoids dampen overall T cell activation by interfering with TCR signaling, and evidence suggests that glucocorticoids preferentially suppresses Th1 and Th17 T cells. Glucocorticoid treatment is also associated with increased circulating Tregs (15). In APCs, glucocorticoids induce tolerogenic features including attenuated DC maturation and reduced expression of MHC class II and co-stimulatory molecules (15, 18).

Tolerogenic vaccines consisting of antigen delivered with dexamethasone have been successful in murine models of experimental autoimmune encephalitis (EAE) (19–22), atherosclerosis (23), antigen-induced arthritis (AIA) (24) and type 1 diabetes (T1D) (25). Other tolerogenic vaccines use dexamethasone to induce tolDCs, which when loaded with disease relevant antigens and injected reduced disease in models of arthritis (26, 27), dust mite allergy (28) and T1D (29). The disease inhibition was often accompanied by an increase in Tregs over effector T cells and increase in anti-inflammatory cytokines. However, tolDC-mediated disease suppression in one arthritis model was independent of antigen (27) and antigen-loaded dexamethasone-derived tolDCs exacerbated a model of T1D while unloaded tolDCs suppressed disease (30) ( Table 1 ).

Dexamethasone is often used together with other adjuvants to induce tolDCs. TolDC vaccines of dexamethasone in combination with one or more other adjuvants suppressed disease in models of EAE (31, 32) and systemic lupus erythematosus (SLE) (33) ( Table 5 ).

Dexamethasone-containing tolerogenic vaccines have been investigated for human use. A phase I clinical trial investigated transfer of antigen-loaded dexamethasone-treated monocyte-derived DCs for treatment of multiple sclerosis and neuromyelitis optica spectrum disorders, by intravenous injection of tolDC every two weeks for a total of three doses. The tolDC vaccine was well tolerated and there was an increase of regulatory Tr1 cells at 12 weeks of follow-up (34). In another phase I trial, antigen-loaded autologous monocyte-derived DCs treated with a combination of dexamethasone, the vitamin D derivative calcitriol, and the TLR4 agonist MPLA were administered into inflamed knee joints. The treatment was well tolerated but did not result in reduction in disease severity or consistent immunoregulatory features (35) ( Table 6 ).

Rapamycin is a small molecule inhibitor of mTOR, a kinase that regulates cell growth and metabolism. Immunosuppressive effects of rapamycin includes suppression of the activation and proliferation of conventional T cells, promotion of T cell anergy or deletion, and enhancement of the development and function of Tregs (36). Additionally, rapamycin can inhibit the differentiation and maturation of DCs and promote tolDC features (37). Rapamycin is clinically used as an immunosuppressant for prevention of organ transplant rejection, as well as an anti-cancer drug.

Tolerogenic nanoparticle vaccines with antigen and rapamycin have successfully reduced disease in multiple models of autoimmunity including arthritis (38), Alzheimer’s disease (39), vitiligo (40), allergic airway disease (41, 42), T1D (43), and EAE (44, 45). Rapamycin-containing nanoparticle vaccines also prevented anti-drug antibody responses toward coagulation factor VIII (FVIII) (45, 46), uricase (47), adalimumab (anti-TNFα) (47), and adenoviral vectors used in gene therapy (48–50). Administration of rapamycin without nanoparticle carrier suppressed OVA-induced skin graft rejection (51) and anti-FVIII anti-drug antibodies in mice (52). Observed immunological alterations in studies of tolerogenic vaccines with rapamycin included increased Tregs, decreased levels of inflammatory cytokines, and/or decreased co-stimulatory molecules on DCs ( Table 1 ).

Two phase Ia and phase Ib clinical trials were conducted investigating administration of rapamycin together with pegadricase, a biological drug used for the treatment of gout, finding that rapamycin suppressed the development of anti-drug antibodies in a dose dependent manner (53). Their following phase III trial demonstrated high response rate, safety, and clinically meaningful reduction in serum urate (54). Another phase I clinical trial administered rapamycin-containing nanoparticles together with AAV8 capsid used in adenoviral gene therapies, leading to reduced development of anti-AAV8 antibodies compared to subjects receiving capsid without rapamycin (press release) ( Table 6 ).

