Exposure to inflammatory cytokines selectively limits GM‐CSF production by induced T regulatory cells

Interest in manipulating the immunosuppressive powers of Foxp3‐expressing T regulatory cells as an immunotherapy has been tempered by their reported ability to produce proinflammatory cytokines when manipulated in vitro, or in vivo. Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important. Here, we have studied this using induced (i) Treg cells in which de novo Foxp3 expression is driven by TCR‐stimulation in vitro in the presence of TGF‐β. We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN‐γ, GM‐CSF and TNF‐α) upon secondary TCR stimulation. GM‐CSF is a critical T‐cell derived cytokine for the induction of EAE in mice. Despite their apparent capacity to produce GM‐CSF, myelin autoantigen‐responsive iTreg cells were unable to provoke EAE. Instead, they maintained strong suppressive function in vivo, preventing EAE induction by their CD4⁺Foxp3^? counterparts. We identified that although iTreg cells maintained the ability to produce IFN‐γ and TNF‐α in vivo, their ability to produce GM‐CSF was selectively degraded upon antigen stimulation under inflammatory conditions. Furthermore, we show that IL‐6 and IL‐27 individually, or IL‐2 and TGF‐β in combination, can mediate the selective loss of GM‐CSF production by iTreg cells.     

Foxp3+ regulatory T cells ensure B lymphopoiesis by inhibiting the granulopoietic activity of effector T cells in mouse bone marrow

Foxp3⁺ Treg cells are crucial for maintaining T‐cell homeostasis, but their role in B‐cell homeostasis remains unclear. Here, we found that Foxp3 mutant scurfy mice had fewer B‐lineage cells and progenitors, including common lymphoid progenitors and lymphoid‐primed multipotent progenitors, but higher myeloid‐lineage cell numbers in BM compared with WT littermates. Homeostasis within the HSC compartment was also compromised with apparent expansion of long‐ and short‐term HSCs. This abnormality was due to the lack of Treg cells, but not to the Treg‐cell extrinsic functions of Foxp3 or cell‐autonomous defects. Among cytokines enriched in the BM of scurfy mice, IFN‐γ affected only B lymphopoiesis, but GM‐CSF, TNF, and IL‐6 collectively promoted granulopoiesis at the expense of B lymphopoiesis. Neutralization of these three cytokines reversed the hematopoietic defects on early B‐cell progenitors in scurfy mice. Treg cells ensured B lymphopoiesis by reducing the production of these cytokines by effector T cells, but not by directly affecting B lymphopoiesis. These results suggest that Treg cells occupy an important niche in the BM to protect B‐lineage progenitor cells from excessive exposure to a lymphopoiesis‐regulating milieu.     

CD8+ Treg cells suppress CD8+ T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg‐cell‐mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3‐GFP transgenic mice, CD8⁺ Foxp3⁺ Treg cells, but not CD4⁺ Foxp3⁺ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8⁺ Foxp3⁺ Treg cells showed a high level of GITR and produced IL‐10. In an adoptive transfer model, CD8⁺ Treg cells suppressed CD8⁺ T‐cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL‐10 and studies with IL‐10R‐deficient mice in vitro and in vivo demonstrated an important role for IL‐10 production in the capacity of CD8⁺ Treg cells to inhibit CD8⁺ T‐cell responses. Our findings identify a previously unrecognized role of CD8⁺ Treg cells in the negative regulation of CD8⁺ T‐cell responses and suggest that modulation of CD8⁺ Treg cells may be a therapeutic strategy to control H5N1 viral infection.     

Expansion of Foxp3+ T‐cell populations by Candida albicans enhances both Th17‐cell responses and fungal dissemination after intravenous challenge

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3⁺ regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3⁺ Treg‐cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4⁺Foxp3⁺ population that correlated positively with fungal burden. Depletion from Foxp3^hCD2 reporter mice in vivo confirmed that Foxp3⁺ cells exacerbated fungal burden and inflammatory renal disease. The CD4⁺Foxp3⁺ population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg‐cell subset, together with conversion of Foxp3^? cells to the induced Treg‐cell form, and to a cell type sharing effector Th17‐cell characteristics, expressing ROR‐γt, and secreting IL‐17A. The expanded Foxp3⁺ T cells inhibited Th1 and Th2 responses, but enhanced Th17‐cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17‐cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3⁺ T cells promotes Th17‐cell responses that drive pathology.     

Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions

Induction of Forkhead‐box p3 (Foxp3) expression in developing T cells upon peptide‐MHC encountering has been proposed to define a lineage of committed Treg cells. However, sustained expression of Foxp3 is required for Treg function and what maintains Foxp3 expression in peripheral Treg remains obscure. To address this issue, we monitored natural Treg phenotype and function upon adoptive transfer into lymphocyte‐deficient mice. We first show that about 50% of Foxp3‐GFP⁺ Treg isolated from Foxp3^gfp KI animals loose Foxp3 expression in severe lymphopenic conditions. We next evidence that the cytokine IL‐2, either produced by co‐transferred conventional T cells or administrated i.v. prevents Foxp3 downregulation. Moreover, we document that Treg that lost Foxp3 expression upon adoptive transfer produce IL‐2 are not suppressive and promote tissue infiltration and damage upon secondary transfer into alymphoid mice. Our findings that Treg convert into pathogenic Th cells in absence of IL‐2 provide new clues to the success of Treg‐based immune therapies.     

Fluorochrome‐based definition of naturally occurring Foxp3+ regulatory T cells of intra‐ and extrathymic origin

Under physiological conditions, studies on the biology of naturally induced Foxp3⁺ Treg cells of intra‐ and extrathymic origin have been hampered by the lack of unambiguous markers to discriminate the mature progeny of such developmental Treg‐cell sublineages. Here, we report on experiments in double‐transgenic mice, in which red fluorescent protein (RFP) is expressed in all Foxp3⁺ Treg cells, whereas Foxp3‐dependent GFP expression is exclusively confined to intrathymically induced Foxp3⁺ Treg cells. This novel molecular genetic tool enabled us to faithfully track and characterize naturally induced Treg cells of intrathymic (RFP⁺GFP⁺) and extrathymic (RFP⁺GFP^?) origin in otherwise unmanipulated mice. These experiments directly demonstrate that extrathymically induced Treg cells substantially contribute to the overall pool of mature Foxp3⁺ Treg cells residing in peripheral lymphoid tissues of steady‐state mice. Furthermore, we provide evidence that intra‐ and extrathymically induced Foxp3⁺ Treg cells represent distinct phenotypic and functional sublineages.     

Plasmodium yoelii infection of BALB/c mice results in expansion rather than induction of CD4+ Foxp3+ regulatory T cells

Recently, we demonstrated elevated numbers of CD4⁺ Foxp3⁺ regulatory T (Treg) cells in Plasmodium yoelii‐infected mice contributing to the regulation of anti‐malarial immune response. However, it remains unclear whether this increase in Treg cells is due to thymus‐derived Treg cell expansion or induction of Treg cells in the periphery. Here, we show that the frequency of Foxp3⁺ Treg cells expressing neuropilin‐1 (Nrp‐1) decreased at early time‐points during P. yoelii infection, whereas percentages of Helios⁺ Foxp3⁺ Treg cells remained unchanged. Both Foxp3⁺ Nrp‐1⁺ and Foxp3⁺ Nrp‐1^? Treg cells from P. yoelii‐infected mice exhibited a similar T‐cell receptor Vβ chain usage and methylation pattern in the Treg‐specific demethylation region within the foxp3 locus. Strikingly, we did not observe induction of Foxp3 expression in Foxp3^? T cells adoptively transferred to P. yoelii‐infected mice. Hence, our results suggest that P. yoelii infection triggered expansion of naturally occurring Treg cells rather than de novo induction of Foxp3⁺ Treg cells.     

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3⁺ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.     

