Lineage Stability Research Articles

The role of AP-1 family transcription factor networks in regulating Th17 cell identity

Abstract CD4 T cells can adopt one of two opposing fates: a helper T cell (Th) specialized in supporting pathogen clearance, or a regulatory T cell (Treg) that attenuates immune responses. IL-17A-producing inflammatory Th17 cells stand out among Th cells, possessing a high level of inherent plasticity in response to altered environments. The underlying mechanisms controlling such flexibility are largely unknown. In this regard, we have identified the AP-1 family of transcription factors (TFs) as key regulators of Th17 cell identity. Our previous work revealed the pioneering role of BATF in supporting global enhancer accessibility and flexibility in CD4 T cells, and here, we describe an antagonistic AP-1 TF, JunB, that limits Th17 plasticity. Indeed, we find that CD4 T cells deficient in JunB exhibit dysregulated effector cytokine and TF expression signatures, and lack disease potential in a mouse model of autoimmunity. In particular, JunB serves to restrain inappropriate Treg and Th1 differentiation. Here, we (i) use fate-mapping reporter mice to dissect the requirement for JunB in maintaining Th17 cell stability, (ii) define the JunB targets that facilitate subset conversion events; (iii) define novel genomic lineage-restriction elements regulated by AP-1 complexes; and (iv) define activating and repressing roles of JunB that support Th17 lineage stability. Such mechanistic insight is key to guiding future work aimed at exploiting the AP-1 balance in Th17 cells to divert damaging inflammatory responses into favorable regulatory responses.

The NF-κB transcription factor RelA is required for the tolerogenic function of Foxp3+ regulatory T cells

The properties of CD4+ regulatory T cell (Treg) subsets are dictated by distinct patterns of gene expression determined by FOXP3 and different combinations of various transcription factors. Here we show the NF-κB transcription factor RelA is constitutively active in naïve and effector Tregs. The conditional inactivation of Rela in murine FOXP3+ cells induces a rapid onset, multi-focal autoimmune disease that depends on RelA being expressed in conventional T cells. In addition to promoting Treg lineage stability, RelA determines the size of the effector Treg population, a function influenced by the presence or absence of RelA in conventional T cells. These findings showing that RelA controls Treg stability and promotes the competitive fitness of effector Tregs highlight the importance of RelA activity in peripheral Treg induced tolerance.

Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis.

Regulatory T (Treg) cells respond to immune and inflammatory signals to mediate immunosuppression, but how functional integrity of Treg cells is maintained under activating environments remains elusive. Here we found that autophagy was active in Treg cells and supported their lineage stability and survival fitness. Treg cell-specific deletion of the essential autophagy gene Atg7 or Atg5 led to loss of Treg cells, increased tumor resistance, and development of inflammatory disorders. Atg7-deficient Treg cells had increased apoptosis and readily lost Foxp3 expression, especially after activation. Mechanistically, autophagy deficiency upregulated mTORC1 and c-Myc function and glycolytic metabolism that contributed to defective Treg function. Therefore, autophagy couples environmental signals and metabolic homeostasis to protect lineage and survival integrity of Treg cells in activating contexts.

Foxp3(+) T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation.

Foxp3 (forkhead box P3 transcription factor)-expressing regulatory T cells (Tregs) are essential for immunological tolerance, best illustrated by uncontrolled effector T-cell responses and autoimmunity upon loss of Foxp3 expression. Tregs can adopt specific effector phenotypes upon activation, reflecting the diversity of functional demands in the different tissues of the body. Here, we report that Foxp3(+)CD4(+) T cells coexpressing retinoic acid-related orphan receptor-γt (RORγt), the master transcription factor for T helper type 17 (Th17) cells, represent a stable effector Treg lineage. Transcriptomic and epigenetic profiling revealed that Foxp3(+)RORγt(+) T cells display signatures of both Tregs and Th17 cells, although the degree of similarity was higher to Foxp3(+)RORγt(-) Tregs than to Foxp3(-)RORγt(+) T cells. Importantly, Foxp3(+)RORγt(+) T cells were significantly demethylated at Treg-specific epigenetic signature genes such as Foxp3, Ctla-4, Gitr, Eos, and Helios, suggesting that these cells have a stable regulatory rather than inflammatory function. Indeed, adoptive transfer of Foxp3(+)RORγt(+) T cells in the T-cell transfer colitis model confirmed their Treg function and lineage stability in vivo, and revealed an enhanced suppressive capacity as compared with Foxp3(+)RORγt(-) Tregs. Thus, our data suggest that RORγt expression in Tregs contributes to an optimal suppressive capacity during gut-specific immune responses, rendering Foxp3(+)RORγt(+) T cells as an important effector Treg subset in the intestinal system.