Cyclosporine A and FK506 (tacrolimus) are calcineurin inhibitors which suppress downstream nuclear factor of activated T cell (NFAT) signaling and IL-2 production (55). Tolerogenic vaccination with cyclosporine A and autoantigens prevented the development of T1D in pre-diabetic mice and promoted Tregs and tolDCs (56). Likewise, tolerogenic vaccination with FK506 co-delivered with DNA encoding autoantigen suppressed EAE (57), and adoptive transfer of antigen-loaded FK506-induced tolDCs reduced disease in a model of CIA (58), in both studies with a reduction in Th17 responses ( Table 1 ).

Nuclear factor kappa B (NFκB) is a transcription factor inducing expression of pro-inflammatory genes including cytokines, cell adhesion molecules, and immunoreceptors. Inhibition of NFκB suppresses T cell responses and induces immunoregulatory features in APCs (59).

Tolerogenic vaccination with antigen-loaded tolDCs treated with NFκB inhibitor BAY 11-7082 suppressed disease in AIA (60), and antigen-loaded tolDCs treated with NFκB inhibitor andrographolide reduced anti-drug antibodies in hemophilia A mice (61). A nanoparticle vaccine loaded with antigen and A20, an anti-inflammatory protein inhibiting NF-κB activation (62), suppressed Th2 responses and reduced disease in an asthma model (63) ( Table 1 ).

A phase I clinical trial investigated antigen-loaded autologous Bay11-7082-treated tolDCs in rheumatoid arthritis. The vaccine reduced effector T cells and increased Tregs, together with a reduction in inflammatory cytokines. The treatment did not induce disease flares and lead to decreased rheumatoid arthritis DAS28 score (64) ( Table 6 ).

Kynurenine is an immunoregulatory tryptophan metabolite signaling via the aryl hydrocarbon receptor (AhR). AhR signaling can influence T cell differentiation and function of APCs to favor expansion of Tregs (65, 66). Tolerogenic vaccination with nanoparticles loaded with autoantigen and the AhR agonist ITE (2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester), suppressed disease in models of EAE (67, 68) and T1D (69), and tolerogenic vaccination with kynurenine and antigen-expressing phages prevented hyperglycemia in a model of T1D (70) ( Table 1 ).

Janus kinase (JAK) and signal transducer and activator of transcription proteins (STAT) signaling induces immune cell activation and cytokine production (71). Tolerogenic vaccination with autoantigen-loaded tolDCs treated with JAK/STAT inhibitors tofacitinib and BD750 suppressed disease, reduced Th1 and Th17 responses, and increased Tregs in models of EAE (72, 73) ( Table 1 ).

Rosiglitazone is an anti-diabetic drug that activates peroxisome proliferator–activated receptor gamma (PPARγ). In immune cells, rosiglitazone can inhibit inflammatory cytokines and promote tolerogenic APCs (74). TolDC vaccination with antigen-loaded, rosiglitazone-treated tolDCs suppressed disease in CIA (75) ( Table 1 ), and autoantigen-loaded tolDCs treated with rosiglitazone in combination with dexamethasone and cobalt (III) protoporphyrin suppressed a murine model of SLE (33) ( Table 5 ).

Inhibition of the protein kinase glycogen synthase kinase 3 (GSK-3) in immune cells leads to reduced inflammatory cytokine production and increased IL-10 (76). Vaccination with antigen-loaded tolDCs treated with GSK-3β inhibitor K313 suppressed disease in EAE (77) ( Table 1 ).

Prostaglandin I2 (PGI2) is a lipid signaling mediator most known for its vasodilating and anti-thrombotic effects. PGI2 also has anti-inflammatory properties and protective effects in allergy and asthma (78, 79). Tolerogenic vaccination with antigen-loaded tolDCs treated with PGI2 analog iloprost reduced disease in ovalbumin-induced asthma (80). Additionally, direct delivery of iloprost and antigen using a hydrogel suppressed antigen-induced lung inflammation and increased the frequency of antigen-specific Tregs (80) ( Table 1 ).

