Inducible IL-7 Hyperexpression Influences Lymphocyte Homeostasis and Function and Increases Allograft Rejection.

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220+ c-kit+ Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8+ T cells in secondary lymphoid organs, and reduced the proportion of CD4+Foxp3+ T regulatory cells. Of relevance to disease, conventional CD4+ T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4+ and CD8+ T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.

Exercise-Induced Activated Platelets Increase Adult Hippocampal Precursor Proliferation and Promote Neuronal Differentiation.

Physical activity is a strong positive physiological modulator of adult neurogenesis in the hippocampal dentate gyrus. Although the underlying regulatory mechanisms are still unknown, systemic processes must be involved. Here we show that platelets are activated after acute periods of running, and that activated platelets promote neurogenesis, an effect that is likely mediated by platelet factor 4. Ex vivo, the beneficial effects of activated platelets and platelet factor 4 on neural precursor cells were dentate gyrus specific and not observed in the subventricular zone. Moreover, the depletion of circulating platelets in mice abolished the running-induced increase in precursor cell proliferation in the dentate gyrus following exercise. These findings demonstrate that platelets and their released factors can modulate adult neural precursor cells under physiological conditions and provide an intriguing link between running-induced platelet activation and the modulation of neurogenesis after exercise.

Critical Role of TGF-ß and IL-2 Receptor Signaling in Foxp3 Induction by an Inhibitor of DNA Methylation.

Under physiological conditions, CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 are generated in the thymus [thymus-derived Foxp3+ Treg (tTregs) cells] and extrathymically at peripheral sites [peripherally induced Foxp3+ Treg (pTreg) cell], and both developmental subsets play non-redundant roles in maintaining self-tolerance throughout life. In addition, a variety of experimental in vitro and in vivo modalities can extrathymically elicit a Foxp3+ Treg cell phenotype in peripheral CD4+Foxp3- T cells, which has attracted much interest as an approach toward cell-based therapy in clinical settings of undesired immune responses. A particularly notable example is the in vitro induction of Foxp3 expression and Treg cell activity (iTreg cells) in initially naive CD4+Foxp3- T cells through T cell receptor (TCR) and IL-2R ligation, in the presence of exogenous TGF-ß. Clinical application of Foxp3+ iTreg cells has been hampered by the fact that TGF-ß-driven Foxp3 induction is not sufficient to fully recapitulate the epigenetic and transcriptional signature of in vivo induced Foxp3+ tTreg and pTreg cells, which includes the failure to imprint iTreg cells with stable Foxp3 expression. This hurdle can be potentially overcome by pharmacological interference with DNA methyltransferase activity and CpG methylation [e.g., by the cytosine nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC)] to stabilize TGF-ß-induced Foxp3 expression and to promote a Foxp3+ iTreg cell phenotype even in the absence of added TGF-ß. However, the molecular mechanisms of 5-aza-dC-mediated Foxp3+ iTreg cell generation have remained incompletely understood. Here, we show that in the absence of exogenously added TGF-ß and IL-2, efficient 5-aza-dC-mediated Foxp3+ iTreg cell generation from TCR-stimulated CD4+Foxp3- T cells is critically dependent on TGF-ßR and IL-2R signaling and that this process is driven by TGF-ß and IL-2, which could either be FCS derived or produced by T cells on TCR stimulation. Overall, these findings contribute to our understanding of the molecular mechanisms underlying the process of Foxp3 induction and may provide a rational basis for generating phenotypically and functionally stable iTreg cells.

T Lymphocytes Contribute to the Control of Baseline Neural Precursor Cell Proliferation but Not the Exercise-Induced Up-Regulation of Adult Hippocampal Neurogenesis.

Cross-talk between the peripheral immune system and the central nervous system is important for physiological brain health. T cells are required to maintain normal baseline levels of neural precursor proliferation in the hippocampus of adult mice. We show here that neither T cells, B cells, natural killer cells nor natural killer T cells are required for the increase in hippocampal precursor proliferation that occurs in response to physical exercise. In addition, we demonstrate that a subpopulation of T cells, regulatory T cells, is not involved in maintaining baseline levels of neural precursor proliferation. Even when applied at supraphysiological numbers, populations of both naive and stimulated lymphocytes had no effect on hippocampal precursor proliferation in vitro. In addition, physical activity had no effect on peripheral immune cells in terms of distribution in the bone marrow, lymph nodes or spleen, activation state or chemokine receptor (CXCR4 and CCR9) expression. Together these results suggest that lymphocytes are not involved in translating the peripheral effects of exercise to the neurogenic niche in the hippocampus and further support the idea that the exercise-induced regulation of adult neurogenesis is mechanistically distinct from its baseline control.

