Induction Of Peripheral Tolerance Research Articles

PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens.

The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3-10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.

295-OR: Mechanisms of Antigen-Specific Immunotherapy following Tolerance Induction to a CD4 Hybrid Insulin Peptide Epitope and the Impact on CD8 T Cell Dysfunction

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

A single-dose, tolerizing vaccine induces antigen-specific regulatory T cells and durable remission while preserving healthy immunity in a mouse model of Multiple sclerosis

Abstract Multiple sclerosis (MS) is an inflammatory disorder that occurs when autoreactive lymphocytes mistakenly attack myelin, resulting in motor deficits and cognitive deterioration. Current drugs are non-curative and require life-long treatment that make compliance challenging, and existing therapeutics can leave patients vulnerable to opportunistic infections. An experimental approach to overcome these hurdles is to drive long-lasting, antigen-specific regulatory T cells (Tregs) that dampen responses to autoantigens. Here, we co-deliver antigen and rapamycin in micron-sized, diffusion-limited, polymer microparticles (MPs) directly to lymph nodes (LNs) where immune responses are orchestrated. Using adoptive transfer of transgenic T cells, we show that MPs generate antigen-specific Tregs in response to two different MP-derived peptides – evidence that MPs do not interfere with induction of peripheral tolerance in LNs. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, single-dose treatment demonstrates potent, long-lasting efficacy (up to three months) following a single treatment. Tolerizing effects are observed during chronic disease – the most challenging for treatment regimens. Efficacy is accompanied by reduced demyelination in spinal cords of treated mice, the cause of paralysis in EAE and MS. Thus, antigen-specific reprogramming of Tregs in LNs reshapes inflammation in distal tissues without need for systemic or repeated dosing. Importantly, MP-treated EAE mice mount healthy immune responses to vaccine challenge. This work advances a strategy to locally reprogram antigen-specific, tolerogenic responses that preserve healthy immunity – a hurdle faced by current MS therapeutics. Supported by grants from NIH (R01 AI169686).

Suppression of IGRP-specific CD8 T cells following induction of tolerance to a Hybrid Insulin Peptide CD4 neoepitope

Abstract An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX 3CR1 +effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction. NIH - T32 5T32DK120520-03, R01 2R01DK081166-11) JDRF 2-SRA-2020-907-S-B

Abstract 4060: Selective Neoantigens Peptide TILs (snapTILs): A next generation tumor infiltrating lymphocytes (TIL) therapy platform

Abstract Background: Immunotherapies using autologous tumor infiltrating lymphocytes (TILs) have become effective treatment modalities for several patients with solid tumors, yielding a response rate of around 49% in patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TILs from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of these administered TILs recognize tumor antigens and most are non-mutated public antigens. Moreover, expression of these antigens on normal cells can trigger central and peripheral tolerance mechanisms. Against this background, cancer neoantigens derived from private mutations represent an ideal class of cancer antigens to target because they are highly tumor specific by nature, therefore reducing the potential induction of central and peripheral tolerance. We recently developed a new technology to enrich TILs with predefined neoantigen specificities, which we termed Selective Neo Antigens Peptides TILs (snapTILs). Here we describe the efficacy studies of a melanoma and pancreatic cancer patient’s snapTILs done in autologous settings both ex vivo and in vivo. Methods: Our workflow consisted of a multistep process performed on a patient-specific basis. Briefly, mutations were called by whole-exome sequencing of tumor vs. normal DNA to generate a peptide library. The peptides were further filtered using an in-silico prediction algorithm and our proprietary peptide-MHC binding assay. Selected peptides were then incubated with TILs isolated from the patient’s tumor biopsy, resulting in TIL population enriched of T cells that recognize the neopeptides (snapTILs). After rapid expansion, snapTIL efficacy was tested by checking their ability to infiltrate in patient-derived tumor organoids, their activation status (IFNγ), cytotoxicity (Granzyme B), and T-cell exhaustion markers. Lastly, snapTILs in vivo efficacy was tested in human melanoma and pancreatic cancer xenograft models using hIL-2 transgenic mice. Results: Our initial data showed that the levels of infiltration of snapTILs into tumor organoids was significantly higher compared to IL2-stimulated TILs in both cancer models. Also, snapTILs recognized tumors with great specificity as they showed significantly less infiltration in organoids generated from generally uninvolved tissues from the same patients. Moreover, snapTILs were found to be more cytotoxic in nature than the IL2-stimulated TILs in terms of IFNγ and Granzyme B secretion after coculture with autologous tumor cells. Our in vivo data confirmed these observations by showing 70% tumor growth inhibition in highly immunogenic melanoma and 50% tumor growth restriction in a poorly immunogenic pancreatic tumor model. In conclusion, our approach proves the potential for personally tailored immunotherapies to yield more effective and safer cancer treatments using snapTILs. Citation Format: Tithi Ghosh Halder, Shelby Rheinschmidt, Sydney Adamson, Anna Larson, Taylor Bargenquast, Ryan Rodriguez del Villar, Serina Ng, Kate Gutowsky, Alexis Weston, Trason Thode, Mohan Kaadige, Erin Kelley, Jorge Soria-Bustos, Jorge Giron, John Altin, Raffaella Soldi, Sunil Sharma. Selective Neoantigens Peptide TILs (snapTILs): A next generation tumor infiltrating lymphocytes (TIL) therapy platform. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4060.