TGF-β is a strongly immunosuppressive cytokine, because genetic deficiency of TGF-β leads to fatal autoimmunity (81). TGF-β is an immunosuppressive cytokine with multiple effects on the immune system: it promotes Treg development and function, inhibits B and T cell proliferation, suppresses differentiation of Th1 and Th2 cells, and induces tolDCs (82). However, when combined with specific other cytokines, TGF-β may trigger T cells to differentiate into non-regulatory phenotypes such as Th17 effectors in presence of IL-6 and Th9 in presence of IL-4 (82).

A tolerogenic nanoparticle vaccine containing autoantigen and TGF-β reduced disease and immune cell activation in EAE (83). TolDC vaccination with autoantigen-loaded tolDCs cultured in presence of TGF-β or TGF-β receptor agonist suppressed disease in a CIA model (84) and reduced anti-drug antibodies toward FVIII (85), together with increases in Tregs and IL-10 ( Table 2 ).

In combination with other adjuvants, tolerogenic vaccines using both TGF-β and IL-10 suppressed disease in CIA (86) and reduced anti-drug antibodies toward FVIII (87). Tolerogenic vaccination with microparticles containing GM-CSF and TGF-β1 alongside nanoparticles with antigen and vitamin D suppressed EAE and T1D (88–92) and microparticles loaded with TGF-β, retinoic acid, and autoantigens suppressed T1D (93) ( Table 5 ).

A tolerogenic DNA vaccination with autoantigen-encoding plasmids in combination with plasmids for either TGF-β, IL-10, and or IL-2 suppressed disease in EAE (94) and T1D (95) ( Table 5 ). A phase I clinical trial is registered for this tolerogenic DNA vaccine to evaluate vaccine safety in patients with T1D ( Table 6 ).

IL-10 is a powerful immunosuppressive and anti-inflammatory cytokine, absence of which causes spontaneous colitis in mice (96). IL-10 suppresses antigen presentation and inflammatory cytokine production by APCs and simultaneously increases their release of anti-inflammatory mediators. In CD4⁺ T cells, IL-10 inhibits proliferation and cytokine production and promotes the development of regulatory Tr1 cells (97).

Tolerogenic vaccination with antigen-loaded tolDCs engineered to express IL-10 suppressed disease in mouse models of T1D and asthma (98, 99), and antigen-loaded tolDCs cultured in presence of IL-10 reduced disease in EAE (100). Tolerogenic vaccination with nanoparticles containing IL-10 and antigen suppressed disease in EAE (101), and DNA vaccines encoding IL-10 and antigen suppressed disease in EAE and T1D models (102, 103) ( Table 2 ).

A probiotic vaccine of Lactococcus lactis (L. lactis) genetically engineered to secrete IL-10 and pro-insulin administered together with anti-CD3 ameliorated disease and increase Tregs in models of T1D (104–106) ( Table 5 ). The L. lactis probiotic vaccine has been studied in human clinical trials where results from phase Ib and IIa studies demonstrated treatment to be safe, metabolic variables were either stabilized or improved, and antigen-specific CD8⁺ T cells were reduced (107) ( Table 6 ).

IL-2 mediates T cell survival, differentiation, and proliferation. IL-2 is specifically required for Treg homeostasis and suppression of autoimmunity and genetic deletion results in systemic autoimmunity in mice (108). In addition, recent studies showed that low-dose IL-2 treatment induces the expansion of Treg cells and had efficacy in numerous mouse models and some early efficacy in clinical trials of T1D, graft-vs-host disease and SLE. Different types of tolerogenic vaccination with IL-2 treatment in combination with antigen exposure suppressed disease in models of EAE (109), experimental autoimmune uveitis (EAU) (110, 111), T1D (112), delayed-type hypersensitivity (DTH) (112), and reduced development of anti-drug antibodies toward FVIII in hemophilia A (113). Overall, the vaccines led to increased Tregs and anti-inflammatory cytokines ( Table 2 ).

