Rohit D. Divekar

The type III histone deacetylase Sirt1 is essential for maintenance of T cell tolerance in mice.

Although many self-reactive T cells are eliminated by negative selection in the thymus, some of these cells escape into the periphery, where they must be controlled by additional mechanisms. However, the molecular mechanisms underlying peripheral T cell tolerance and its maintenance remain largely undefined. In this study, we report that sirtuin 1 (Sirt1), a type III histone deacetylase, negatively regulates T cell activation and plays a major role in clonal T cell anergy in mice. In vivo, we found that loss of Sirt1 function resulted in abnormally increased T cell activation and a breakdown of CD4+ T cell tolerance. Conversely, upregulation of Sirt1 expression led to T cell anergy, in which the activity of the transcription factor AP-1 was substantially diminished.Furthermore, Sirt1 interacted with and deacetylated c-Jun, yielding an inactive AP-1 factor. In addition, Sirt1-deficient mice were unable to maintain T cell tolerance and developed severe experimental allergic encephalomyelitis as well as spontaneous autoimmunity. These findings provide insight into the molecular mechanisms of T cell activation and anergy, and we suggest that activators of Sirt1 may be useful as therapeutic agents for the treatment and/or prevention of autoimmune diseases.

Innocuous IFNγ induced by adjuvant-free antigen restores normoglycemia in NOD mice through inhibition of IL-17 production

The role of Th17 cells in type I diabetes (TID) remains largely unknown. Glutamic acid decarboxylase (GAD) sequence 206–220 (designated GAD2) represents a late-stage epitope, but GAD2-specific T cell receptor transgenic T cells producing interferon γ (IFNγ) protect against passive TID. Because IFNγ is known to inhibit Th17 cells, effective presentation of GAD2 peptide under noninflammatory conditions may protect against TID at advanced disease stages. To test this premise, GAD2 was genetically incorporated into an immunoglobulin (Ig) molecule to magnify tolerance, and the resulting Ig-GAD2 was tested against TID at different stages of the disease. The findings indicated that Ig-GAD2 could not prevent TID at the preinsulitis phase, but delayed TID at the insulitis stage. More importantly, Ig-GAD2 sustained both clearance of pancreatic cell infiltration and β-cell division and restored normoglycemia when given to hyperglycemic mice at the prediabetic stage. This was dependent on the induction of splenic IFNγ that inhibited interleukin (IL)-17 production. In fact, neutralization of IFNγ led to a significant increase in the frequency of Th17 cells, and the treatment became nonprotective. Thus, IFNγ induced by an adjuvant free antigen, contrary to its usual inflammatory function, restores normoglycemia, most likely by localized bystander suppression of pathogenic IL-17–producing cells.

Specific T Regulatory Cells Display Broad Suppressive Functions against Experimental Allergic Encephalomyelitis upon Activation with Cognate Antigen

To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F1 (SJL/J × C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.

A Sudden Decline in Active Membrane-Bound TGF-β Impairs Both T Regulatory Cell Function and Protection against Autoimmune Diabetes

Autoimmunity presumably manifests as a consequence of a shortfall in the maintenance of peripheral tolerance by CD4+CD25+ T regulatory cells (Tregs). However, the mechanism underlying the functional impairment of Tregs remains largely undefined. In this study a glutamic acid decarboxylase (GAD) diabetogenic epitope was expressed on an Ig to enhance tolerogenic function, and the resulting Ig-GAD expanded Tregs in both young and older insulitis-positive, nonobese diabetic (NOD) mice, but delayed autoimmune diabetes only in the former. Interestingly, Tregs induced at 4 wk of age had significant active membrane-bound TGF-β (mTGF-β) and sustained protection against diabetes, whereas Tregs expanded during insulitis had minimal mTGF-β and could not protect against diabetes. The Tregs probably operate suppressive function through mTGF-β, because Ab blockade of mTGF-β nullifies protection against diabetes. Surprisingly, young Tregs that modulated pathogenic T cells maintained stable frequency over time in the protected animals, but decreased their mTGF-β at the age of 8 wk. More strikingly, these 8-wk-old mTGF-β-negative Tregs, which were previously protective, became unable to confer resistance against diabetes. Thus, a developmental decline in active mTGF-β nullifies Treg function, leading to a break in tolerance and the onset of diabetes.

