Transcriptional regulation and development of regulatory T cells

Regulatory T (Treg) cells are a distinct subset of CD4⁺ T cells. Instead of triggering adaptive immunity, they suppress immune responses. Small numbers of Treg cells reside within lymphoid organs and peripheral tissues, but their contribution to immune tolerance is so significant that defects in Treg cell function cause catastrophic immune disorders. Since they were first discovered 20 years ago, efforts have been made to understand the differences in developmental processes between Treg cells and conventional T cells that determine the ultimate fate of the overall T-cell population. Transcription factor Foxp3 is crucial for Treg cell differentiation, but it is not the whole story. Owing to recent advances in Treg cell research, we are now on the verge of appreciating the comprehensive mechanisms underlying Treg cell generation. Here, we discuss major discoveries, active study topics and remaining questions regarding Treg cell development.

The transcription factor Batf3 inhibits the differentiation of regulatory T cells in the periphery

Naive CD4 T cells activated by antigen-presenting cells (APCs) undergo terminal differentiation in the periphery. Multiple mechanisms determine their fates, that is, whether they differentiate into conventional T (Tconv) cells or regulatory T (Treg) cells. The key event during Treg generation is expression of the transcription factor Foxp3, which is the lineage-determining regulator for Treg differentiation and function. Here we show that the transcription factor Batf3 acts as a fate-decision factor with respect to Tconv versus Tregs by restraining Treg differentiation. Batf3 was preferentially expressed in effector CD4 T cells but not in Treg cells, and ectopic expression of Batf3 inhibited Foxp3 induction. Batf3-deficient CD4 T cells favorably differentiated into Treg cells in vitro and in colonic lamina propria. Batf3 KO mice also showed enhanced Treg function in gut-associated immune disease models (for example, ovalbumin tolerance and inflammatory bowel disease models). Batf3 bound to the CNS1 region of the Foxp3 locus and reduced expression of the gene. Thus, Batf3 is a transcriptional suppressor of Treg differentiation.

Casein kinase 2 is a critical determinant of the balance of Th17 and Treg cell differentiation

Th17 cells promote inflammatory reactions, whereas regulatory T (Treg) cells inhibit them. Thus, the Th17/Treg cell balance is critically important in inflammatory diseases. However, the molecular mechanisms underlying this balance are unclear. Here, we demonstrate that casein kinase 2 (CK2) is a critical determinant of the Th17/Treg cell balance. Both the inhibition of CK2 with a specific pharmacological inhibitor, CX-4945, and its small hairpin RNA (shRNA)-mediated knockdown suppressed Th17 cell differentiation but reciprocally induced Treg cell differentiation in vitro. Moreover, CX-4945 ameliorated the symptoms of experimental autoimmune encephalomyelitis and reduced Th17 cell infiltration into the central nervous system. Mechanistically, CX-4945 inhibited the IL-6/STAT3 and Akt/mTOR signaling pathways. Thus, CK2 has a crucial role in regulating the Th17/Treg balance.

The requirement of natural killer T-cells in tolerogenic APCs-mediated suppression of collagen-induced arthritis

TGF-beta-induced tolerogenic-antigen presenting cells (Tol-APCs) could induce suppression of autoimmune diseases such as collagen-induced arthritis (CIA) and allergic asthma. In contrast, many studies have shown that NKT cells are involved in the pathogenesis of Th1-mediated autoimmune joint inflammation and Th2-mediated allergic pulmonary inflammation. In this study, we investigated the effect of CD1d-restricted NKT cells in the Tol-APCs-mediated suppression of autoimmune disease using a murine CIA model. When CIA-induced mice were treated with Tol-APCs obtained from CD1d+/- or CD1d-/- mice, unlike CD1d+/- APCs, CD1d-/- Tol-APCs failed to suppress CIA. More specifically, CD1d-/- Tol-APCs failed to suppress the production of inflammatory cytokines and the induction of Th2 responses by antigen-specific CD4 T cells both in vitro and in vivo. Our results demonstrate that the presence of CD1d-restricted NKT cells is critical for the induction of Tol-APCs-mediated suppression of CIA.

TGF-beta-treated antigen presenting cells suppress collagen-induced arthritis through the promotion of Th2 responses

Collagen-induced arthritis (CIA) is mediated by self-reactive CD4+ T cells that produce inflammatory cytokines. TGF-beta2-treated tolerogenic antigen-presenting cells (Tol-APCs) are known to induce tolerance in various autoimmune diseases. In this study, we investigated whether collagen-specific Tol-APCs could induce suppression of CIA. We observed that Tol-APCs could suppress the development and severity of CIA and delay the onset of CIA. Treatment of Tol-APCs reduced the number of IFN-gamma- and IL-17-producing CD4+ T cells and increased IL-4- and IL-5-producing CD4+ T cells upon collagen antigen stimulation in vitro. The suppression of CIA conferred by Tol-APCs correlated with their ability to selectively induce IL-10 production. We also observed that treatment of Tol-APCs inhibited not only cellular immune responses but also humoral immune responses in the process of CIA. Our results suggest that in vitro-generated Tol-APCs have potential therapeutic value for the treatment of rheumatoid arthritis as well as other autoimmune diseases.

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8+ iNKT cells in mice raised the question of whether CD8+ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8+ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Valpha14+ transgenic mice, where the Valpha14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8+ iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8+ iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8+ iNKT cells in wild type mice. Our results suggest that CD8+ NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.