Tolerogenic vaccines using IL-2 in combination with rapamycin expanded Tregs and suppressed disease in models of T1D (114, 115), EAE (116), and primary biliary cholangitis (114). Furthermore IL-2 in combination with Retinoic Acid suppressed EAE and EAU (117). These combination vaccines expanded Tregs or induced antigen-specific Tr1 cells ( Table 5 ).

Interferon beta (IFN-β) is a type I interferon with immunomodulatory properties, used therapeutically for multiple sclerosis (MS). IFN-β reduces T cell activation, promotes Tregs and induces tolDCs (118, 119). Tolerogenic vaccines comprised of autoantigen and IFN-β suppressed murine and rat models of EAE via the induction of neuroantigen-specific, suppressive CD25⁺ Tregs (120, 121) ( Table 2 ). In addition, mice treated with IFN-β while receiving autoantigen-loaded vitamin D-treated tolDCs, further suppressed disease in a model of EAE (122) ( Table 5 ).

In addition to being a growth factor and chemokine, GM-CSF possesses anti-inflammatory effects. Administration of GM-CSF leads to a reduction in disease severity in several animal models of autoimmune disease (123), and GM-CSF promotes development and function of both tolDCs and Tregs (123, 124).

Tolerogenic vaccines with antigen-GM-CSF conjugates using neuropeptide autoantigens have been used to treat EAE in mice and rats, accompanied by increased Tregs (125–129). Additionally, GM-CSF is used for differentiation of DCs for most tolDC vaccines. Although most tolDC vaccines use additional adjuvants, also without other adjuvants transfer of antigen-loaded GM-CSF differentiated tolDCs suppressed murine EAU (130) ( Table 2 ). A hydrogel/microparticle vaccine incorporating GM-CSF and TLR9 agonist CpG with autoantigen suppressed disease in a model of T1D (131) ( Table 5 ).

IL-35 is a potent inducer of Tregs and regulatory B cells, and it can inhibit the proliferation and function of effector Th1 and Th17 cells. IL-35 has been shown to be protective against autoimmune disease and IL-35 treatment have been able to suppress disease in multiple models of autoimmunity and chronic inflammation (132). In tolerogenic vaccines, tolDCs engineered to overexpress IL-35 and loaded with disease relevant antigen suppressed EAE and DTH (133, 134) ( Table 2 ).

IL-27 is an immunoregulatory cytokine which can promote tolerance by supporting development of Tregs and Tr1, antagonizing development of Th2 and Th17 cells, and by increasing co-inhibitory receptor expression on APCs (135). Tolerogenic vaccination by adoptive transfer of IL-27-conditioned antigen-loaded DCs led to a significant amelioration of disease and reduction in Th1 and Th17 cells in murine EAE (136) ( Table 2 ).

IL-4 promotes type 2 immunity and suppresses Th1 polarization. Treatment with IL-4 suppressed disease severity in models of EAE and arthritis (137, 138). Tolerogenic DNA vaccines encoding IL-4 and antigen suppressed murine models of CIA, EAE, and T1D (139–142) ( Table 2 ).

Hepatocyte growth factor (HGF) is a cytokine with pleiotropic effects, including the promotion of tolDCs (143). In a model of EAE, systemic HGF ameliorated disease and tolerogenic vaccination with HGF-treated antigen-loaded DCs mediated functional recovery in mice with established EAE and suppressed T cell mediated inflammation (144) ( Table 2 ).

Vasoactive intestinal peptide (VIP) is a peptide functioning as a neurotransmitter in the central and peripheral nervous systems and has multiple effects, including immune modulation. VIP reduces the release of inflammatory cytokines, stimulates production of IL-10 and TGF-β, and decreases the co-stimulatory activity of APCs (145). Tolerogenic vaccination with antigen-loaded VIP-treated tolDCs led to amelioration of CIA and EAE, accompanied by increased levels of Tr1 cells (146) ( Table 2 ).