Thecamoebians (Testate Amoebae) Straddling the Permian-Triassic Boundary in the Guryul Ravine Section, India: Evolutionary and Palaeoecological Implications.

Exceptionally well-preserved organic remains of thecamoebians (testate amoebae) were preserved in marine sediments that straddle the greatest extinction event in the Phanerozoic: the Permian-Triassic Boundary. Outcrops from the Late Permian Zewan Formation and the Early Triassic Khunamuh Formation are represented by a complete sedimentary sequence at the Guryul Ravine Section in Kashmir, India, which is an archetypal Permian-Triassic boundary sequence [1]. Previous biostratigraphic analysis provides chronological control for the section, and a perspective of faunal turnover in the brachiopods, ammonoids, bivalves, conodonts, gastropods and foraminifera. Thecamoebians were concentrated from bulk sediments using palynological procedures, which isolated the organic constituents of preserved thecamoebian tests. The recovered individuals demonstrate exceptional similarity to the modern thecamoebian families Centropyxidae, Arcellidae, Hyalospheniidae and Trigonopyxidae, however, the vast majority belong to the Centropyxidae. This study further confirms the morphologic stability of the thecamoebian lineages through the Phanerozoic, and most importantly, their apparent little response to an infamous biological crisis in Earth’s history.

Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.

Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226.

Regulatory T cells (Tregs) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4(+)FOXP3(+)Helios(+) thymic-derived Tregs and CD4(+)FOXP3(+)Helios(-) T cells, followed by comparison with CD4(+)FOXP3(-)Helios(-) T conventional cells. These analyses revealed that the coinhibitory receptor T cell Ig and ITIM domain (TIGIT) was highly expressed on thymic-derived Tregs. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4(+)CD25(+)CD127(lo/-) T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Tregs after activation and in vitro expansion, and is associated with lineage stability and suppressive capacity. Conversely, the CD226(+)TIGIT(-) population was associated with reduced Treg purity and suppressive capacity after expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Tregs in health and disease.

Produtividade de grãos, adaptabilidade e estabilidade de genótipos de soja de ciclos precoce e médio

The work was developed with objective of evaluate the grain yield, the adaptability and phenotypic stability of soybean cultivars and lineages with earlier and medium cycles. The experiments were carried out in Viçosa, Florestal, São Gotardo and Rio Paranaíba, in the Minas Gerais State, in two agricultural years. We used the experimental design of blocks completely with treatments allocate at random, in four repetitions. Wasobtain the production relative index to the patterns (cultivar) and employed the Annicchiarico and Centroid methods for adaptability and stability fenotipic analyzes. The genotypes x environments x crop seasonsinteraction (GxLxA) for yield grains was significant and, for the R2, it was detected that the differential performance of the genotypes in relation to the environments (R2 = 50,17%) it was bigger than in to the crop seasons (R2 = 13,81%). The lineage CS 801 showed adaptability to high-tech environments, surpassing the productivity of standards cultivars used. The CAC-1, UFV-16, UFV-19 cultivars and CS 801, CS 802 and UFV98 700,739 lineage were classified as general adaptability.

Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2.

The ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2-mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1(high)PDGFRα(-). Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2-dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability.

Retinoic Acid Is Essential for Th1 Cell Lineage Stability and Prevents Transition to a Th17 Cell Program

SummaryCD4+ T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4+ T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells.

Memory T follicular helper CD4 T cells.

