Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Adenosine and Its Receptors in the Pathogenesis and Treatment of Inflammatory Skin Diseases.

Inflammatory skin diseases highlight inflammation as a central driver of skin pathologies, involving a multiplicity of mediators and cell types, including immune and non-immune cells. Adenosine, a ubiquitous endogenous immune modulator, generated from adenosine triphosphate (ATP), acts via four G protein-coupled receptors (A1, A2A, A2B, and A3). Given the widespread expression of those receptors and their regulatory effects on multiple immune signaling pathways, targeting adenosine receptors emerges as a compelling strategy for anti-inflammatory intervention. Animal models of psoriasis, contact hypersensitivity (CHS), and other dermatitis have elucidated the involvement of adenosine receptors in the pathogenesis of these conditions. Targeting adenosine receptors is effective in attenuating inflammation and remodeling the epidermal structure, potentially showing synergistic effects with fewer adverse effects when combined with conventional therapies. What is noteworthy are the promising outcomes observed with A2A agonists in animal models and ongoing clinical trials investigating A3 agonists, underscoring a potential therapeutic approach for the management of inflammatory skin disorders.

Antigen targeting to dendritic cells: Still a place in future immunotherapy?

The hallmark of DCs is their potent and outstanding capacity to activate naive resting T cells. As such, DCs are the sentinels of the immune system and instrumental for the induction of immune responses. This is one of the reasons, why DCs became the focus of immunotherapeutical strategies to fight infections, cancer, and autoimmunity. Besides the exploration of adoptive DC-therapy for which DCs are generated from monocytes or purified in large numbers from the blood, alternative approaches were developed such as antigen targeting of DCs. The idea behind this strategy is that DCs resident in patients' lymphoid organs or peripheral tissues can be directly loaded with antigens in situ. The proof of principle came from mouse models; subsequent translational studies confirmed the potential of this therapy. The first clinical trials demonstrated feasibility and the induction of T-cell immunity in patients. This review will cover: (i) the historical aspects of antigen targeting, (ii) briefly summarize the biology of DCs and the immunological functions upon which this concept rests, (iii) give an overview on attempts to target DC receptors with antibodies or (glycosylated) ligands, and finally, (iv) discuss the translation of antigen targeting into clinical therapy.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

The onset of active disease in systemic lupus erythematosus patients is characterised by excessive regulatory CD4+-T-cell differentiation.

OBJECTIVES: An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in systemic lupus erythematosus (SLE) patients. Both cell subsets consist of highly proliferating Tregs/Tresps expressing inducible T-cell co-stimulatory molecule (ICOS) and less proliferating ICOS--Tregs/Tresps. METHODS: Six-colour-flow-cytometric analysis was used to examine the effect of ICOS+- and ICOS--Treg/Tresp cell differentiation on the composition of the total CD4+-T-helper cell pool with ICOS+- and ICOS--Tregs/Tresps. Functionality of Tregs was examined using suppression assays. RESULTS: In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE patients (SLEDAI <7), disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In these patients, the functional activity of ICOS+-Tregs, but not of ICOS--Tregs, was preserved. In 13 markedly active disease patients (SLEDAI >7), the percentage of both ICOS+-Tregs and ICOS+-Tresps, was strongly increased within total CD4+-T-helper cells. However, the increased ratio of ICOS+-Tregs/ICOS+-Tresps was not maintained in these patients, due to terminal differentiation and accumulation of naïve cells within total ICOS+-Tregs. Despite increased differentiation of both ICOS--Tregs and ICOS--Tresps, the percentage of ICOS--Tregs increased within CD4+-T-helper cells, while that of ICOS--Tresps decreased, resulting in a significantly increased ratio of ICOS--Tregs/ICOS--Tresps independent of age. CONCLUSIONS: Our data reveal a crucial role of Treg immune senescence for the occurrence of disease flares in SLE patients, with ICOS+-Treg cells being most affected.

End-stage renal disease, dialysis, kidney transplantation and their impact on CD4+ T-cell differentiation.