TNF-related apoptosis-inducing ligand (TRAIL) is a cytokine that induces apoptosis and activation of NFκB. TRAIL has immunoregulatory effects demonstrated by the exacerbated development of autoimmunity in TRAIL-deficient mice (147). Tolerogenic vaccination with DCs engineered to co-express TRAIL and antigen reduced antigen-specific T cell responses and disease symptoms in models of EAE (148, 149) ( Table 2 ).

Just as the addition of anti-inflammatory or immunoregulatory cytokines can promote tolerogenic responses, silencing of inflammatory cytokines can also be effective. Silencing of B cell activating factor (BAFF), an essential cytokine for both T and B cell activation (150), in TolDCs using siRNA suppressed murine CIA and promoted Tregs (151) ( Table 2 ).

Vitamin D is primarily known for its role in calcium homeostasis and bone health, but it is also immunoregulatory. Having low levels of vitamin D is associated with increased susceptibility to a variety of infectious and autoimmune diseases. Vitamin D suppresses T cell activation, skews T cell differentiation away from Th17 while promoting Tregs and Th2 responses, and promotes tolerogenic DC features, including low surface expression of co-stimulatory molecules and decreased production of inflammatory cytokines (10, 152). Vitamin D signals via a nuclear receptor and exert immunoregulatory effects by regulating gene expression (10).

Many tolerogenic vaccines using vitamin D are tolDC vaccines. Vitamin D-treated tolDCs loaded with antigen or antigen-encoding mRNA reduced disease and promoted immunoregulatory cells and cytokines in murine models of EAE (153–156). The vitamin D-treated tolDCs had reduced expression of MHC class II, co-stimulatory molecules, and pro-inflammatory cytokines, and induced less T cell proliferation compared to DCs that were untreated (153, 154) ( Table 2 ). TolDC vaccines using a combination of vitamin D and dexamethasone suppressed murine models of arthritis (26) and T1D (29) ( Table 5 ).

Tolerogenic nanoparticle vaccines with vitamin D have been examined in T1D (157, 158), where the nanoparticles reduced disease incidence and increased Tregs or tolDCs in vivo. Separate delivery studies of vitamin D and antigen have been studied in EAE (159, 160) and DTH (161), in all studies reducing disease severity and inflammation ( Table 2 ).

Vitamin D-containing tolerogenic vaccines have been investigated in human clinical trials. In a phase IIa trial of latent autoimmune diabetes in adult patients were treated with daily oral Vitamin D and monthly injections of antigen-alum. The trial demonstrated safety and stable β-cell function and metabolic control at 5 months follow-up (162). Two phase I clinical trials have been registered to test tolerogenic vaccination with antigen-loaded vitamin D-treated monocyte-derived DCs in multiple sclerosis (163). Likewise, a phase I clinical trial tested monocyte-derived DC loaded with proinsulin and treated with Vitamin D and dexamethasone in T1D. The study showed the treatment was safe and lead to reduce proinsulin specific CD8+ T cells and increased ICOS⁺ CCR4⁺ TIGIT⁺ Tregs (164). Another phase I trial investigated liposomes containing collagen II and calcitriol, the active form of vitamin D, for the treatment of rheumatoid arthritis. The calcitriol-antigen-liposomes led to reduced pathogenic T cells and expansion of antigen-specific PD1⁺ T cells (165) ( Table 6 ).

Retinoic acid is an immunoregulatory vitamin A metabolite, which like vitamin D signals via a nuclear receptor (10). Retinoic acid promotes the development and function of Tregs while inhibiting the differentiation and activation of effector Th1 and Th17 cells. DCs and macrophages can produce retinoic acid to support Treg induction and maintenance, and the retinoic acid-producing capacity of DCs is further enhanced upon retinoic acid exposure (10, 166).