T follicular helper (Tfh) cells are the subset of CD4 T helper cells that are required for generation and maintenance of germinal center reactions and the generation of long-lived humoral immunity. This specialized T helper subset provides help to cognate B cells via their expression of CD40 ligand, IL-21, IL-4, and other molecules. Tfh cells are characterized by their expression of the chemokine receptor CXCR5, expression of the transcriptional repressor Bcl6, and their capacity to migrate to the follicle and promote germinal center B cell responses. Until recently, it remained unclear whether Tfh cells differentiated into memory cells and whether they maintain Tfh commitment at the memory phase. This review will highlight several recent studies that support the idea of Tfh-committed CD4 T cells at the memory stage of the immune response. The implication of these findings is that memory Tfh cells retain their capacity to recall their Tfh-specific effector functions upon reactivation to provide help for B cell responses and play an important role in prime and boost vaccination or during recall responses to infection. The markers that are useful for distinguishing Tfh effector and memory cells, as well as the limitations of using these markers will be discussed. Tfh effector and memory generation, lineage maintenance, and plasticity relative to other T helper lineages (Th1, Th2, Th17, etc.) will also be discussed. Ongoing discoveries regarding the maintenance and lineage stability versus plasticity of memory Tfh cells will improve strategies that utilize CD4 T cell memory to modulate antibody responses during prime and boost vaccination.

Role of retinoic acid in the stability of the T-helper-type 1 lineage and implications for autoimmunity

BackgroundCD4 T cells with features of both T-helper-type 1 (Th1) and 17 (Th17) cells have been implicated in several autoimmune diseases suggesting that plasticity among CD4 T-cell lineages is potentially pathogenic. However, the factors that regulate T-cell lineage stability are largely unknown. Retinoic acid (RA) is synthesised at sites of inflammation. We hypothesised that retinoic acid, a profound epigenetic modifier, could regulate T-cell lineage stability. MethodsWe used a mouse model in which retinoic acid signalling is specifically ablated within the T-cell compartment through overexpression of a dominant negative retinoic acid receptor α (RARα) (dnRARα mice) to investigate its role in the regulation of Th1 lineage stability. Genome-wide ChIP-seq analysis was done to identify RARα targets. In parallel, we performed global mapping of regulatory regions, termed enhancers, to gain mechanistic insight into retinoic acid regulation of T-cell fate. The in-vivo relevance of our findings was determined in a model of oral antigen-induced intestinal inflammation. FindingsWe found that retinoic acid is crucial for maintenance of the Th1 lineage. Abrogation of retinoic acid signalling in Th1 cells resulted in loss of T-bet expression and STAT4 activity. Th1 cells from dnRARα mice showed enhanced plasticity with the emergence of hybrid Th1–Th17 and Th17 effector cells. Global analysis of RARα binding and enhancer mapping revealed that RA–RARα directly regulated enhancer activity at Th1 lineage defining genes while repressing genes that regulate Th17 cell fate. Retinoic acid inhibition of Th1 plasticity was essential for maintaining appropriate Th cell responses in vivo and preventing autoimmune intestinal inflammation. InterpretationOur study has identified RA–RARα as a key component of the regulatory network governing maintenance and plasticity of Th1 cells and defines a new pathway for the development of pathogenic Th17 cells. Retinoids might be novel therapeutic agents for Th17-associated autoimmune diseases. FundingWellcome Trust.

Regulatory T cells. The PTEN stabilizer.

PTEN maintains regulatory T cell lineage stability and immune homeostasis.

Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability.

Foxp3+ regulatory T cells (Tregs) are required for immune homeostasis. One notable distinction between conventional T cells (Tconv) and Tregs is differential phosphatidylinositol 3-kinase (PI3K) activity: only Tconv downregulate PTEN, the primary negative regulator of PI3K, upon activation. Here, we show that control of PI3K in Tregs is essential for lineage homeostasis and stability. Mice lacking Pten in Tregs developed an autoimmune-lymphoproliferative disease characterized by excessive TH1 responses and B cell activation. Diminished control of PI3K activity in Tregs led to reduced CD25 expression, accumulation of Foxp3+CD25− cells and ultimately, loss of Foxp3 expression in these cells. Collectively, these data demonstrate that control of PI3K signaling by PTEN in Tregs is critical to maintain their homeostasis, function and stability.

In Vivo Maintenance of Human Regulatory T Cells during CD25 Blockade

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.

Backcrossing to increase meiotic stability in triticale.

