In T cells, processes such as migration and immunological synapse formation are accompanied by the dynamic reorganization of the actin cytoskeleton, which has been suggested to be mediated by regulators of RhoGTPases and by F-actin bundlers. SWAP-70 controls F-actin dynamics in various immune cells, but its role in T cell development and function has remained incompletely understood. CD4+ regulatory T (Treg) cells expressing the transcription factor Foxp3 employ diverse mechanisms to suppress innate and adaptive immunity, which is critical for maintaining immune homeostasis and self-tolerance. Here, we propose Swap-70 as a novel member of the Foxp3-dependent canonical Treg cell signature. We show that Swap-70-/- mice have increased numbers of Foxp3+ Treg cells with an effector/memory-like phenotype that exhibit impaired suppressor function in vitro, but maintain overall immune homeostasis in vivo. Upon formation of an immunological synapse with antigen presenting cells in vitro, cytosolic SWAP-70 protein is selectively recruited to the interface in Treg cells. In this context, Swap-70-/- Treg cells fail to downregulate CD80/CD86 on osteoclast precursor cells by trans-endocytosis and to efficiently suppress osteoclastogenesis and osteoclast function. These data provide first evidence for a crucial role of SWAP-70 in Treg cell biology and further highlight the important non-immune function of Foxp3+ Treg cells in bone homeostasis mediated through direct SWAP-70-dependent mechanisms.

Human T lymphotropic virus type -1 (HTLV-1) infections are important causes of mortality and morbidity in endemic areas worldwide. There is neither a vaccine specific for the virus nor satisfactory treatment for the associated malignancy or inflammatory syndromes. HTLV-1 associated infective dermatitis (IDH) is a chronic dermatitis that has been observed in a variable proportion of HTLV-1 infected children. IDH may serve as an early clinical marker for HTLV-1 and an indicator of increased risk for developing other HTLV-1 associated conditions such as adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy or transient spastic paraparesis (HAM/TSP). However the mechanisms underlying IDH and the relationships with HAM/TSP and ATLL are poorly understood. We undertook skin biopsies from 14 cases with IDH, and controls which included five asymptomatic carriers (ACs) and 18 healthy uninfected individuals from South Africa. We conducted clinical assessments, proviral load, allergen-specific IgE, peripheral blood and cutaneous T cell and virological analyses. We obtained relevant clinical history and examined all cases and controls based on a pre-formed questionnaire. Despite the partial clinical similarities with atopic dermatitis, the individuals with IDH did not have an increased incidence of atopic disease including asthma or rhinitis. Furthermore house dust mite-specific IgE levels were not elevated in the cases compared to the controls, suggesting that atopy is not a predisposing factor for the development of IDH in HTLV-1 infected individuals. Circulating proviral load was significantly higher in those with IDH compared to asymptomatic carriers and skin biopsy revealed acanthosis, and lymphocytic epidermotropism associated with a superficial perivascular and periadnexal lymphocytic infiltration of CD8+, and CD4+ T cells. Furthermore IDH associated with an infiltrate of epidermal and dermal FoxP3+ T cells and lesional down-regulation of filaggrin expression compared to non-lesional skin. We did not observe an elevation of pro-inflammatory cytokines in the sera of individuals with IDH compared to the controls. We investigated integration patterns in the skin and blood of 10 cases with IDH, and two asymptomatic carrier (AC) individuals from South Africa. We first showed that the virus is present in the skin at high levels (total mean levels of 47.09 proviral copies per 1000 cells) as comparable to that which has been observed in blood (total mean levels 137 proviral copies per 1000 cells). Using a high throughput Illumina sequencing system in collaboration with Professor Bangham, we mapped and quantified the relationship between oligoclonal proliferation of HTLV-1 infected T cells in the skin and blood of IDH patients. It was found that in IDH, a selective outgrowth of certain clones is favoured, supporting the possibility of skin-specific factors exerting positive selection on proliferation. In IDH, there was not a preferential integration of the provirus in transcriptionally active regions of the gene sites, as had been observed in other HTLV-1 associated conditions. These observations imply that the selection forces that favour oligoclonal proliferation of HTLV-1+ T cells differ fundamentally between simple HTLV-1 infection and other events associated with the dermatitis. In conclusion, these data show that HTLV-1 is not associated with an atopic diathesis. Given the lack of elevated pro-inflammatory cytokines and presence of a cutaneous infiltrate of FoxP3+ T cells, the findings suggest that high levels of HTLV-1 replication promotes a regulatory environment leading to filaggrin down-regulation, cutaneous susceptibility to infection, and secondary inflammatory skin disease. Viral integration patterns would support the presence of skin-specific positive selection, perhaps eventually leading to expansion of particular clones with the potential to develop towards ATLL. It remains to be explained whether the high viral load in IDH changes over time, more specifically in the steps leading to ATLL.