Tolerogenic vaccination with liposomes incorporating retinoic acid and autoantigen converted pathogenic autoantigen specific Th17 cells to Tr1 cells and suppressed disease in EAE (167). Tolerogenic vaccination with retinoic acid, IL-2, and autoantigen suppressed EAE and EAU and pathogenic Th17 and Th1 responses (117). Prophylactic tolerogenic vaccination with retinoic acid, TGF-β and autoantigen inhibited the incidence of T1D in mice (93), and in another study treatment of mice with autoantigen and retinoic acid in combination with IL-4 suppressed EAE (168) ( Tables 2 , 6 ).

CD3 is the invariant chain of the TCR. Anti-CD3 monoclonal antibodies suppresses disease in numerous animal models of autoimmunity, and anti-CD3 is an FDA approved treatment to delay early onset type 1 diabetes. The exact mechanism of anti-CD3-mediated immune suppression is unclear but proposed mechanisms include prevention of T cells from recognizing their antigens and the induction of anergy or apoptosis in activated T cells while sparing Tregs (169). Tolerogenic vaccination with anti-CD3 treatment in combination with an antigen-expressing lentiviral vector suppressed T1D and induced autoantigen-specific Tregs (170). Anti-CD3 is also a component of the previously described probiotic vaccine (104–106) ( Tables 3 , 5 ).

CD4 is a glycoprotein on helper T cells which primarily functions as a TCR co-receptor. Non-depleting anti-CD4 therapy has been shown to suppress autoimmunity and graft rejection by modulating the function of CD4⁺ T cells by blocking T cell activation and promoting Treg differentiation and suppressor functions (171). A tolerogenic vaccine comprised of aluminum hydroxide (alum), FVIII, and non-depleting anti-CD4 prevented development of anti-drug antibodies in mice (172). Another tolerogenic vaccine using treatment with depleting anti-CD4 antibodies followed by antigen administration suppressed disease in a murine model of EAU via the induction of antigen-specific Tregs (173) ( Table 3 ).

“Tregitopes” are peptides derived from IgG that are recognized by a subset of natural Tregs. When presented in MHCII, these peptides activate Tregitope-specific Tregs and suppression of effector T cell responses to co-delivered antigens. Administration of nanoparticles with antigen and tregitopes decreased incidence and severity of T1D in mice (174). Likewise, co-administration of autontigen with Tregitope-albumin fusion proteins decreased incidence and reverse mild T1D (175) ( Table 3 ).

Invariant natural killer T cells (iNKT cells) are immunoregulatory T cells important for preventing autoimmune reactions. The glycolipid α-galactosylceramide (α-GalCer) is a strong inducer of iNKT cells and has been shown to suppress disease in multiple animal models of autoimmunity (176). A tolerogenic vaccine comprised of lipid nanoparticles carrying autoantigen and α-GalCer prevented the development of diabetes in prediabetic mice (177) ( Table 3 ).

Including MHC class II-targeting molecules in a tolerogenic vaccine ensures delivery to APCs and may disrupt the immunological synapse. A tolerogenic vaccine with antigen conjugated to antibody fragments (nanobodies) targeting MHC class II suppressed disease in EAE. When combined with dexamethasone, the vaccine also overcame the inflammation associated with antigen exposure (178) ( Table 5 ).

Co-stimulatory signals, such as CD80 and CD86, are necessary for T cell activation by APCs. T cell recognition of antigen on MHC II without co-stimulation results in anergy or apoptosis. Abatacept, a cytotoxic T-lymphocyte associated protein 4 (CTLA-4)-Fc fusion protein blocks CD80 and CD86 and is FDA approved for the treatment of autoimmune arthritis (179). Tolerogenic vaccination with nanoparticles displaying abatacept and carrying autoantigen and dexamethasone suppressed EAE (180) ( Table 5 ).

Signaling via CD40-CD40L induces activation and pro-inflammatory cytokine production in both B cells, T cells, and APCs, and CD40L-blockade reduced disease in numerous animal models of autoimmunity (181). Tolerogenic vaccination with FVIII and anti-CD40 prevented subsequent development of anti-FVIII antibodies during rechallenge (182) ( Table 3 ).

