Peripherally induced regulatory T cells contribute to the control of autoimmune diabetes in the NOD mouse model

Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic beta cells and is partly caused by deficiencies in the Foxp3⁺ regulatory T‐cell (Treg) compartment. Conversely, therapies that increase Treg function can prevent autoimmune diabetes in animal models. The majority of Tregs develop in the thymus (tTregs), but a proportion of Foxp3⁺ Tregs is generated in the periphery (pTregs) from Foxp3^?CD4⁺ T‐cell precursors. Whether pTregs play a distinct role in T1D has not yet been explored. We report here that pTregs are a key modifier of disease in the nonobese diabetic (NOD) mouse model for T1D. We generated NOD mice deficient for the Foxp3 enhancer CNS1 involved in pTreg induction. We show that CNS1 knockout decreased the frequency of pTregs and increased the risk of diabetes. Our results show that pTregs fulfill an important non‐redundant function in the prevention of beta cell autoimmunity that causes T1D.     

The transcriptional repressor Bcl6 controls the stability of regulatory T cells by intrinsic and extrinsic pathways

Foxp3⁺ regulatory T (Treg) cells are essential to maintain immune homeostasis, yet controversy exists about the stability of this cell population. Bcl6-deficient (Bcl6^−/−) mice develop severe and spontaneous T helper type 2 (Th2) inflammation and Bcl6-deficient Treg cells are ineffective at controlling Th2 responses. We used a lineage tracing approach to analyse the fate of Treg cells in these mice. In the periphery of Bcl6^−/− mice, increased numbers of Foxp3-negative ‘exTreg’ cells were found, particularly in the CD25⁺ population. ExTreg cells from Bcl6^−/− mice expressed increased interleukin-17 (IL-17) and extremely elevated levels of Th2 cytokines compared with wild-type exTreg cells. Although Treg cells normally express only low levels of cytokines, Treg cells from Bcl6^−/− mice secreted higher levels of IL-4, IL-5, IL-13 and IL-17 than wild-type conventional T cells. Next, Treg-specific conditional Bcl6-deficient (Bcl6^Foxp3−/−) mice were analysed. Bcl6^Foxp3−/− mice do not develop inflammatory disease, indicating a requirement for non-Treg cells for inflammation in Bcl6^−/− mice, and have normal numbers of exTreg cells. We induced Th2-type allergic airway inflammation in Bcl6^Foxp3−/− mice, and found that while exTreg cytokine expression was normal, Bcl6-deficient Treg cells expressed higher levels of the Th2-specific regulator Gata3 than Bcl6⁺ Treg cells. Bcl6^Foxp3−/− mice had increased numbers of Th2 cells after induction of airway inflammation and increased T cells in the bronchoalveolar lavage fluid. These data show both Treg-intrinsic and Treg-extrinsic roles for Bcl6 in controlling Treg cell stability and Th2 inflammation, and support the idea that Bcl6 expression in Treg cells is critical for controlling Th2 responses.     

IL-2 Family of Cytokines in T Regulatory Cell Development and Homeostasis

Introduction  Interleukin 2 (IL-2) induces an essential signal for T regulatory (Treg) cells. Without a functional IL-2R, only immature CD4⁺ Foxp3^low CD25^neg T cells develop, and these cells fail to suppress autoreactive T cells in the periphery. Discussion  IL-2 functions during Treg cell development by upregulating Foxp3 and CD25 and by increasing the number of thymic Treg cells. Upon exiting the thymus during neonatal life, IL-2 is responsible for rapid amplification of the number of Treg cells in peripheral lymph nodes to insure suppression of autoreactive T cells that escape negative selection, thereby maintaining tolerance. The homeostasis of Treg cells in mature immunocompetent mice also depends on IL-2. However, there is an alternative mechanism for Treg cells homeostasis that may represent a minor IL-2-independent pathway or the consequence of weak and very transient IL-2R signaling. Conclusion  Thus, IL-2 provides importance signals for Treg cell development and for their homeostasis in peripheral immune tissues.     