Delayed maturation of an IL-12–producing dendritic cell subset explains the early Th2 bias in neonatal immunity

Primary neonatal T cell responses comprise both T helper (Th) cell subsets, but Th1 cells express high levels of interleukin 13 receptor α1 (IL-13Rα1), which heterodimerizes with IL-4Rα. During secondary antigen challenge, Th2-produced IL-4 triggers the apoptosis of Th1 cells via IL-4Rα/IL-13Rα1, thus explaining the Th2 bias in neonates. We show that neonates acquire the ability to overcome the Th2 bias and generate Th1 responses starting 6 d after birth. This transition was caused by the developmental maturation of CD8α+CD4− dendritic cells (DCs), which were minimal in number during the first few days of birth and produced low levels of IL-12. This lack of IL-12 sustained the expression of IL-13Rα1 on Th1 cells. By day 6 after birth, however, a significant number of CD8α+CD4− DCs accumulated in the spleen and produced IL-12, which triggered the down-regulation of IL-13Rα1 expression on Th1 cells, thus protecting them against IL-4–driven apoptosis.

IL-10 Diminishes CTLA-4 Expression on Islet-Resident T Cells and Sustains Their Activation Rather Than Tolerance

IL-10, a powerful anti-Th1 cytokine, has shown paradoxical effects against diabetes. The mechanism underlying such variable function remains largely undefined. An approach for controlled mobilization of endogenous IL-10 was applied to the NOD mouse and indicated that IL-10 encounter with diabetogenic T cells within the islets sustains activation, while encounter occurring peripheral to the islets induces tolerance. Insulin β-chain (INSβ) 9-23 peptide was expressed on an Ig, and the aggregated (agg) form of the resulting Ig-INSβ triggered IL-10 production by APCs, and expanded IL-10-producing T regulatory cells. Consequently, agg Ig-INSβ delayed diabetes effectively in young NOD mice whose pathogenic T cells remain peripheral to the islets. However, agg Ig-INSβ was unable to suppress the disease in 10-wk-old insulitis-positive animals whose diabetogenic T cells have populated the islets. This is not due to irreversibility of the disease because soluble Ig-INSβ did delay diabetes in these older mice. Evidence is provided indicating that upon migration to the islet, T cells were activated and up-regulated CTLA-4 expression. IL-10, however, reverses such up-regulation, abolishing CTLA-4-inhibitory functions and sustaining activation of the islet T lymphocytes. Therefore, IL-10 supports T cell tolerance in the periphery, but its interplay with CTLA-4 sustains activation within the islets. As a result, IL-10 displays opposite functions against diabetes in young vs older insulitis-positive mice.

Fetal Exposure to High-Avidity TCR Ligand Enhances Expansion of Peripheral T Regulatory Cells

Lately, it has become clear that regulatory T cells (Tregs) play a major role in the maintenance of peripheral tolerance and control of autoimmunity. Despite these critical functions, the process underlying the development of Tregs remains largely undefined. Herein, altered peptide ligand (APL) variants derived from the proteolipid protein-1 (PLP1) epitope were expressed on immunoglobulins (Igs) and the resulting Ig-APLs were used to deliver the APLs from mother to fetus through the maternal placenta to influence thymic T cell selection. This delivery system was then adapted to the SJL/J mouse, a strain that expresses only the DM20 form of PLP, which lacks the dominant PLP1 epitope in the thymus during fetal and neonatal development. This model, which restores thymic T cell selection for PLP1, was then used to determine whether affinity plays a role in the development of Tregs. The findings show that fetal exposure to low-affinity peptide ligand was unable to drive development of Tregs while variants with higher affinity to the TCR resulted in significant seeding of the periphery with mature, naive Tregs. Thus, contrary to pathogenic T cells, Tregs require avid TCR-ligand interaction to undergo thymic development and maturation.