Triticale (X Triticosecale Wittmack) is an intergeneric hybrid derived from a cross between wheat and rye. As a newly created allopolyploid, the plant shows instabilities during the meiotic process, which may result in the loss of fertility. This genomic instability has hindered the success of triticale-breeding programs. Therefore, strategies should be developed to obtain stable triticale lines for use in breeding. In some species, backcrossing has been effective in increasing the meiotic stability of lineages. To assess whether backcrossing has the same effect in triticale, indices of meiotic abnormalities, meiotic index, and pollen viability were determined in genotypes from multiple generations of triticale (P1, P2, F1, F2, BC1a, and BC1b). All analyzed genotypes exhibited instability during meiosis, and their meiotic index values were all lower than normal. However, the backcrosses BC1a and BC1b showed the lowest mean meiotic abnormalities and the highest meiotic indices, demonstrating higher stability. All genotypes showed a high rate of pollen viability, with the backcrosses BC1a and BC1b again exhibiting the best values. Statistical analyses confirmed that backcrossing positively affects the meiotic stability of triticale. Our results show that backcrossing should be considered by breeders aiming to obtain triticale lines with improved genomic stability.

In vivo maintenance of human regulatory T cells during CD25 blockade

Regulatory T cells (Tregs) mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction, and instability are implicated in the pathogenesis of numerous autoimmune diseases. There is considerable interest in therapeutic modulation of the IL-2 pathway to treat autoimmunity, facilitate transplantation tolerance, or potentiate tumor immunotherapy. Daclizumab is a humanized mAb that binds the IL-2 receptor a subunit (IL-2R a or CD25) and prevents IL-2 binding. In this study, we investigated the effect of daclizumab-mediated CD25 blockade on Treg homeostasis in patients with relapsing-remitting multiple sclerosis. We report that daclizumab therapy caused an ~50% decrease in Tregs over a 52-wk period. Remaining FOXP3+ cells retained a demethylated Treg-specific demethylated region in the FOXP3 promoter, maintained active cell cycling, and had minimal production of IL-2, IFN- g, and IL-17. In the presence of daclizumab, IL-2 serum concentrations increased and IL-2R bg signaling induced STAT5 phosphorylation and sustained FOXP3 expression. Treg declines were not associated with daclizumab-related clinical benefit or cutaneous adverse events. These results demonstrate that Treg phenotype and lineage stability can be maintained in the face of CD25 blockade.

Direct Genesis of Functional Rodent and Human Schwann Cells from Skin Mesenchymal Precursors

SummaryRecent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs), a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

PTU-140 Intrahepatic Tregs Are Plastic But Functional And Biliary Epithelial Cells Support Their Fate

IntroductionRegulatory T cells (Tregs) are crucial in maintaining peripheral tolerance. Tregs control T effector CD8, CD4, Th1 cells along with other immune cells to maintain hepatic tolerance. They are implicated...

TGFβ converts Th1 cell into Th17 cells through stimulation of Runx1 expression under inflammatory conditions in intestines (MUC2P.819)

Abstract Although accumulating evidence demonstrates that differentiated CD4 cells preserve plasticity to alter phenotypes under various conditions, it is still unclear how stable Th1 cells are and whether Th1 cells can convert into Th17 cells. The high stability of Th1 is supported by some epigenetic studies and numerous reports of Treg and Th17 cells conversion into Th1 cells but not vice versa. However, recent reports of Th1 cell conversion to Treg, Th2 and Tfh cells argue the absolute stability of Th1 lineage. By using IFNγThy1.1 CBir1 TCR transgenic reporter mice, whose TCR is specific for an immunodominant microbiota antigen, we investigate the stability of Th1 cells under intestinal inflammatory conditions. Transfer of purified CBir1 specific IFNγ+Th1 cells induces colitis in RAG-/- mice and IFNγ+Th1 cells convert into IL-17+ Th17 but not Foxp3+ Treg cells in the inflamed intestines. TGFβ, IL-6 and IL-2, but not hypoxia factors, differentially regulate Th1 to Th17 conversion. TGFβ induction of transcriptional factors, Runx1 and RORγt, is crucial for the conversion, in that silencing Runx1 by siRNA inhibits Th1 conversion into Th17 cells. Furthermore, using ChIP assay, we show that TGFβ enhances histone acetylation but inhibits trimethylation of Runx1 and RORγt binding sites on il-17 or rorc gene in Th1 cells. In conclusion, our data demonstrate that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is mediated by TGFβ-induction of Runx1.