Low-level regulatory T-cell activity is essential for functional type-2 effector immunity to expel gastrointestinal helminths

Helminth infection is frequently associated with the expansion of regulatory T cells (Tregs) and suppression of immune responses to bystander antigens. We show that infection of mice with the chronic gastrointestinal helminth Heligmosomoides polygyrus drives rapid polyclonal expansion of Foxp3⁺Helios⁺CD4⁺ thymic (t)Tregs in the lamina propria and mesenteric lymph nodes while Foxp3⁺Helios⁻CD4⁺ peripheral (p)Treg expand more slowly. Notably, in partially resistant BALB/c mice parasite survival positively correlates with Foxp3⁺Helios⁺CD4⁺ tTreg numbers. Boosting of Foxp3⁺Helios⁺CD4⁺ tTreg populations by administration of recombinant interleukin-2 (rIL-2):anti-IL-2 (IL-2C) complex increased worm persistence by diminishing type-2 responsiveness in vivo, including suppression of alternatively activated macrophage and granulomatous responses at the sites of infection. IL-2C also increased innate lymphoid cell (ILC) numbers, indicating that Treg functions dominate over ILC effects in this setting. Surprisingly, complete removal of Tregs in transgenic Foxp3-DTR mice also resulted in increased worm burdens, with “immunological chaos” evident in high levels of the pro-inflammatory cytokines IL-6 and interferon-γ. In contrast, worm clearance could be induced by anti-CD25 antibody–mediated partial depletion of early Treg, alongside increased T helper type 2 responses and without incurring pathology. These findings highlight the overarching importance of the early Treg response to infection and the non-linear association between inflammation and the prevailing Treg frequency.     

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice

Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3⁺ regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3⁺CD103⁺CD38^? stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3⁺CD103^?CD38⁺ labile-phenotype Tregs in the thymus and increased autoreactive CD4⁺ T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4⁺ T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.     

Role of CD4+ Foxp3+ Regulatory T Cells in Protection Induced by a Live Attenuated,Replicating Type I Vaccine Strain of Toxoplasma gondii

Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii type I and type II strains. The involvement of regulatory T cells (Tregs) in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4⁺ Foxp3⁺ T regulatory cells followed by recruitment/expansion of CD4⁺ Foxp3⁻ CD25⁺ effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Tregs were of the TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4⁺ Foxp3⁺ cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of the TH1/TH17-like type. In addition, in vivo Treg induction in RH-infected mice with interleukin-2 (IL-2)/anti-IL-2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Tregs may contribute to the protective response after vaccination with Mic1.3KO.     

Schistosoma mansoni Larvae Do Not Expand or Activate Foxp3+ Regulatory T Cells during Their Migratory Phase

Foxp3⁺ regulatory T (Treg) cells play a key role in suppression of immune responses during parasitic helminth infection, both by controlling damaging immunopathology and by inhibiting protective immunity. During the patent phase of Schistosoma mansoni infection, Foxp3⁺ Treg cells are activated and suppress egg-elicited Th2 responses, but little is known of their induction and role during the early prepatent larval stage of infection. We quantified Foxp3⁺ Treg cell responses during the first 3 weeks of murine S. mansoni infection in C57BL/6 mice, a time when larval parasites migrate from the skin and transit the lungs en route to the hepatic and mesenteric vasculature. In contrast to other helminth infections, S. mansoni did not elicit a Foxp3⁺ Treg cell response during this early phase of infection. We found that the numbers and proportions of Foxp3⁺ Treg cells remained unchanged in the lungs, draining lymph nodes, and spleens of infected mice. There was no increase in the activation status of Foxp3⁺ Treg cells upon infection as assessed by their expression of CD25, Foxp3, and Helios. Furthermore, infection failed to induce Foxp3⁺ Treg cells to produce the suppressive cytokine interleukin 10 (IL-10). Instead, only CD4⁺ Foxp3⁻ IL-4⁺ Th2 cells showed increased IL-10 production upon infection. These data indicate that Foxp3⁺ Treg cells do not play a prominent role in regulating immunity to S. mansoni larvae and that the character of the initial immune response invoked by S. mansoni parasites contrasts with the responses to other parasitic helminth infections that promote rapid Foxp3⁺ Treg cell responses.     