Macrophage‐mediated psoriasis can be suppressed by regulatory T lymphocytes

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis‐like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro‐inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis‐like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF‐mediated psoriasis, ihTNFtg mice were crossbred into RAG1^KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1^KO or wild‐type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild‐type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3‐positive cells into RAG1^KO or wild‐type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro‐inflammatory activity of macrophages, which are the major immune effector cells in hTNF‐mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Transient attenuated Foxp3 expression on CD4⁺ T cells treated with 7D4 mAb contributes to the control of parasite burden in DBA / 2 mice infected with lethal Plasmodium chabaudi chabaudi AS

CD4⁺ CD25⁺ regulatory T (Treg) cells expressing Foxp3⁺ play a critical role in maintaining immune homoeostasis and controlling excessive immune responses. However, controversy about the immunoregulatory role of Treg cells exists in malaria studies. Given the role of maintenance of Foxp3 expression in Treg cells’ activities, we investigated whether anti‐CD25 mAb (7D4 clone) treatment affects Foxp3 expression in CD4⁺ T cells in DBA/2 mice infected with Plasmodium chabaudi chabaudi AS (P. c. chabaudi AS). We found that DBA/2 mice succumbed to P. c. chabaudi AS infection, which was accompanied by increased expression of Foxp3 in CD4⁺ T cells at the peak parasitemia. In contrast, Foxp3 expression was impaired in CD25‐depleted mice with 7D4 mAb treatment, leading to delayed parasitemia and extended survival of infected mice. Production of IFN‐γ, TNF‐α and IL‐6, as well as NO was significantly enhanced in CD25‐depleted mice. The majority of CD4⁺ CTLA‐4⁺ cells expressed high levels of Foxp3 (Foxp3^hi cells) in control mice with P. c. chabaudi AS infection. However, the number of CD4⁺ Foxp3^hiCTLA‐4⁺ cells was reduced in CD25‐depleted mice. Together, these data suggest that CD4⁺ Foxp3^hiCTLA‐4⁺ cells may be involved in regulating the intensity of pro‐inflammatory responses via CTLA‐4.     

CD4+ Foxp3+ regulatory T‐cell number increases in the gastric tissue of C57BL/6 mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori), one of the most common infections, is associated with various clinical outcomes. In addition to inducing inflammation, immunological clearance of the pathogen is often incomplete. Regulatory T cells (Treg cells) have been recently demonstrated to play an important role in H. pylori infection and the final clinical outcome. The aim of this study was to investigate the number and localization of CD4⁺Foxp3⁺ Treg cells in stomachs and spleens of H. pylori‐infected mice. The expression levels of Foxp3 as well as anti‐ and pro‐inflammatory cytokines before and after H. pylori triple eradication therapy were examined. We found that the percentages of CD4⁺Foxp3⁺ Treg cells out of the lamina propria lymphocytes (LPLs) and spleen lymphocytes in the infection group were higher than the PBS negative control group and the treatment group. H. pylori antigen stimulation was associated with an increased number of Treg cells in vitro. Furthermore, compared with the PBS and treatment groups, a higher mRNA expression level of Foxp3 in the gastric tissue was detected in the infection group. IL‐10 and TGF‐β1 contents were increased significantly in the culture supernatant of spleen lymphocyte stimulated with H. pylori antigen. A marked elevation in serum IFN‐γ level was observed in H. pylori‐infected mice. In addition, gastric tissues of the infection group contained more Foxp3⁺ cells. These results indicate that the percentage of CD4⁺Foxp3⁺ Treg cells are increased in H. pylori‐infected mice, suggesting a role of Treg cells in H. pylori‐induced pathologies, even at the early stages of chronic gastritis and gastric tumorigenesis.     

Donor bone marrow cells are essential for iNKT cell‐mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti‐CD40 ligand (anti‐CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub‐lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3‐positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)‐γ‐producing CD8⁺T cells, expand host Ki67⁺CD4⁺CD25⁺Foxp3⁺ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC‐recipients accepted skin grafts in an allo‐specific manner. Myeloid‐derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.     