Other approaches include genetic modification of co-stimulatory signals. Antigen delivered with a CRISPR-Cas9 plasmid and guide RNAs toward CD80, CD86, and CD40 disrupted co-stimulation by DCs, reduced inflammatory cytokines, increased Tregs, and suppressed disease in a model of T1D (183) ( Table 5 ). Administration of a DNA vector encoding membrane-bound autoantigen together with a B7.1/CD40L mutant fusion protein binding to CTLA-4 but not CD28, providing co-inhibitory but not co-stimulatory signals, reduced disease incidence in murine model of T1D (184) ( Table 3 ).

Intercellular adhesion molecule 1 (ICAM-1) is a cell surface glycoprotein most known for its role in leukocyte migration. Inhibition of ICAM-1 can block T cell activation and induce tolerance by disrupting T cell-APC interactions, inhibiting co-stimulation, promoting PD-L1 expression, and by inducing T cell anergy or exhaustion (185). A tolerogenic vaccine comprised of hyaluronic acid with autoantigen and an ICAM-1 inhibitory peptide suppressed disease in EAE (186–188). In another tolerogenic vaccine, fusion molecules of antigen and ICAM-1 inhibitory peptide prevented the development of T1D (189) and suppressed EAE (190) ( Table 3 ).

OX40 is a TNF receptor superfamily member expressed by activated T cells and resting Tregs, which acts as a co-stimulatory molecule promoting cell proliferation (191). Tolerogenic vaccination of prediabetic mice with antigen and an OX40 agonistic antibody reduced diabetes incidence and increased antigen specific Tregs (192) ( Table 3 ).

Programmed cell death protein 1 (PD-1) is an immune checkpoint molecule that induces T cell apoptosis and suppresses conventional T cell activation and favors development of Tregs (193). Treatment with tolDCs engineered to express PD-L1 and MOG reduced antigen-specific T cell responses and suppressed MOG induced EAE but not MBP induced EAE (148, 149) ( Table 3 ).

B and T lymphocyte attenuator (BTLA) is an inhibitory receptor structurally related to PD-1, activation of which leads to the suppression of T cell activation (194), and BTLA-expressing DCs promoted Treg development (195). Adoptive transfer of bone marrow derived DCs treated with a nanoparticle containing antigen and a BTLA-encoding plasmid suppressed EAE (196) ( Table 3 ).

Fas is a death receptor inducing apoptosis upon binding to Fas ligand (FasL). A tolerogenic vaccine using FasL-conjugated microparticles containing monocyte chemotactic protein-1 (MCP-1) recruited T cells and then induced their apoptosis. When coupled with respective antigens, the microparticles could suppress EAE and prevented development of T1D in pre-diabetic mice (197). ( Table 5 ) A tolerogenic vaccine aiming for engagement of multiple inhibitory receptors for immune suppression used microparticles displaying surface PD-L1-Fc, anti-Fas, and self-marker CD47, and containing TGF-β. The vaccine led to reduced T cell infiltration and EAE suppression (198, 199) ( Table 5 ).

CD22 and siglec G are inhibitory receptors inhibiting B cell receptor (BCR) signaling, thus suppressing B cell responses (200). Tolerogenic vaccination with liposomes displaying antigen and CD22/siglec G ligands induced antigen-specific tolerance in mice and reduce development of anti-drug antibodies toward FVIII in a model of hemophilia A (201) and reduced antigen-specific antibody production (202) ( Table 3 ). Encapsulation of rapamycin in CD22L autoantigen liposomes suppressed arthritis in mice (203–205) ( Table 5 ).

Apoptotic cells are cleared by phagocytic cells via anti-inflammatory mechanisms, which in part is mediated by phosphatidylserine exposed on the apoptotic cell surface (206). Therefore, phosphatidylserine liposomes, O-phospho-L-serine (OPLS), or pro-apoptotic factors have been used as tolerogenic adjuvants to reduce antigen immunogenicity.