Premature aging of both CD4+ regulatory T (Treg) and CD4+ responder-T (Tresp) cells in patients with end-stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T-cell populations are released from the thymus as inducible T-cell co-stimulator-positive (ICOS+ ) and ICOS- recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS+ and ICOS- RTE Treg/Tresp cells into ICOS+  CD31- or ICOS-  CD31- memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS+ and ICOS- Treg cells in co-culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS+ RTE Treg/Tresp cells and ICOS- RTE Treg cells through CD31+ memory Treg/Tresp cells into CD31- memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS- RTE Tresp cells showed an increased differentiation via ICOS- mature naive (MN) Tresp cells into CD31- memory Tresp cells. Thereby, the ratio of ICOS+ Treg/ICOS+ Tresp cells was not changed, whereas that of ICOS- Treg/ICOS- Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS+ and ICOS- RTE Tresp cells proceeded, whereas that of ICOS+ RTE Treg cells ceased and that of ICOS- RTE Treg cells switched to an increased differentiation via ICOS- MN Treg cells. Consequently, the ratios of ICOS+ Treg/ICOS+ Tresp cells and of ICOS- Treg/ICOS- Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance-inducing differentiation of ICOS+ and ICOS- Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long-term renal replacement therapy.

The role of age-related T-cell differentiation in patients with renal replacement therapy.

Dialysis patients have deficiencies regarding the generation of immune responses and show an increased susceptibility for infections. Persisting uremic conditions are made responsible for the increased aging of their immune system. In this study, we analyzed whether age-related differences in the differentiation of both recent-thymic-emigrant-(RTE)-regulatory (Tregs) and RTE-responder T cells (Tresps) into CD31--memory Tregs/Tresps led to differences in the suppressive activity of naive and memory Tregs on autologous Tresps between healthy volunteers and dialysis patients. We found that regardless of age, the differentiation of RTE-Treg/Tresps into CD31--memory-Treg/Tresps was significantly increased in dialysis patients. By analyzing the age-related differences in the differentiation of Tregs/Tresps, we saw that in healthy volunteers RTE-Tregs differentiate via CD31+-memory Tregs into CD31--memory Tregs, which may strengthen the suppressive activity of the total Treg pool. In contrast RTE-Tresps of healthy volunteers differentiate via mature naive (MN)-Tresps into CD31--memory-Tresps, which may weaken the reactivity of the total Tresp pool. Our data revealed that this normal differentiation via MN-Tresps was lost in dialysis patients, suggesting that their Tresps are less sensitive to Treg-mediated immunosuppression. Functional analysis of Tregs on autologous Tresps showed an increasing suppressive activity with age in healthy individuals, who therefore may have a lower risk of developing autoimmune diseases but owing to decreased reactivity of their Tresps are more likely to suffer from infections. In contrast, dialysis patients exhibited a decreasing suppressive activity with age, owing to strengthened Tresp reactivity, which could explain the higher prevalence of chronic inflammatory conditions in these patients.

Constitutive crosspresentation of tissue antigens by dendritic cells controls CD8+ T cell tolerance in vivo.

Immature dendritic cells (DCs) sample tissue-specific antigens (TSAs) and process them for "crosspresentation" via major histocompatibility complex (MHC) class I and II molecules. Findings with adoptively transferred T cell receptor (TCR)-transgenic CD8+ T cells in transgenic mice expressing model TSA indicate that this process contributes to tolerance induction of CD8+ T cells, a phenomenon termed "crosstolerance." However, up to now it has been unknown whether "crosstolerance" can also control autoimmune T cells specific for physiological nontransgenic TSA. Here, we showed that a DC-specific deficiency in uptake of apoptotic material inhibits crosspresentation in vivo. This defect allowed the accumulation of fully functional autoreactive CD8+ T cells that could be activated for autoimmune attack in peripheral lymphoid organs. Thus, our data demonstrate the importance of crosstolerance induction by DCs as a vital instrument for controlling self-reactive T cells from the peripheral repertoire and preventing autoimmune disease.

