Danielle M. Tartar

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.

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.

FoxP3+RORγt+ T Helper Intermediates Display Suppressive Function against Autoimmune Diabetes

Recently, traces of double-positive FoxP3+RORγt+ T cells were identified and viewed as dual programming differentiation intermediates geared toward development into T regulatory or Th17 cells. In this study, we report that FoxP3+RORγt+ intermediates arise in the NOD mouse T cell repertoire prior to inflammation and can be expanded with tolerogen without further differentiation. Furthermore, FoxP3+RORγt+ cells express both CD62L and membrane-bound TGFβ and use the former to traffic to the pancreas and the latter to suppress effector T cells both in vitro and in vivo. The cells perform these functions as FoxP3+RORγt+ intermediates, despite being able to terminally differentiate into either FoxP3+RORγt− T regulatory or FoxP3−RORγt+ Th17 cells on polarization. These previously unrecognized observations extend plasticity to both differentiation and function and indicate that the intermediates are poised to traffic to sites of inflammation and target diverse pathogenic T cells, likely without prior conditioning by effector T cells, thus broadening efficacy against autoimmunity.

Coordinate Regulation of GATA-3 and Th2 Cytokine Gene Expression by the RNA-Binding Protein HuR

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate–rich elements (AREs) present in the 3′ untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3′UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

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.

Mechanisms Underlying Antigen-Specific Tolerance of Stable and Convertible Th17 Cells During Suppression of Autoimmune Diabetes

Type 1 diabetes involves both T helper (Th)1 and Th17 cells. While the mechanisms underlying the control of Th1 cells are relatively well defined, those operating modulation of Th17 cells remain unknown. Moreover, given that Th17 cells are plastic and can drive disease as stable or convertible T cells, effective approaches to counter type 1 diabetes would have to alter Th17 function under both circumstances. Herein, we genetically incorporated the BDC2.5-reactive p79 mimotope into an Ig molecule, and the resulting Ig-p79 was used to investigate Th17 tolerance. Accordingly, diabetogenic BDC2.5 Th17 cells were transferred into NOD mice under convertible or stable conditions and their fate was evaluated upon induction of tolerance and disease suppression by Ig-p79. The findings show that convertible (Th17 to Th1) cells display downregulation of the chemokine (C-X-C motif) receptor 3 that was associated with diminished T-box transcription factor T-bet expression, retention in the spleen, and inhibition of trafficking to the pancreas. In contrast, stable Th17 cells downregulated orphan nuclear receptor ROR-γt but increased Fas ligand expression and died by apoptosis. Thus, the final signature transcription factor shapes the mechanism of tolerance in plastic Th17 cells. These findings suggest that effective strategies against type 1 diabetes will require regimens that could drive both mechanisms of tolerance to overcome the disease.

Bone Marrow-Derived IL-13Ra1-Positive Thymic Progenitors Are Restricted to the Myeloid Lineage

The earliest thymic progenitors (ETPs) were recently shown to give rise to both lymphoid and myeloid cells. Whereas the majority of ETPs are derived from IL-7Rα–positive cells and give rise exclusively to T cells, the origin of the myeloid cells remains undefined. In this study, we show both in vitro and in vivo that IL-13Rα1+ ETPs yield myeloid cells with no potential for maturation into T cells, whereas IL-13Rα1− ETPs lack myeloid potential. Moreover, transfer of lineage-negative IL-13Rα1+ bone marrow stem cells into IL-13Rα1–deficient mice reconstituted thymic IL-13Rα1+ myeloid ETPs. Myeloid cells or macrophages in the thymus are regarded as phagocytic cells whose function is to clear apoptotic debris generated during T cell development. However, the myeloid cells derived from IL-13Rα1+ ETPs were found to perform Ag-presenting functions. Thus, IL-13Rα1 defines a new class of myeloid restricted ETPs yielding APCs that could contribute to development of T cells and the control of immunity and autoimmunity.

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.