CRISPR Activation Screening Identifies VGLL3-TEAD1-RUNX1/3 as a Transcriptional Complex for PD-L1 Expression.

The PD-L1/2-PD-1 immune checkpoint is essential for the proper induction of peripheral tolerance and limits autoimmunity, whereas tumor cells exploit their expression to promote immune evasion. Many different cell types express PD-L1/2, either constitutively or upon stimulation, but the factors driving this expression are often poorly defined. In this study, using genome-wide CRISPR activation screening, we identified three factors that upregulate PD-L1 expression: GATA2, MBD6, and transcription cofactor vestigial-like protein 3 (VGLL3). VGLL3 acts as a transcriptional regulator, and its expression induced PD-L1 in many different cell types. Conversely, loss of VGLL3 impaired IFN-γ-induced PD-L1/2 expression in human keratinocytes. Mechanistically, by performing a second screen to identify proteins acting in concert with VGLL3, we found that VGLL3 forms a complex with TEAD1 and RUNX1/3 to drive expression of PD-L1/2. Collectively, our work identified a new transcriptional complex controlling PD-L1/2 expression and suggests that VGLL3, in addition to its known role in the expression of proinflammatory genes, can balance inflammation by upregulating the anti-inflammatory factors PD-L1 and PD-L2.

Nanoparticle-Based Tolerogenic Vaccines for the Treatment of Autoimmune Diseases: A Review

Current treatments for autoimmune diseases can only ameliorate the symptoms of the disease and may cause immunocompromised or opportunistic diseases. Recent advances in nanotechnology and immunology have facilitated the development of tolerogenic immunotherapies against autoimmune diseases, which show more promising outcomes in animal models and early clinical trials than conventional therapies. Tolerogenic immunotherapies employ the characteristics and functions of immune cells to downregulate the autoreactivity of immune responses that damage tissues and organs. Here, we review the utilization of nanoparticles as a tolerogenic vaccine platform to induce peripheral immune tolerance against self-antigen via the induction of tolerogenic dendritic cells and regulatory T cells. After introduction of the general mechanism for the induction of peripheral immune tolerance, the materials design of recent nanoparticle-based tolerogenic vaccines, their delivering strategies, and regulation of the autoreactive immune responses in disease models are reviewed. Last, the opportunities and challenges of these approaches are discussed.

Double-negative T cells: Setting the stage for disease control or progression.

Double-negative (DN) T cells are present at relatively low frequencies in human peripheral blood, and are characterized as expressing the alpha-beta or gamma-delta T-cell receptor (TCR), but not the CD4 nor the CD8 co-receptors. Despite their low frequencies, these cells are potent producers of cytokines and, thus, are key orchestrators of immune responses. DN T cells were initially associated with induction of peripheral immunological tolerance and immunomodulatory activities related to disease prevention. However, other studies demonstrated that these cells can also display effector functions associated with pathology development. This apparent contradiction highlighted the heterogeneity of the DN T-cell population. Here, we review phenotypic and functional characteristics of DN T cells, emphasizing their role in human diseases. The need for developing biomarkers to facilitate the translation of studies from animal models to humans will also be discussed. Finally, we will examine DN T cells as promising therapeutic targets to prevent or inhibit human disease development.