Regulatory T cell-derived adenosine induces dendritic cell migration through the Epac-Rap1 pathway.

Dendritic cells (DC) are one target for immune suppression by regulatory T cells (Treg), because their interaction results in reduced T cell stimulatory capacity and secretion of inhibitory cytokines in DC. We show that DC in the presence of Treg are more mobile as compared with cocultures with conventional CD4(+) T cells and form DC-Treg aggregates within 2 h of culture. The migration of DC was specifically directed toward Treg, as Treg, but not CD4(+) T cells, attracted DC in Boyden chambers. Treg deficient for the ectonucleotidase CD39 were unable to attract DC. Likewise, addition of antagonists for A2A adenosine receptors abolished the formation of DC-Treg clusters, indicating a role for adenosine in guiding DC-Treg interactions. Analysis of the signal transduction events in DC after contact to Treg revealed increased levels of cAMP, followed by activation of Epac1 and the GTPase Rap1. Subsequently activated Rap1 localized to the subcortical actin cytoskeleton in DC, providing a means by which directed locomotion of DC toward Treg is facilitated. In aggregate, these data show that Treg degrade ATP to adenosine via CD39, attracting DC by activating Epac1-Rap1-dependent pathways. As a consequence, DC-Treg clusters are formed and DC are rendered less stimulatory. This adenosine-mediated attraction of DC may therefore act as one mechanism by which Treg regulate the induction of immune responses by DC.

The role of recent thymic emigrant-regulatory T-cell (RTE-Treg) differentiation during pregnancy.

During pregnancy, regulatory T cells (Tregs) have a key role in maternal immune tolerance to the semi-allogeneic fetus. Our previous results showed that the naive CD45RA(+)-Treg pool is functionally improved in pregnant women compared with non-pregnant women. Therefore, we examined the thymic output and differentiation of CD45RA(+)CD31(+) recent thymic emigrant (RTE)-Tregs during normal pregnancy and in the presence of preeclampsia. With the onset of pregnancy, the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg pool changed in the way that its percentage of RTE- and CD45RA(-)CD31(+)-memory Tregs decreased strongly, whereas that of the CD45RA(+)CD31(-)-mature naive (MN)-Tregs did not change and that of the CD45RA(-)CD31(-)-memory Tregs increased complementary. Thereby, the ratio of RTE-/MN-Tregs decreased from 1.0 to 0.7 leading to a significant increase in the suppressive activity of the naive CD45RA(+)-Treg pool. This effect was confirmed by re-assembling separated RTE- and MN-Tregs from non-pregnant women in the ratio of pregnant women. The suppressive activity of both separated naive Treg subsets was equally high in non-pregnant and pregnant women, but considerably reduced in preeclampsia patients, who showed significantly increased percentages of CD45RA(-)CD31(+)-memory Tregs, but decreased percentages of RTE- and MN-Tregs. Our results suggest a reduced thymic output of RTE-Tregs during pregnancy, which causes a decrease in the ratio of RTE-/MN-Tregs and thus an increase in the differentiation of RTE-Tregs towards CD45RA(-)CD31(-)-memory Tregs. Presumably, this differentiation of RTE-Tregs, which was impaired in preeclampsia patients, ensures the improved suppressive activity of the CD45RA(+)-naive Treg pool and thus retains the maintenance of pregnancy.

Targeting of autoantigens to DEC205⁺ dendritic cells in vivo suppresses experimental allergic encephalomyelitis in mice.