Upregulated IL-17A secretion and CCR6 co-expression in Treg subsets are related to the imbalance of Treg/Th17 cells in active UC patients

Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disease, which is characterized with overactive immune response. It is well established that the imbalance between Tregs and Th17 cells plays a pivotal role in pathogenesis of UC. In this study, we investigated the impact of functional changes in Treg subsets on Treg/Th17 ratio and further explored their clinical significance in the activity of UC. Treg subsets were comprehensively analysed using flow cytometry and in vitro cultured in both active and remission UC patients, of which nine active UC patients were further followed up. The correlation analyses were performed to explore the potential associations between Treg subsets and clinical indicators, as well as the impact of serum cytokines, detected by ELISA, on IL-17A secretion and CCR6 co-expression of Treg subsets. In active UC patients, we found CD45RA^-FoxP3^hiTregs were obviously decreased and inversely correlated with disease activity, while CD45RA⁺FoxP3^loTregs were increased and positively correlated with disease activity. Meanwhile, IL-17A secretion and CCR6 co-expression levels in Tregs were significantly increased in active UC. Moreover, Tregs co-expressing CCR6 possesses higher level of IL-17A secretion. In nine followed up patients, we observed downregulated IL-17A secreting and CCR6 co-expression when achieving remission from active stage. In addition, IL-17A⁺FoxP3⁺ and IL-17A⁺FoxP3⁺CCR6⁺Tregs were positively correlated with serum IL-21 and disease activity, respectively. These findings suggested that upregulated IL-17A secretion and CCR6 co-expression in Treg subsets may be related to the imbalance between Tregs and Th17 cells and associated with the disease activity in UC patients.     

A distinct chemokine axis does not account for enrichment of Foxp3+ CD4+ T cells in carcinogen‐induced fibrosarcomas

The frequency of CD4⁺ Foxp3⁺ regulatory T (Treg) cells is often significantly increased in the blood of tumour-bearing mice and people with cancer. Moreover, Treg cell frequencies are often higher in tumours compared with blood and lymphoid organs. We wished to determine whether certain chemokines expressed within the tumour mass selectively recruit Treg cells, thereby contributing to their enrichment within the tumour-infiltrating lymphocyte pool. To achieve this goal, the chemokine profile of carcinogen-induced fibrosarcomas was determined, and the chemokine receptor expression profiles of both CD4⁺ Foxp3⁻ and CD4⁺ Foxp3⁺ T cells were compared. These analyses revealed that the tumours are characterized by expression of inflammatory chemokines (CCL2, CCL5, CCL7, CCL8, CCL12, CXCL9, CXCL10 and CX3CL1), reflected by an enrichment of activated Foxp3⁻ and Foxp3⁺ T cells expressing T helper type 1-associated chemokine receptors. Notably, we found that CXCR3⁺ T cells were significantly enriched in the tumours although curiously we found no evidence that CXCR3 was required for their recruitment. Instead, CXCR3 marks a population of activated Foxp3⁻ and Foxp3⁺ T cells, which use multiple and overlapping ligand receptor pairs to guide their migration to tumours. Collectively, these data indicate that enrichment of Foxp3⁺ cells in tumours characterized by expression of inflammatory chemokines, does not occur via a distinct chemokine axis, thus selective chemokine blockade is unlikely to represent a meaningful therapeutic strategy for preventing Treg cell accumulation in tumours.     

A B-1a cell subset induces Foxp3? T cells with regulatory activity through an IL-10-independent pathway

Regulatory T (Treg) cells play a critical role in the maintenance of tolerance. B-1a cells belong to a specific and functionally important B-cell subset that exerts its regulatory role through the production of IL-10. While IL-10 has been correlated with the induction of type 1 Treg (Tr1) cells or Tr1-like cells, whether IL-10-producing B-1a cells are able to induce Treg cells, especially the Tr1 lineage, is poorly understood. We have demonstrated that, similar to the reported B-2 cells, B-1a cells are able to convert naïve CD4⁺CD25⁻ T cells into a subset of T cells with suppressive function, which we called ‘Treg-of-B1a'' cells. Treg-of-B1a cells do not express Foxp3, but upregulate the Treg markers OX40, programmed death 1 (PD-1), inducible costimulator (ICOS) and IL-10R. Moreover, Treg-of-B1a cells do not express Foxp3 and produce high levels of IFN-γ and IL-10, but minimal amounts of IL-4; therefore, they resemble Tr1 cells. However, utilizing IL-10^−/− mice, we showed that IL-10 was not involved in the induction of Treg-of-B1a cells. On the contrary, CD86-mediated costimulation was essential for B-1a cells to drive the induction of Treg-of-B1a cells. Finally, we demonstrated that, in contrast to the Treg cells generated by B-2 cells that mediate contact-dependent suppression, Treg-of-B1a cells suppress through secreting soluble factors. While Tr1 cells mediate suppression mainly through IL-10 or TGF-β secretion, Treg-of-B1a cells mediate suppression through an IL-10- and TGF-β-independent pathway. Together, these findings suggest that B-1a cells induce a functionally and phenotypically distinct Treg population that is dissimilar to the reported Foxp3⁺ Treg or Tr1 cells.     