Affinity for self antigen selects Treg cells with distinct functional properties.

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

Induced miR-99a expression represses Mtor cooperatively with miR-150 to promote regulatory T-cell differentiation.

Peripheral induction of regulatory T (Treg) cells provides essential protection from inappropriate immune responses. CD4(+) T cells that lack endogenous miRNAs are impaired to differentiate into Treg cells, but the relevant miRNAs are unknown. We performed an overexpression screen with T-cell-expressed miRNAs in naive mouse CD4(+) T cells undergoing Treg differentiation. Among 130 candidates, the screen identified 29 miRNAs with a negative and 10 miRNAs with a positive effect. Testing reciprocal Th17 differentiation revealed specific functions for miR-100, miR-99a and miR-10b, since all of these promoted the Treg and inhibited the Th17 program without impacting on viability, proliferation and activation. miR-99a cooperated with miR-150 to repress the expression of the Th17-promoting factor mTOR. The comparably low expression of miR-99a was strongly increased by the Treg cell inducer "retinoic acid", and the abundantly expressed miR-150 could only repress Mtor in the presence of miR-99a. Our data suggest that induction of Treg cell differentiation is regulated by a miRNA network, which involves cooperation of constitutively expressed as well as inducible miRNAs.

Continuous T cell receptor signals maintain a functional regulatory T cell pool.

Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.

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.

Myelin-specific T helper 17 cells promote adult hippocampal neurogenesis through indirect mechanisms.

CD4 + T cells provide a neuro-immunological link in the regulation of adult hippocampal neurogenesis, but the exact mechanisms underlying enhanced neural precursor cell proliferation and the relative contribution of different T helper (Th) cell subsets have remained unclear. Here, we explored the pro-proliferative potential of interleukin 17-producing T helper (Th17) cells, a developmentally and functionally distinct Th cell subset that is a key mediator of autoimmune neurodegeneration. We found that base-line proliferation of hippocampal precursor cells in a T cell-deficient mouse model of impaired hippocampal neurogenesis can be restored upon adoptive transfer with homogeneous Th17 populations enriched for myelin-reactive T cell receptors (TCR). In these experiments, enhanced proliferation was independent of direct interactions of infiltrating Th17 cells with precursor cells or neighboring cells in the hippocampal neurogenic niche. Complementary studies in immunocompetent mice identified several receptors for Th17 cell-derived cytokines with mRNA expression in hippocampal precursor cells and dentate gyrus tissue, suggesting that Th17 cell activity in peripheral lymphoid tissues might promote hippocampal neurogenesis through secreted cytokines.

Foxp3(+) regulatory T cells in mouse models of type 1 diabetes.

Studies on human type 1 diabetes (T1D) are facilitated by the availability of animal models such as nonobese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, as well as a variety of genetically engineered mouse models with reduced genetic and pathogenic complexity, as compared to the spontaneous NOD model. In recent years, increasing evidence has implicated CD4(+)CD25(+) regulatory T (Treg) cells expressing the transcription factor Foxp3 in both the breakdown of self-tolerance and the restoration of immune homeostasis in T1D. In this paper, we provide an overview of currently available mouse models to study the role of Foxp3(+) Treg cells in the control of destructive ß cell autoimmunity, including a novel NOD model that allows specific and temporally controlled deletion of Foxp3(+) Treg cells.

Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus.

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.

Vagaries of fluorochrome reporter gene expression in Foxp3+ regulatory T cells.

CD4(+)CD25(+) regulatory T (Treg) cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(?) single-positive stages of thymic development, as well as in postthymic CD4(+) T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3(+) Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC)-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3(+) DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4(+) T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3(+) Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3(+) Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene.

Targeted antigen delivery to DEC-205⁺ dendritic cells for tolerogenic vaccination.

Dendritic cells (DCs) and Foxp3-expressing CD4⁺ regulatory T (Treg) cells play non-redundant roles in the maintenance of peripheral tolerance to self-antigens, thereby preventing fatal autoimmunity. A common hallmark of intra- and extra-thymic Treg cell lineage commitment is the induction of Foxp3 expression as a consequence of appropriate T cell receptor engagement with MHC class II:agonist ligand. It has now become increasingly clear that agonist ligand presentation by immature DCs in the steady state induces T cell tolerance by both recessive and dominant mechanisms, rather than promoting productive T helper cell responses. In this context, the ability of steady-state DCs to promote the extrathymic conversion of initially naïve CD4⁺Foxp3⁻ T cells into Foxp3⁺ Treg cells is of particular interest as it provides novel perspectives to enhance antigen-specific Treg cell function in clinical settings of unwanted immunity, such as ß-cell autoimmunity.

Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells.

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205-mediated delivery of the self-peptide proteolipid protein (PLP)139-151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139-151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4(+) Vß6(+) TCR T cells specific for PLP139-151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4(+) T cell-mediated suppressor mechanism was obtained.

Dendritic cell-targeted pancreatic beta-cell antigen leads to conversion of self-reactive CD4(+) T cells into regulatory T cells and promotes immunotolerance in NOD mice.

Studies employing T cell receptor transgenic T cells have convincingly shown that selective delivery of non-self model antigens to DEC-205(+) dendritic cells (DCs) in the steady-state can induce Foxp3-expressing CD4(+)CD25(+) regulatory T (Treg) cells from conventional CD4(+)CD25(-)Foxp3(-) T cells. Although of considerable clinical interest, the concept of DC-targeted de novo generation of antigen-specific Treg cells has not yet been evaluated for self-antigens and self-reactive CD4(+) T cells in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Here, we show in proof-of-principle experiments that targeting a mimotope peptide to the endocytic receptor DEC-205 on DCs in NOD mice induces efficient conversion of pancreatic beta-cell-reactive BDC2.5 CD4(+) T cells into long-lived Foxp3(+) Treg cells. Of note, conversion efficiency in normoglycemic and hyperglycemic mice with early diabetes onset was indistinguishable. While de novo generation of BDC2.5 Treg cells did not interfere with disease progression, anti-DEC-205-mediated targeting of whole proinsulin in prediabetic NOD mice substantially reduced the incidence of diabetes. These results suggest that promoting antigen-specific Treg cells in vivo might be a feasible approach towards cellular therapy in T1D.

Identification of an immediate Foxp3(-) precursor to Foxp3(+) regulatory T cells in peripheral lymphoid organs of nonmanipulated mice.

CD4(+)CD25(+) regulatory T cells (T reg cells) expressing the transcription factor Foxp3 can be induced from peripheral T cell receptor (TCR) transgenic CD4(+)CD25(-)Foxp3(-) T cells stimulated with noninflammatory dendritic cells presenting low amounts of agonist cognate antigen. However, limited evidence exists for extra-thymic T reg cell generation from non-TCR transgenic T cells in unmanipulated mice. We compared events early during agonist-driven generation of Foxp3(+) TCR transgenic T cells to polyclonal CD4(+) T cell populations in unmanipulated mice. We identified an interleukin-2- and phosphatidylinositol-3-kinase-dependent precommitted Foxp3(-) precursor to Foxp3(+) T reg cells in peripheral lymphoid organs. Transforming growth factor beta signaling played a minor role in the generation and subsequent differentiation of these T reg precursor cells.

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation.

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.

Depletion of CD4+CD25+ human regulatory T cells in vivo: kinetics of Treg depletion and alterations in immune functions in vivo and in vitro.

The aim of this study was to investigate whether depletion of CD4(+)CD25(+) regulatory T cells (Treg) from melanoma patients affects immune responses against tumors. By application of recombinant IL-2-diphteria toxin fusion protein, also known as ONTAK, we were able to significantly reduce the frequency of Treg in peripheral blood, whereas other cell populations remained unaffected. The reduction of Treg started immediately after the first bolus of ONTAK with a dose of 5 microg ONTAK per kg bodyweight and lasted for 13 days with subsequent recovery thereafter. Successive ONTAK treatments further reduced the number of circulating Treg. Using the contact sensitizer DCP we show that all patients developed vast eczema after Treg depletion, whereas no or only mild eczematous reactions were detectable before ONTAK treatment. Corresponding induction of DCP-specific CD4(+) and CD8(+) T cells were detectable. Moreover, after immunization of ONTAK treated patients with tumor antigen peptides, MelanA/MART-1 and gp100, significant induction of peptide specific CD8(+) T cells could be observed in 90% of the patients treated. These cells displayed effector functions, as they were able to lyse peptide-pulsed target cells and secreted IFNgamma upon restimulation. In aggregate, our data indicate that ONTAK depletes Treg in vivo significantly, resulting in enhanced immune functions and substantial development of antigen-specific CD8(+) T cells in vaccinated individuals.