Les cellules T régulatrices Foxp3+ (Treg) représentent une sous-population T CD4 cruciale pour le maintient de l'immuno-tolerance. Mieux comprendre la biologie des Treg, leur hétérogénéité, leur développement, leur mécanisme d’action et les facteurs assurant leur survie en périphérie reste un objectif majeur. L'objectif de ce travail de thèse était de mieux définir les mécanismes impliqués dans le contrôle de l’homéostasie Treg et d’évaluer l’éventuelle contribution des perturbations de l’homéostasie Treg en pathologie humaine.Dans la première partie de ce travail de thèse nous avons essayé de finalement définir dans le modèle murin le rôle joué par l'IL-7 dans le contrôle de l’homéostasie Treg. Nous avons montré que l'expression de CD127, la chaîne alpha du récepteur à l'IL-7, est finement régulée à la surface des Treg et qu'elle dépend de leur activation ainsi que de leur localisation tissulaire. Nous avons démontré que l’expression de CD127 par les Treg activées est fonctionnelle, identifiant ces cellules comme cibles potentiels de l'IL-7. En utilisant des modèles murins présentant une altération de la voie de signalisation IL-7/IL-7R et des modèles de transfert adoptif, nous avons obtenu une démonstration définitive du rôle direct de l'IL-7 dans la régulation du nombre de cellules Treg. Enfin, nous avons démontré que la signalisation IL-7 optimise la capacité de ces cellules de réagir à l'IL-2, une cytokine importante dans la régulation de l’homéostasie Treg. Dans la deuxième partie de ce travail, nous avons étudié l’homéostasie Treg dans le contexte de l'infection par le VIH. Cette étude a bénéficié de l’accès à des patients au cours de la primo infection et des HIV contrôleurs. Nous avons montré que les Treg effecteurs plus que les Treg naïves sont affectés par l'infection par le VIH. De plus, nous avons montré que le nombre des effecteurs Treg corrélant inversement avec les réponses T CD8 spécifiques, offrant une preuve ex vivo de l'implication des Treg dans l'immunité anti-VIH. / Regulatory T cells (Treg) represent a crucial CD4 T cells subset involved in maintenance of immune-tolerance. Since their first description important efforts have been undertaken to better understand their biology, their development and their mechanisms of action. However, little is known about factors controlling Treg peripheral homeostasis. The aim of this thesis work was to better define mechanisms involved in governing Treg homeostasis and to investigate the eventual contribution of perturbation of Treg homeostasis in human disease. In the first part of this thesis work we tried to define in the murine system the role played by IL-7 in governing Treg homeostasis. We showed that Treg surface expression of CD127, the IL-7 receptor alpha chain, is finely regulated as it depends on their activation as well as on their tissue localization. More importantly, we demonstrated that Treg do express functional levels of CD127, identifying these cells as potential target of IL-7. Using both genetically modified murine models of altered IL-7 signaling and adoptive transfer models, we obtained definitive evidence for a direct role of IL-7 in governing Treg cell numbers. Finally, we demonstrated that IL-7 signaling in Treg optimizes their capacity to react to IL-2 an important cytokine regulating Treg homeostasis. In the second part of this work we investigated Treg homeostasis in the context of HIV infection. Employing for the first time in HIV infection a novel consensus Treg identification strategy and applying it to different groups of HIV infected patients, including primary infected patients and HIV controllers, we showed that HIV infection is characterized by an early and long lasting alteration of Treg homeostasis. In particular we demonstrated that effector rather than naive Treg are affected by HIV infection. Moreover, we showed that effector Treg numbers inversely correlated with HIV specific CD8 T cells responses, providing ex vivo evidence of Treg involvement in HIV immunity.