The imbalance of Th17/Th1/Tregs in patients with type 2 diabetes: relationship with metabolic factors and complications

Immune disorders are linked to the development of type 2 diabetes (T2D) and its complications. The relationship of CD4⁺CD25^hi T regulatory cells (Treg) and pro-inflammatory Th17 and Th1 subsets in T2D patients with metabolic disorders and complications need to be determined. The ratios of CD4⁺CD25^hi Treg/Th17 cells and CD4⁺CD25^hi Treg/Th1 cells, but not Th17/Th1 cells, were significantly decreased in T2D patients. The thymic output CD4⁺Foxp3⁺Helios⁺ Tregs were normal but peripheral induced CD4⁺Foxp3⁺Helios⁻ Tregs were decreased in T2D patients. The Bcl-2/Bax ratio decreased in CD4⁺CD25^hi Tregs in T2D patients, supporting the increased sensitivity to cell death of these cells in T2D. CD4⁺CD25^hiCD127⁻ Tregs in T2D patients with microvascular complications were significantly less than T2D patients with macrovascular complications. Importantly, CD4⁺CD25^hiCD127⁻ Tregs were positively correlated with plasma IL-6, whereas IL-17⁺CD4⁺cells were negatively related to high-density lipoprotein (HDL). Our data offered evidence for the skewed balance of anti- and pro-inflammatory T cell subsets in T2D patients and identified that HDL closely modulate T cell polarization. These results opened an alternative explanation for the substantial activation of immune cells as well as the development of T2D and complications, which may have significant impacts on the prevention and treatment of T2D patients.     

Analysis of miR‐146a and miR‐142‐3p as Potential Markers of Freshly Isolated or In Vitro‐Expanded Human Treg cells

Regulatory CD4⁺ T cells (Tregs) are pivotal for prevention of autoimmunity. The use of Tregs is therefore of increasing interest in in vitro drug screening assays as well as for a cytotherapy per se against autoimmune disorders. For both purposes, in vitro expansion of peripheral blood Tregs is necessary and there is an increasing need to identify novel markers that can discriminate natural thymic‐derived Tregs (tTregs) from other T cell subsets, and ideally, such markers should be stably expressed during in vitro expansion procedures. We screened for novel miRNAs differentially expressed in tTregs and identified miR‐146a and 142‐3p as possible candidates. We analysed freshly isolated naïve and activated tTregs and non‐Treg subsets after or prior to in vitro expansion. We observed a tTreg‐specific profile of these miRNAs together with FOXP3 and Helios in freshly isolated tTregs, but observed a decline in the same markers in activated tTregs as opposed to naïve tTregs. In vitro‐expanded Tregs could be identified based on FOXP3 expression, but with loss of a discriminate profile for miRNA candidates and a decline in FOXP3 when activated tTregs were expanded. Our data demonstrate miR‐146a and 142‐3p as potential miRNA markers for discrimination between non‐Treg cells and tTregs, but these miRNAs are not stable markers for in vitro‐expanded Treg cells. In addition, the loss of FOXP3 in expansion of activated tTregs has implication for in vitro use of this cell subset in immunopharmacological assays and cytotherapy as FOXP3 is pivotal for suppressive function.     

Identification of a CD4+ T cell line with Treg-like activity

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4⁺ CD25^high Foxp3⁺ Tregs and demonstrate they suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3⁺ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4⁺ PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4⁺ PBMC proliferation by MoT cells and Tregs, suggesting cell–cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity. We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner, suggesting that a Foxp3⁺ Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.     