Regulatory T cells in acute and chronic human Chikungunya infection

Chikungunya virus (CHIKV) infection generates strong immune responses that are associated with the disease pathophysiology. Regulatory T cells (Treg-cluster of differentiation (CD)-4+CD25highforkhead box P3 (FOXP3+)) are essential for the induction and maintenance of peripheral tolerance. Thus, they play key roles in determining the patient prognosis by preventing excessive immune responses via different suppression immune mechanisms. However, the regulatory mechanisms involved in human CHIKV infection are still poorly understood. Here, we characterize for the first time the Treg cell molecule-associated-mechanism during acute and chronic human Chikungunya disease. Here, we assessed the Treg cell population and molecule-associated mechanism in the peripheral blood samples of acute and chronic patients with Chikungunya. Our results indicate that CHIKV infection is associated with reduced frequency of Tregs, along with the impaired expression and production of Treg functional markers, including CD39, CD73, perforin, granzyme, programmed death 1 (PD-1), cytotoxic T lymphocyte antigen (CTLA)-4, and transforming growth factor (TGF)-β. This observation suggests that Treg cells possess the poor regulatory capacity in both acute and chronic phases of the disease. Taken together, these data provide significant evidence that the imbalanced response of Treg cells plays an essential role in establishing the pathogenesis of Chikungunya.

Enhancing Antigen Presentation and Inducing Antigen-Specific Immune Tolerance with Amphiphilic Peptides.

Ag-specific immunotherapy to restore immune tolerance to self-antigens, without global immune suppression, is a long-standing goal in the treatment of autoimmune disorders such as type 1 diabetes (T1D). However, vaccination with autoantigens such as insulin or glutamic acid decarboxylase have largely failed in human T1D trials. Induction and maintenance of peripheral tolerance by vaccination requires efficient autoantigen presentation by APCs. In this study, we show that a lipophilic modification at the N-terminal end of CD4+ epitopes (lipo-peptides) dramatically improves peptide Ag presentation. We designed amphiphilic lipo-peptides to efficiently target APCs in the lymph nodes by binding and trafficking with endogenous albumin. Additionally, we show that lipophilic modification anchors the peptide into the membranes of APCs, enabling a bivalent cell-surface Ag presentation. The s.c. injected lipo-peptide accumulates in the APCs in the lymph node, enhances the potency and duration of peptide Ag presentation by APCs, and induces Ag-specific immune tolerance that controls both T cell- and B cell-mediated immunity. Immunization with an amphiphilic insulin B chain 9-23 peptide, an immunodominant CD4+ T cell epitope in NOD mice, significantly suppresses the activation of T cells, increases inhibitory cytokine production, induces regulatory T cells, and delays the onset and lowers the incidence of T1D. Importantly, treatment with a lipophilic β-cell peptide mixture delays progression to end-stage diabetes in acutely diabetic NOD mice, whereas the same doses of standard soluble peptides were not effective. Amphiphilic modification effectively enhances Ag presentation for peptide-based immune regulation of autoimmune diseases.

CD27 expression on Treg cells limits immune responses against tumors

Regulatory T cells (Tregs) suppress immune responses and thus contribute to immune homeostasis. On the downside, Tregs also limit immune responses against tumors promoting the progression of cancer. Among the many mechanisms implied in Treg-mediated suppression, the inhibition of dendritic cells (DCs) has been shown to be central in peripheral tolerance induction as well as in cancers. We have shown previously that the maintenance of peripheral T cell tolerance critically depends on cognate interactions between Tregs and DCs and that the CTL priming by unsuppressed steady state DCs is mediated via CD70. Here, we have investigated whether the CD70/CD27 axis is also involved in Treg-mediated suppression of anti-tumor immunity. Using a mixed bone marrow chimeric mouse model in which we can deplete regulatory T cells in a temporally controlled fashion, we show that Treg-expressed CD27 prevents the breakdown of peripheral tolerance and limits anti-tumor immunity. Furthermore, ablation of Treg expressed CD27 acts synergistically with PD-1 checkpoint inhibition to improve CTL mediated immunity against a solid tumor. Our data thus identify Treg-expressed CD27 as a potential target in cancer immunotherapy.Key messagesTreg expressed CD27 maintains steady state DC tolerogenicTreg expressed CD27 limits anti-tumor immunityAblation of Treg expressed CD27 synergizes with PD-1 blockade to improve CTL mediated tumor control

NKG2D Enhances Double-Negative T Cell Regulation of B Cells.

Numerous studies reported a small subpopulation of TCRαβ+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

The Therapeutic Potential of Regulatory T Cells: Challenges and Opportunities.