Activation of immunity to self-antigens is the goal in cancer immunotherapy, whereas blocking such responses is the goal in autoimmune disease. Thus, it is not surprising that investigation into cancer immunotherapy might also produce insights for the treatment of autoimmune disease. Heat shock protein, gp96, based therapies lead to the robust activation of CD8+ cytotoxic T cells that can slow tumor growth in 60-70% of mice, but only lead to the elimination of tumors in 30-40% of animals. The primary goal of the current studies was to understand why vaccination with a secreted gp96 vaccine was not efficacious in a larger proportion of animals, and identify combination therapies that enhanced the anti-tumor activity of gp96-Ig vaccination. It was found that in mice bearing established tumors, some mice responded well to vaccination with gp96-Ig, and that the induction of CD8+ T cells was found to correlate with tumor rejection; indicating that the proportion of mice that failed to reject tumors had established mechanisms of tumor-mediated suppression of anti-tumor immunity. The mechanism of this suppression was found to differ between various tumor models, so combination therapy sought to amplify CD8+ T cell responses directly, rather than by indirectly inhibiting suppressive factors induced by established tumors. It was found that antibody-based therapies leading to the stimulation of TNFRSF25, a powerful T cell co-stimulatory receptor, caused synergistic expansion of tumor-specific T cells when given in combination with gp96-Ig vaccination and led to enhanced rejection of multiple tumor types. Interestingly, TNFRSF25 agonistic antibodies were also found to directly stimulate the proliferation of natural CD4+FoxP3+ regulatory T cells. This activity was found to be beneficial in the prevention of allergic lung inflammation when administered prior to antigen challenge. These studies have therefore identified the conditions required for successful tumor elimination following gp96-Ig vaccination, and discovered that a TNFRSF25 agonistic antibody may be used to enhance anti-tumor immunity induced by gp96-Ig. These studies have also identified TNFRSF25 stimulation as the most powerful, and physiologically relevant, method to selectively induce Treg proliferation in vivo ever discovered, with important consequences for the treatment of autoimmune inflammation.

T regulatory cells (Tregs) have been extensively studied in patients with cancer or autoimmunity. These cells hamper the immune system’s ability to clear tumor cells in cancer patients. In autoimmune diseases, on the other hand, they are not able to restrain autoreactive immune responses. If we manage to understand Tregs and their role in health and diseases we may be able to develop better immunomodulatory therapies. Early studies demonstrated that tolerance was maintained by a subset of CD25+ T-cells. CD25 was the earliest marker for Tregs and is still often used to define these cells. Several Treg-associated markers have been suggested throughout the years. However, these markers can be upregulated by activated T-cells as well. The most specific marker for Tregs is currently the transcription factor forkhead box P3 (FoxP3). In this thesis, we investigated the presence of CD25- Tregs in patients with B-cell malignancies and in patients with autoimmunity. These cells were identified in both patient groups. Further, patients with B-cell malignancies often have high levels of soluble CD25 (sCD25) in the periphery. In our patient cohorts, the level of peripheral Tregs correlated with the level of sCD25 in patients with lymphoma. Tregs were shown to release sCD25 in vitro and sCD25 had a suppressive effect on T-cell proliferation. These data show that Tregs may release CD25 to hamper T-cell proliferation and that this may be an immune escape mechanism in cancer patients. Previous studies have demonstrated that an increased infiltration of FoxP3+ cells into lymphoma-affected lymph nodes is associated with a better patient outcome. This is in contrast to studies from non-hematological cancers where an increased presence of Tregs is associated with a poor prognosis. Since previous studies have shown that Tregs are able to kill B-cells, we wanted to investigate if Tregs are cytotoxic in patients with B-cell tumors. In the subsequent studies, Tregs from patients with B-cell lymphoma and B-cell chronic lymphocytic leukemia (CLL) were phenotyped to investigate the presence of cytotoxic markers on these cells. FoxP3-expressing T-cells from both patients with CLL and B-cell lymphoma displayed signs of cytotoxicity by upregulation of FasL and the degranulation marker CD107a. Tregs from CLL patients could further kill their autologous B-cells in in vitro cultures. Taken together the studies in this thesis have demonstrated two possible new functions of Tregs in patients with B-cell malignancies and the presence of CD25- Tregs in both cancer and autoimmunity.