Natural and inducible Tregs in swine: Helios expression and functional properties

Within the population of regulatory T cells (Tregs) natural Tregs (nTregs) and inducible Tregs (iTregs) can be distinguished. Although information about Tregs in swine exists, porcine iTregs were not under investigation yet. In this study, Foxp3⁺ iTregs were generated from CD4⁺Foxp3⁻ T cells by in vitro stimulation in the presence of IL-2 and TGF-β. In comparison to ex vivo Tregs these iTregs had a similar suppressive capacity on the proliferation of CD3-stimulated PBMC, caused higher levels of IL-10 in PBMC/Treg co-cultures, but did not suppress IFN-γ levels. The Ikaros family member Helios is currently discussed to distinguish iTregs and nTregs or to serve as an activation marker of Tregs. In this study, we demonstrate the cross-reactivity of an anti-mouse/human Helios mAb with porcine Helios. Flow cytometric analyses with this antibody showed that porcine iTregs do not express Helios after in vitro iTreg induction. Nevertheless, thymic Foxp3⁺ T cells, which arise at the CD4/CD8α single-positive stage of T-cell development and are defined as nTregs, entirely expressed Helios. Although this might suggest the suitability of Helios as an nTreg–iTreg differentiation marker we also found that Helios⁻ Tregs displayed a phenotype of naive CD4⁺ T cells in vivo. Since iTregs are by definition activated/differentiated Tregs, this finding precludes that all Helios⁻ Tregs are iTregs and thus also the use of Helios as a selection marker for porcine nTregs. Furthermore, Helios⁺ Tregs displayed a more differentiated phenotype indicating that Helios might rather serve as a Treg activation/differentiation marker.     

Ssu72 is a T-cell receptor-responsive modifier that is indispensable for regulatory T cells

The homeostatic balance between effector T cells and regulatory T cells (Tregs) is crucial for adaptive immunity; however, epigenetic programs that inhibit phosphorylation to regulate Treg development, peripheral expression, and suppressive activity are elusive. Here, we found that the Ssu72 phosphatase is activated by various T-cell receptor signaling pathways, including the T-cell receptor and IL-2R pathways, and localizes at the cell membrane. Deletion of Ssu72 in T cells disrupts CD4⁺ T-cell differentiation into Tregs in the periphery via the production of high levels of the effector cytokines IL-2 and IFNγ, which induce CD4⁺ T-cell activation and differentiation into effector cell lineages. We also found a close correlation between downregulation of Ssu72 and severe defects in mucosal tolerance in patients. Interestingly, Ssu72 forms a complex with PLCγ1, which is an essential effector molecule for T-cell receptor signaling as well as Treg development and function. Ssu72 deficiency impairs PLCγ1 downstream signaling and results in failure of Foxp3 induction. Thus, our studies show that the Ssu72-mediated cytokine response coordinates the differentiation and function of Treg cells in the periphery.     

On integrity in immunity during ontogeny or how thymic regulatory T cells work

The Standard model of T cell recognition asserts that T cell receptor (TCR) specificities are positively and negatively selected during ontogeny in the thymus and that peripheral T cell repertoire has mild self‐major histocompatibility complex (MHC) reactivity, known as MHC restriction of foreign antigen. Thus, the TCR must bind both a restrictive molecule (MHC allele) and a peptide reclining in its groove (pMHC ligand) in order to transmit signal into a T cell. The Standard and Cohn's Tritope models suggest contradictory roles for complementarity‐determining regions (CDRs) of the TCRs. Here, I discuss both concepts and propose a different solution to ontogenetic mechanism for TCR‐MHC–conserved interaction. I suggest that double (CD4⁺CD8⁺)‐positive (DP) developing thymocytes compete with their αβTCRs for binding to self‐pMHC on cortical thymic epithelial cells (cTECs) that present a selected set of tissue‐restricted antigens. The competition between DPs involves TCR editing and secondary rearrangements, similar to germinal‐centre B cell somatic hypermutation. These processes would generate cells with higher TCR affinity for self‐pMHC, facilitating sufficiently long binding to cTECs to become thymic T regulatory cells (tTregs). Furthermore, CD4⁺ Foxp3⁺ tTregs can be generated by mTECs via Aire‐dependent and Aire‐independent pathways, and additionally on thymic bone marrow–derived APCs including thymic Aire‐expressing B cells. Thymic Tregs differ from the induced peripheral Tregs, which comprise the negative feedback loop to restrain immune responses. The implication of thymocytes’ competition for the highest binding to self‐pMHC is the co‐evolution of species‐specific αβTCR V regions with MHC alleles.