Regulatory T cells (Tregs) are an immunosuppressive subgroup of CD4+ T cells which are identified by the expression of forkhead box protein P3 (Foxp3). The modulation capacity of these immune cells holds an important role in both transplantation and the development of autoimmune diseases. These cells are the main mediators of self-tolerance and are essential for avoiding excessive immune reactions. Tregs play a key role in the induction of peripheral tolerance that can prevent autoimmunity, by protecting self-reactive lymphocytes from the immune reaction. In contrast to autoimmune responses, tumor cells exploit Tregs in order to prevent immune cell recognition and anti-tumor immune response during the carcinogenesis process. Recently, numerous studies have focused on unraveling the biological functions and principles of Tregs and their primary suppressive mechanisms. Due to the promising and outstanding results, Tregs have been widely investigated as an alternative tool in preventing graft rejection and treating autoimmune diseases. On the other hand, targeting Tregs for the purpose of improving cancer immunotherapy is being intensively evaluated as a desirable and effective method. The purpose of this review is to point out the characteristic function and therapeutic potential of Tregs in regulatory immune mechanisms in transplantation tolerance, autoimmune diseases, cancer therapy, and also to discuss that how the manipulation of these mechanisms may increase the therapeutic options.

Treg cell therapy: How cell heterogeneity can make the difference.

CD4+ CD25high CD127low/- FOXP3+ T regulatory cells are responsible for maintaining immune tolerance and controlling excessive immune responses. Treg cell use in pre-clinical animal models showed the huge therapeutic potential of these cells in immune-mediated diseases and laid the foundations for their applications in therapy in humans. Currently, there are several clinical trials utilizing the adoptive transfer of Treg cells to reduce the morbidity in autoimmune disorders, allogeneic HSC transplantation, and solid organ transplantation. However, a large part of them utilizes total Treg cells without distinction of their biological variability. Many studies on the heterogeneity of Treg cell population revealed distinct subsets with different functions in the control of the immune response and induction of peripheral tolerance. Some of these subsets also showed a role in controlling the general homeostasis of non-lymphoid tissues. All these Treg cell subsets and their peculiar properties can be therefore exploited to develop novel therapeutic approaches. This review describes these functionally distinct subsets, their phenotype, homing properties and functions in lymphoid and non-lymphoid tissues. In addition, we also discuss the limitations in using Treg cells as a cellular therapy and the strategies to enhance their efficacy.

Influence of Innate Immunity on Immune Tolerance.

This review mainly focuses on the mechanisms of peripheral immune tolerance within the perspectives of innate immunity. Healthy immune response requires balanced interaction of the highly specialized elements of immunity within a harmony. Innate immunity supported by microbial pattern recognition receptors, physical anatomical barriers and soluble effectors stands as the first line of defense against non-self-antigens. Innate receptors recognize major classes of pathogens and trigger immediate immune/inflammatory responses. The decisive action has been the key issue in skewing of immune reactivity to a pathogen or to tolerate self- and non-self-antigens. Non-responsiveness to self- or to harmless foreign antigens with means of multiple mechanisms is known as immune tolerance; a non-inflammatory, non-proliferative and suppressive response linked to suppressor molecules as CTLA-4 and cytokines like IL-10, TGF-β and IL-35, and also to non-inflammatory blocking antibody isotypes as IgG4. Regulatory cells ascertain both induction and maintenance of peripheral tolerance. Allergic diseases, autoimmunity and transplant rejection are the best illustrations of immune tolerance loss. Adaptive immunity responsible for both establishment and maintenance of a long-lasting immune responsiveness is mainly fine-tuned by actions of innate immunity. Better understanding of the relationship between innate immunity and immune tolerance is a prerequisite both for better understanding of pathogenesis of tolerance-related diseases and also for development of novel therapeutic options. CONCLUSION: Recent evidences point the important roles of innate immunity for establishment of immune tolerance with decisive role in central mechanisms. In a peremptory way, a 'balanced tolerance' is essential for the survival.

Increased Protein Stability and Interleukin-2 Production of a LATG131D Variant With Possible Implications for T Cell Anergy.

The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-γ activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LATG131D) increases phosphorylation of Tyr132, PLC-γ activation and calcium influx generation. Interestingly, the LATG131D mutant has a slower protein turnover while being equally sensitive to Fas-mediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LATG131D secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LATG131D mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance.

Type I interferons induce peripheral T regulatory cell differentiation under tolerogenic conditions.

The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/β is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/β play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/β on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.