In hemophilia A, both co-delivery of FVIII with OPLS and tolerogenic nanoparticle vaccination with FVIII encapsulated in phosphatidylserine liposomes led to a reduction in anti-drug antibodies toward FVIII in mice (207, 208), and antigen-containing phosphatidylserine liposomes reduced disease incidence in a model of T1D (209). In addition, phosphatidylserine liposomes loaded with collagen peptide and the immunomodulator leflunomide, which inhibits the mitochondrial enzyme dihydroorotate dehydrogenase preventing uridine synthesis, suppressed CIA in mice (210). Furthermore, a DNA vaccine encoding antigen and the pro-apoptotic protein BAX, promoting apoptosis in cells expressing antigen, suppressed T1D via modulation of APC function and promotion of Treg development (211, 212) ( Table 4 ).

TLR agonists are well known inflammatory stimuli and often used as adjuvants in immunogenic vaccines to enhance the immune response to the target antigen (213). However, signaling through microbial pattern recognizing receptors might also protect from development of autoimmunity. Dose and administration of the TLR agonist affects if the response is immunogenic or tolerogenic, it is believed that a short-term, high-dose stimulation will result in immunogenic responses whereas low-dose, repeated stimulation results in tolerance (214).

TLR4 agonist LPS is often used in cultures of DCs to induce maturation and enhance their antigen presenting capacity. Antigen-loaded LPS-treated DCs suppressed EAE while non-treated DCs did not (215). A recombinant fusion protein of autoantigen and flagellin A, TLR5 agonist, induced production of IL-6 and IL-10 in DCs and reduced T cell-driven inflammation in a murine model of intestinal allergy (216, 217). Co-delivery of flagellin B and antigen reduced disease in models of allergy (218, 219) and a fusion protein of antigen and flagellin B reduced disease and IgE responses in allergy (220) ( Table 4 ).

TLR9 agonist CpG DNA has been used in different tolerogenic vaccines in combination with other adjuvants. Treatment with a hydrogel vaccine containing CpG DNA, antigen, and GM-CSF prevented and delayed disease onset in pre-diabetic mice, and the inclusion of CpG DNA enhanced efficacy compared to GM-CSF alone (131). Tolerogenic vaccination with antigen, CpG DNA, and heat shock protein 60 induced an antigen-specific increase in IL-10 production and reduced disease severity in a model of arthritis (221) ( Table 5 ).

The use of glycans in tolerogenic vaccine design can mediate tolerance by targeting antigen to APCs expressing receptors for these glycans, promoting its uptake and processing, while concurrently inducing immunoregulatory effects in DCs (14, 222, 223).

β-glucan is a polysaccharide naturally occurring in cell walls of plants, bacteria, and fungi, and binds to Dectin-1 on myeloid cells. In a model of T1D, treatment with β-glucan and antigen led to increased protection from disease compared to treatment with β-glucan or antigen alone, and promoted tolDC features and increased Tregs (224). Conjugates of allergens to mannan, targeted these antigens to APCs expressing mannose and C-type lectin receptors, and promoted a tolerogenic response in comparison to native allergens in vitro and in vivo (222, 223). Skin-prick tests with mannan-conjugated grass pollen allergoids caused less inflammation than native allergens in patients with grass pollen allergy (225), and immunization of mice with mannan-allergoid conjugates led to tolerogenic responses and increase in Foxp3⁺ Tregs compared to native antigen (225, 226) ( Table 4 ). Mannan-allergoid conjugates have been tested for dust mite and grass pollen allergens in two phase II clinical trials, showing improvement in nasal provocation test (227, 228) ( Table 6 ).

Galectin-1 is a glycan-binding protein with diverse functions, including modulation of DCs and T cell responses (229). In a study of EAE, treatment of DCs with galectin-1 led to tolDC differentiation that suppressed EAE when loaded with relevant autoantigen (230). The disease suppression was dependent on IL-27 and IL-10 induced by galectin-1 (230) ( Table 4 ).