The dendritic and epithelial cell receptor with a m.w. of 205 kDa (DEC205) is expressed by dendritic cells (DCs) and facilitates Ag presentation. After injection of Ags coupled to Abs specific for DEC205 into mice, Ag presentation occurs by nonactivated DCs, which leads to induction of regulatory T cells (Tregs). To test this system for tolerance induction in experimental allergic encephalomyelitis (EAE), we created single-chain fragment variables (scFv) specific for DEC205 and fused the scFv to the self-Ag myelin oligodendrocyte glycoprotein (MOG; scFv DEC:MOG). An anti-ß-galactosidase scFv:MOG fusion protein (scFv GL117:MOG) served as isotype control. After staining of DCs in vitro with purified scFv DEC:MOG, binding to DCs and colocalization with MHC class II was apparent, whereas isotype controls did not bind. We next injected scFv DEC:MOG into mice and observed elevated numbers of highly activated, IL-10-producing CD4⁺CD25⁺Foxp3⁺ Tregs (17% of CD4) in spleens, as compared with isotype controls and uninjected mice (12% of CD4). Furthermore, DCs isolated from scFv DEC:MOG-injected animals produced significantly increased levels of TGF-ß. Most importantly, when EAE was induced in scFv DEC:MOG-injected mice, 90% of the mice were protected from EAE, whereas all mice in the isotype controls (scFv GL117:MOG) experienced development of EAE. When applying scFv DEC:MOG to mice that had already experienced EAE symptoms, abrogation of the disease in 90% of the animals was apparent, whereas all animals in the control groups experienced development of severe EAE. Thus, these data indicate that targeting of MOG to "steady-state" DCs in vivo may provide a tool to prevent and to treat EAE by a DC/Treg-driven mechanism.

Adenosine, bridging chronic inflammation and tumor growth.

Adenosine (Ado) is a well-known immunosuppressive agent that may be released or generated extracellularly by cells, via degrading ATP by the sequential actions of the ectonucleotides CD39 and CD73. During inflammation Ado is produced by leukocytes and tissue cells by different means to initiate the healing phase. Ado downregulates the activation and the effector functions of different leukocyte (sub-) populations and stimulates proliferation of fibroblasts for re-establishment of intact tissues. Therefore, the anti-inflammatory actions of Ado are already intrinsically triggered during each episode of inflammation. These tissue-regenerating and inflammation-tempering purposes of Ado can become counterproductive. In chronic inflammation, it is possible that Ado-driven anti-inflammatory actions sustain the inflammation and prevent the final clearance of the tissues from possible pathogens. These chronic infections are characterized by increased tissue damage, remodeling and accumulating DNA damage, and are thus prone for tumor formation. Developing tumors may further enhance immunosuppressive actions by producing Ado by themselves, or by "hijacking" CD39+/CD73+ cells that had already developed during chronic inflammation. This review describes different and mostly convergent mechanisms of how Ado-induced immune suppression, initially induced in inflammation, can lead to tumor formation and outgrowth.

Tolerance to 2,4-Dinitrofluorobenzene‒Induced Contact Hypersensitivity Is Mediated by CD73-Expressing Tissue-Homing Regulatory T Cells.

Regulatory T cells (Tregs) express CD73, an ectonucleotidase that converts adenosine (Ado) monophosphate to Ado, which has been shown to suppress immune reactions. To investigate the role(s) of CD73+ Tregs during the induction of tolerance, we used a 2,4-dinitrofluorobenzene‒driven contact hypersensitivity model, in which tolerance can be induced by pretreating wild type mice with 2,4-dinitrothiocyanobenzene. CD73-deficient mice were unable to acquire tolerance. Likewise, transfer of CD73‒/‒ Tregs failed to suppress 2,4-dinitrofluorobenzene‒induced ear swelling in wild type mice, whereas transfer of wild type‒derived Tregs into CD73‒/‒ mice re-established tolerance. This indicates a crucial role of CD73+ Tregs for skin-induced tolerance. Furthermore, we found that 2,4-dinitrothiocyanobenzene induces more activated CD73+ tissue-homing Tregs (marked by Ki-67, CTLA4, CCR4, CD103, CCR6, and CD49b expression) in draining lymph nodes and blood, eventually accumulating in the skin. The application of anti-CD73 antibodies that block CD73-derived Ado production as well as the injection of Ado deaminase, which degrades Ado in tissues, abrogated tolerance induction. Thus, our data indicate that CD73+ Ado-producing Tregs are crucial for the regulation of contact hypersensitivity reactions and tolerance induction in the skin and that manipulating the function(s) of CD73 in tissues may offer a tool to influence autoimmunity and inflammation in vivo.