Inhibitors-Recent insights.

The development of inhibitory antibodies to therapeutic factor VIII (FVIII) in haemophilia A (HA) patients is the major complication in treatment/prevention of haemorrhages. The reasons some HA patients develop inhibitors while others do not remain unclear. This review briefly summarizes our understanding of anti-FVIII immune responses, the roles of T cells, both effector and regulatory, and generally discusses the interplay between FVIII and the immune system, both in factor replacement therapy and gene therapy, with some comparisons to factor IX and haemophilia B therapies. Notably, we propose that the prevailing observed active tolerance to FVIII in both HA and non-HA individuals rests to greater or lesser extents on peripherally induced immune tolerance. We also propose that the immune systems of inhibitor-negative HA patients do not merely ignore therapeutic FVIII, but rather have immunologically assessed and actively tolerized the patients to exogenous FVIII. Induction of such peripheral immune tolerance may further be triggered in HA patients who failed to tolerize upon initial FVIII exposure by 'appropriate' stimulation of their immune system, eg by immune tolerance induction therapy via intensive FVIII therapy, by oral administration of FVIII, by cellular therapies or by gene therapy directed to immuno-tolerogenic sites such as the liver.

312 Activation of AhR in Langerhans cells by a microbial metabolite of tryptophan maintains skin homeostasis

Skin commensal bacteria play important roles in maintenance of skin homeostasis, and Langerhans cells (LCs) are critical in induction of peripheral tolerance; however, the role of LCs' response to the skin microbiota, specifically the microbial metabolites, in skin homeostasis is not clear. In the present study, LCs that derived from CD34+ hematopoietic stem cells in the cord blood cells were utilized, and we found that a microbial metabolite of tryptophan (Trp), indole-3-aldehyde (IAId), induced the production of indoleamine 2,3-dioxygenase and IL-10 in LCs through activation of aryl hydrocarbon receptor (AhR). Moreover, IAId promoted the expression of receptor activator of NF-κB and receptor activator of NF-κB ligand on LCs and keratinocytes in an AhR-dependent manner respectively, which might result in the activation of NF-κB signaling and production of IL-10. Additionally, despite a mature phenotype of LCs induced by IAId, IAId-activated LCs inhibited CD4+ T cell proliferation and induced IL-10 secretion. Our study revealed a negatively regulatory function of a Trp microbial metabolite on LCs through activation of AhR, and microbial metabolites could be developed as a new strategy for the treatment of inflammatory skin diseases.

Intravenous tolerance induces T cell anergy/apoptosis via IFN-γ/IL-27/PD-L1 axis

Abstract The induction of peripheral tolerance by intravenous injection (i.v.) of auto-Ags has shown to suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis, by eliciting tolerogenic APCs, Tregs, and suppressing self-reactive T cells. The programmed cell death protein (PD)-1 and its ligands, PD-L1 and PD-L2, regulate the balance between T cell activation and immune tolerance. Although PD-L1 and PD-L2 are expressed on a variety of cell types, however, the mechanisms related to peripheral tolerance that induce these molecules are still unclear. Here we show that PD-L1, but not PD-L2, is critical for inducing peripheral tolerance in EAE, and that PD-L1-dependent tolerance relies on both IFN-γ and IL-27. I.v. administration of auto-Ag(s) attenuates the progression of EAE, by inducing PD-L1 expression in monocyte-derived DCs (moDCs) in the CNS, via IFN-γ/ IL-27-dependent mechanism. Indeed, when PD-L1 is blocked, autoantigen(s)/i.v treatment becomes ineffective. Following IFN-γ−/− CD4+ T cell transfer into Rag1−/− mice, i.v. tolerance treatment failed to suppress EAE. Moreover, loss of IFN-γR signaling in classical DCs type I (cDCs I) compromised IL-27 production and consequent PD-L1 expression by moDCs. We also show that IL-27 stimulates moDCs to over-express PD-L1, in a STAT-3-dependent way. Indeed, in CCR2−/−/WSX−/− (CD45.2) → CD45.1 radiation-induced bone marrow chimera mice, auto-Ag(s)/i.v. treatment failed to suppress EAE. Similarly, EAE mice that expressed mutant STAT-3 (muSTAT-3) in CCR2+ cells were also insensitive to i.v. tolerance induction. Our data reveal a mechanism whereby IFN-γ and IL-27 are the key factors in PD-L1 induction and subsequent peripheral tolerance restoration.