Sumoylation of RORγt regulates TH17 differentiation and thymocyte development.

RORγt controls the differentiation of TH17 cells, which are mediators of autoimmune conditions such as experimental autoimmune encephalomyelitis (EAE). RORγt also regulates thymocyte development and lymph node genesis. Here we show that the function of RORγt is regulated by its sumoylation. Loss of Sumo3, but not Sumo1, dampens TH17 differentiation and delays the progression of thymic CD8+ immature single-positive cells (ISPs). RORγt is SUMO3-modified by E3 ligase PIAS4 at lysine 31 (K31), and the mutation of K31 to arginine in mice prevents RORγt sumoylation, leading to impaired TH17 differentiation, resistance to TH17-mediated EAE, accumulation of thymic ISPs, and a lack of Peyer's patches. Mechanistically, sumoylation of RORγt-K31 recruits histone acetyltransferase KAT2A, which stabilizes the binding of SRC1 to enhance RORγt transcription factor activity. This study thus demonstrates that sumoylation is a critical mechanism for regulating RORγt function, and reveals new drug targets for preventing TH17-mediated autoimmunity.

Bacteria and their cell wall components uniformly co-activate interleukin-17-producing thymocytes.

Interleukin (IL)-17-producing T cells play a critical role in the immune response against microbial pathogens. Traditionally, experimental studies have focused upon understanding the activity of IL-17-producing T cells which differentiate from naive T cells in the peripheral immune system. However, we have demonstrated previously that IL-17-producing T cells are also present in the thymus of naive wild-type mice and can be co-activated there by microbial stimuli. Other studies have supported the concept that IL-17-producing thymocytes have a specific role in the immediate defence against microbial pathogens, which is independent from the development of an adaptive immune response. Given an important role of the thymus in systemic bacterial infection and sepsis, in this study we investigate the effect of a broad spectrum of bacteria and cell wall components on thymocyte cytokine production. Surprisingly, we find that all types of bacteria investigated (including non-pathogenic species) uniformly activate IL-17-producing thymocytes upon α-CD3 stimulation. In contrast, there is a heterogeneous effect on IL-6 and interferon (IFN)-γ-production with Gram-negative bacteria inducing far higher frequencies of IL-6- and IFN-γ-producing thymocytes than Gram-positive bacteria. We conclude that IL-17-producing thymocytes constitute a 'first line of recognition', but not a 'first line of defence' against bacteria in general. Their activity might lead to immune activation, but not necessarily to a pathological inflammatory disease condition. The difference between these two states might be determined by other immunological effector molecules, such as IL-6 and IFN-γ.

Staphylococcus aureus convert neonatal conventional CD4(+) T cells into FOXP3(+) CD25(+) CD127(low) T cells via the PD-1/PD-L1 axis.

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4(+) T cells for the induction of CD25(+) CD127(low) Treg cells in vitro. The proportion of circulating CD25(+) CD127(low) Treg cells and their expression of FOXP3, Helios and CTLA-4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet-stained non-Treg cells from cord or peripheral blood from adults were co-cultured with autologous CD25(+) CD127(low) Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25(+) CD127(low) Treg cells than adults, but these cells were Helios(+) and CTLA-4(+) to a higher extent than in adults. FOXP3(+) CD25(+) CD127(low) T cells were induced mainly in neonatal CellTrace-stained non-Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25(+) CD127(low) T cells only if sorted naive CD45RA(+) non-Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25(+) CD127(low) T cells from S. aureus-stimulated cultures were still suppressive. Finally, blocking PD-L1 during stimulation reduced the induction of FOXP3(+) CD25(+) CD127(low) T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios(+) Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4(+) T cells into FOXP3(+) CD25(+) CD127(low) Treg cells via the PD-1/PD-L1 axis.

Interferon-γ- and glucocorticoid-mediated pathways synergize to enhance death of CD4(+) CD8(+) thymocytes during Salmonella enterica serovar Typhimurium infection.

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S. typhimurium). Significant death of CD4(+) CD8(+) thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4(+) CD8(+) thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon-γ (IFN-γ), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifnγ(-/-) mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN-γ and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifnγ(-/-) mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifnγ(-/-) mice. Importantly, the number of CD4(+) CD8(+) thymocytes is significantly higher in Ifnγ(-/-) mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN-γ-mediated pathways are parallel but synergize in an additive manner to induce death of CD4(+) CD8(+) thymocytes during S. typhimurium infection. The implications of this study for host responses during infection are discussed.

Foxp4 is dispensable for T cell development, but required for robust recall responses.

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4(+) and CD8(+) T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3(+) T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.