Topical Application of Adenosine A2-Type Receptor Agonists Prevents Contact Hypersensitivity Reactions in Mice by Affecting Skin Dendritic Cells.

Adenosine (Ado) produced by skin and skin migratory CD73+ dendritic cells is critically involved in tolerance to haptens. We therefore investigated the use of Ado receptor agonists for the treatment of contact hypersensitivity reactions. A2A- 4-[2-[[6-Amino-9-(N-ethyl-ß-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl]benzenepropanoic acid hydrochloride (CGS) and A2B- 2-[[6-Amino-3,5-dicyano-4-[4-[cyclopropylmethoxy]phenyl]-2-pyridinyl]thio]-acetamide (BAY) specific Ado receptor agonists were epicutaneously applied to the skin before sensitization and challenge with DNFB. Both agonists reduced ear swelling compared with solvent controls. This was accompanied by fewer activated T cells in the skin after the challenge and by higher numbers of T cells expressing anergic markers such as LAG-3, CD137, PD-1, CD272, and TIM-3 in the lymph nodes of CGS-treated groups. In ear tissue, Ado receptor agonist treatment reduced the production of proinflammatory cytokines and chemokines as well as the infiltration by neutrophils after sensitization. Moreover, reduced numbers of skin migratory dendritic cells producing less IL-12 and exhibiting lower expression of CD86 were recorded in lymph nodes after sensitization. In cocultures of skin migratory dendritic cells from CGS-treated mice with T cells, reduced proliferation of T cells and decreased secretion of proinflammatory cytokines compared with that of solvent controls were apparent. In conclusion, topical application of Ado receptor agonists to the skin prevents sensitization of T cells against haptens by reducing the migration and activation of skin migratory dendritic cells.

Term and preterm labor: decreased suppressive activity and changes in composition of the regulatory T-cell pool.

Regulatory T cells (Tregs) exert a key role in tolerance induction to the semi-allogeneic fetus. Currently, it is not known whether immunological rejection processes are involved in the induction of normal term or irresistible preterm labor. In this study, we examined whether there were differences in the percentage of the total CD4(+)CD127(low+/-)CD25(+)FoxP3(+)-Treg-cell pool, its suppressive activity and its composition with distinct Treg subsets (HLA-DR(low+)-, HLA-DR(high+)-, HLA-DR(-)- and naive CD45RA(+)-Tregs) between preterm and term laboring women. We found that its percentage was decreased neither in term nor in preterm laboring women. Its suppressive activity was strongly diminished in preterm laboring women and to a lesser extent in spontaneously term laboring women. During the normal course of pregnancy, its composition changed in such a way that the percentage of naive CD45RA(+)-Tregs increased while the percentage of the highly suppressive HLA-DR(low+)- and HLA-DR(high+)-Tregs decreased significantly until term. With the onset of spontaneous term labor this phenomenon was reversed and reached significant values postpartum. In addition, we confirmed that both the decreased percentage of HLA-DR(+)-Tregs within the total Treg-cell pool and their decreased level of HLA-DR expression (depending on the percentage of HLA-DR(low+)- and HLA-DR(high+)-Tregs) had a reducing effect on the suppressive activity of the total Treg cell pool in preterm laboring women. However, spontaneous term delivery was associated with increasing percentages of HLA-DR(+)-Tregs and increasing HLA-DR expression of this Treg subset. Therefore, it becomes apparent that the mechanisms inducing term or preterm labor may be completely different.

Targeting of the hNC16A collagen domain to dendritic cells induces tolerance to human type XVII collagen.

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.