Few Foxp3+ regulatory T cells are sufficient to protect adult mice from lethal autoimmunity

Foxp3 specifies the Treg cell lineage and is indispensable for immune tolerance. Accordingly, rare Foxp3 mutations cause lethal autoimmunity. The mechanisms precipitating more prevalent human autoimmune diseases are poorly understood, but involve a combination of genetic and environmental factors. Many autoimmune diseases associate with a partial Treg‐cell dysfunction, yet mouse models reflecting such complex pathophysiological processes are rare. Around 95% of Foxp3⁺ Treg cells can be specifically depleted in bacterial artifical chromosome (BAC)‐transgenic Depletion of REGulatory T cells (DEREG) mice through diphtheria toxin (DT) treatment. However, Treg‐cell depletion fails to cause autoimmunity in adult DEREG mice for unclear reasons. By crossing Foxp3^GFP knock‐in mice to DEREG mice, we introduced additional genetic susceptibility that does not affect untreated mice. Strikingly, DT treatment of DEREG × Foxp3^GFP mice rapidly causes autoimmunity characterized by blepharitis, tissue damage, and autoantibody production. This inflammatory disease is associated with augmented T‐cell activation, increased Th2 cytokine production and myeloproliferation, and is caused by defective Treg‐cell homeostasis, preventing few DT‐insensitive Treg cells from repopulating the niche after Treg‐cell depletion. Our study provides important insights into self‐tolerance. We further highlight DEREG × Foxp3^GFP mice as a model to investigate the role of environmental factors in precipitating autoimmunity. This may help to better understand and treat human autoimmunity.     

ICOS controls Foxp3+ regulatory T‐cell expansion,maintenance and IL‐10 production during helminth infection

Foxp3⁺ regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co‐stimulatory environment that favours the development of Foxp3⁺ Treg‐cell responses during helminth infections. As recent evidence implicates the co‐stimulatory receptor ICOS in defining Foxp3⁺ Treg‐cell functions, we investigated the role of ICOS in helminth‐induced Foxp3⁺ Treg‐cell responses. Infection of ICOS^?/? mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3⁺ Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3⁺ Treg‐cell apoptosis, a Foxp3⁺ Treg‐cell specific impairment in IL‐10 production, and a failure to mount putatively adaptive Helios^?Foxp3⁺ Treg‐cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3⁺ Treg‐cell responses were associated with increased production of IL‐4 and IL‐13 by CD4⁺ T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2‐promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue‐specific manner, and plays a key role in driving Foxp3⁺ Treg‐cell expansion and function during helminth infections.     

Expansion of activated regulatory T cells by myeloid‐specific chemokines via an alternative pathway in CSF of bacterial meningitis patients

Previous studies have demonstrated that activation/expansion by certain cytokines as well as recruitment by specific chemokines is involved in enrichment of regulatory T (Treg) cells in local tissues or organs under pathological conditions. Recent evidence indicates that human Treg cells are a heterogeneous population that comprises three distinct subpopulations: CD25⁺CD45RA⁺ resting Treg (rTreg) cells, CD25^hiCD45RA^? activated Treg (aTreg) cells, which are both suppressive, and CD25⁺CD45RA^? cytokine‐secreting T cells with proinflammatory capacity. Moreover, rTreg cells can proliferate and convert to aTreg cells. Here, we found an increase in aTreg‐cell frequency in the cerebrospinal fluid (CSF) of patients with postneurosurgery bacterial meningitis. We revealed that such an increased aTreg‐cell frequency in the CSF was not due to enhanced chemotaxis. Instead of a classic conversion pathway from rTreg to aTreg cells, we identified an alternative route of Treg‐cell conversion from cytokine‐secreting cells to aTreg cells induced by myeloid‐specific chemokine CXC chemokine receptor (CXCR) ligand 5 via CXCR1 and CXCR2 receptors, or by CSF myeloid cells in a cell–cell contact manner. Our results reveal a different view of how the immune system controls overwhelming local immune responses during infection, and provide evidence of how innate immunity negatively regulates adaptive immunity.