T Cell Dynamics during Induction of Tolerance and Suppression of Experimental Allergic Encephalomyelitis

The cell dynamics associated with induction of peripheral T cell tolerance remain largely undefined. In this study, an in vivo model was adapted to two-photon microscopy imaging, and T cell behavior was analyzed on tolerogen-induced modulation. FcγR-deficient (FcγR−/−) mice were unable to resist or alleviate experimental allergic encephalomyelitis when treated with Ig-myelin oligodendrocyte glycoprotein (MOG) tolerogen, an Ig carrying the MOG35–55 peptide. However, when FcγR+/+ dendritic cells (DCs) are adoptively transferred into FcγR−/− mice, uptake and presentation of Ig-MOG occurs and the animals were able to overcome experimental allergic encephalomyelitis. We then fluorescently labeled FcγR+/+ DCs and 2D2 MOG-specific TCR-transgenic T cells, transferred them into FcγR−/− mice, administered Ig-MOG, and analyzed both T cell–DC contact events and T cell motility. The results indicate that tolerance takes place in lymphoid organs, and surprisingly, the T cells do not become anergic but instead have a Th2 phenotype. The tolerant Th2 cells displayed reduced motility after tolerogen exposure similar to Th1 cells after immunization. However, the Th2 cells had higher migration speeds and took longer to exhibit changes in motility. Therefore, both Th1 immunity and Th2 tolerance alter T cell migration on Ag recognition, but the kinetics of this effect differ among the subsets.

Early Effector T Cells Producing Significant IFN-γ Develop into Memory

Currently, transition of T cells from effector to memory is believed to occur as a consequence of exposure to residual suboptimal Ag found in lymphoid tissues at the waning end of the effector phase and microbial clearance. This led to the interpretation that memory arises from slightly activated late effectors producing reduced amounts of IFN-γ. In this study, we show that CD4 T cells from the early stage of the effector phase in which both the Ag and activation are optimal also transit to memory. Moreover, early effector T cells that have undergone four divisions expressed significant IL-7R, produced IFN-γ, and yielded rapid and robust memory responses. Cells that divided three times that had marginal IL-7R expression and no IFN-γ raised base level homeostatic memory, whereas those that have undergone only two divisions and produced IFN-γ yielded conditioned memory despite low IL-7R expression. Thus, highly activated early effectors generated under short exposure to optimal Ag in vivo develop into memory, and such transition is dependent on a significant production of the cell’s signature cytokine, IFN-γ.

In Trans T Cell Tolerance Diminishes Autoantibody Responses and Exacerbates Experimental Allergic Encephalomyelitis

A number of Ag-specific approaches have been developed that ameliorate experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis. Translation to humans, however, remains a consideration, justifying the search for more insight into the mechanism underlying restoration of self-tolerance. Ig-proteolipid protein (PLP) 1 and Ig-myelin oligodendrocyte glycoprotein (MOG) are Ig chimeras carrying the encephalitogenic PLP 139–151 and MOG 35–55 amino acid sequence, respectively. Ig-PLP1 ameliorates EAE in SJL/J (H-2s) mice while Ig-MOG modulates the disease in C57BL/6 (H-2b) animals. In this study, we asked whether the chimeras would suppress EAE in F1 mice expressing both parental MHC alleles and representing a polymorphism with more relevance to human circumstances. The results show that Ig-MOG modulates both PLP1 and MOG peptide-induced EAE in the F1 mice, whereas Ig-PLP1 counters PLP1 EAE but exacerbates MOG-induced disease. This in trans aggravation of MOG EAE by Ig-PLP1 operates through induction of PLP1-specific T cells producing IL-5 that sustained inhibition of MOG-specific Abs leading to exacerbation of EAE. Thus, in trans T cell tolerance, which should be operative in polymorphic systems, can aggravate rather than ameliorate autoimmunity. This phenomenon possibly takes place through interference with protective humoral immunity.