ATP activates regulatory T Cells in vivo during contact hypersensitivity reactions.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) require activation to develop their full suppressive capacity. Similar to conventional T cells, Tregs can be activated via their TCRs; however, other means may be in place. We injected naive and nonactivated Tregs, being CD69(-)CD44(low)CD62L(+) into mice, and analyzed their phenotype after sensitization or challenge with the contact sensitizer 2,4,6-trinitro-1-chlorobenzene. We found that Tregs acquired an activated phenotype (CD69(+)CD44(high)CD62L(-)) in the draining lymph node after sensitization. In contrast, Ag challenge activated Tregs in the blood. This tissue-specific activation was induced by ATP, which was released at the respective tissue sites after sensitization or challenge, respectively. To demonstrate that activation was also essential for the induction of the suppressive function of Tregs, Tregs were treated with ATP receptor antagonists. In this study, we show that ATP receptor antagonists abrogated the suppressive effects of injected naive Tregs in contact hypersensitivity reactions. Thus, these data indicate that activation of Tregs via ATP in vivo provides a novel pathway of stimulating the suppressive function of Tregs.

Endothelial cells augment the suppressive function of CD4+ CD25+ Foxp3+ regulatory T cells: involvement of programmed death-1 and IL-10.

Blood endothelial cells (ECs) act as gatekeepers to coordinate the extravasation of different T cell subpopulations. ECs express defined panels of adhesion molecules, facilitating interaction with blood circulating T cells. In addition to the mere adhesion, this cellular interaction between ECs and transmigrating T cells may also provide signals that affect the phenotype and function of the T cells. To test the effects of ECs on regulatory T cells (T(reg)) we set up cocultures of freshly isolated murine T(reg) and primary ECs and assessed the phenotype and function of the T(reg). We show that T(reg) upregulate programmed death-1 (PD-1) receptor expression, as well IL-10 and TGF-beta secretion after contact to ECs. These changes in phenotype were accompanied by an increased suppressive capacity of the T(reg). Blockade of the PD-1 and/or the IL-10 secretion in the in vitro suppression assays abrogated the enhanced suppressive capacity, indicating relevance of these molecules for the enhanced suppressive activity of T(reg). In aggregate, our data show, that ECs increase the immunosuppressive potential of activated T(reg) by upregulation of PD-1 and stimulation of the production of high levels of IL-10 and TGF-beta. Therefore, one can speculate that T(reg) during transendothelial transmigration become "armed" for their suppressive function(s) to be carried out in peripheral tissues sites.

The Multifaceted Actions of CD73 During Development and Suppressive Actions of Regulatory T Cells.

Adenosine (Ado) has been shown to have immunosuppressive effects in a variety of diseases. It can either be released directly into the extracellular environment by cells, or it can be produced by degradation of ATP within the extracellular spaces. This extracellular pathway is facilitated by the concerted actions of the ectoenzymes CD39 and CD73. In a first step CD39 dephosphorylates ATP to ADP and AMP, respectively, and in a second step CD73 converts AMP to Ado. Thus, activity of CD73 on the cell surface of cells is the rate limiting step in the generation of extracellular Ado. Among T cells, CD73 is most abundantly expressed by regulatory T cells (Tregs) and is even upregulated after their activation. Functionally, the generation of Ado by CD73+ Tregs has been shown to play a role in immune suppression of dendritic cells, monocytes and T cells, and the defined expression of CD73 by Tregs in immunosuppressive environments, such as tumors, made CD73 a novel checkpoint inhibitor. Therefore, therapeutical intervention by anti-CD73 antibodies or by chemical inhibitors of the enzymatic function is currently under investigation in some preclinical animal models. In the following we summarize the expression pattern and the possible functions of CD73 in T cells and Tregs, and exemplify novel ways to manipulate CD73 functions in Tregs to stimulate anti-tumor immunity.

Impaired Treg-DC interactions contribute to autoimmunity in leukocyte adhesion deficiency type 1.

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common ß-chain of the ß2-integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. Patients with LAD-1 are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ Treg are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity, we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here, we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell-DC interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extralymphatic organs, but it resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.