In recent years, there has been a worldwide increase in obesity, which parallels a rise in pathologies, including type 2 diabetes, collectively termed the metabolic syndrome. Chronic, low-grade inflammation has been implicated as a major link between these diseases. Recent work showed the presence of a unique subset of CD4+Foxp3+ regulatory T cells residing in visceral adipose tissue (VAT Treg) with PPAR-g being the key transcription factor responsible for their phenotype and function in controlling adipose tissue inflammation and, thereby, insulin sensitivity. VAT Tregs inversely correlated with insulin resistance. In contrast, there was a dramatic age-associated increase in frequency of VAT Tregs in lean animals, correlating with continued insulin sensitivity, despite significant increases in body and adipose tissue weights. This increase in Treg frequencies was not observed in other lymphoid and non-lymphoid tissues, including the subcutaneous fat depot. We characterized this unique age-associated increase in VAT Tregs through the use of adoptive transfer models, in vivo labeling and tracking systems, parabiosis, and analysis of the T cell receptor (TCR) repertoire used by VAT Tregs. Our findings indicate that the progressive increase in VAT Tregs is not due to conversion of conventional CD4+ T cells nor to substantial infiltration of Tregs from the circulation and secondary lymphoid organs. However, by analyzing the TCR repertoire on a single-cell level we uncovered a striking oligo-clonal expansion of VAT Tregs, suggesting their accumulation results from in situ proliferation. We further showed that this accumulation is dependent on major histocompatibility complex (MHC) class II, but not on CD1d. Finally, we showed that IL-33 was able to induce proliferation of VAT Tregs. In parallel, we extended our analysis of TCR repertoire to the Treg population residing in skeletal muscle. In acute and chronic models of muscle injury, muscle-resident Tregs underwent a substantial clonal expansion, with a particular clone being detected in multiple individuals. Taken together these studies highlight the importance of proliferation as a mechanism of Treg accumulation in tissues in response to acute and chronic inflammation.

Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.

Les lymphocytes B régulent la réponse immunitaire par la sécrétion de facteurs proinflammatoires ou immunosuppresseurs. Dans la Leucémie Lymphoïde Chronique (LLC),une sous-population de lymphocytes B CD5⁺ présente des propriétés immunosuppressives qui l’apparente aux lymphocytes B régulateurs notamment par la production d’IL-10. La survie des cellules leucémiques est, elle, associée à la réponse antigénique et à la production de cytokines dont l’IL-6. L’objectif de ce travail a été de caractériser dans la pathologie les populations produisant les cytokines pro-survie ou immunorégulatrices et d’analyser la relevance fonctionnelle de leur sécrétion. Nous avons identifié des sous-populations de cellules B leucémiques exprimant trois facteurs immunorégulateurs l’IL-10, le TGFβ1 et pour la première fois le facteur de transcription FOXP3, La proportion augmentée de cellules exprimant l’IL10 est associée à une diminution des cellules exprimant l’IL6. De manière importante, ce travail a identifié une boucle autocrine de stimulation de l’activité métabolique des cellules par l’IL10. La cytokine en se fixant à son récepteur permet l’activation des facteurs STAT3 et induit l’expression à la fois de protéines anti- apoptotiques de la famille Bcl2 mais surtout sa propre expression. Un blocage de cette boucle au niveau du récepteur à l’IL10 suspend l’avantage de survie des cellules tumorales. L’IL-6 ne déclenche pas ces mécanismes de maintien des cellules de LLC. Ce travail montre qu’en plus de son rôle sur les cellules du microenvironnement tumoral, l’IL-10 participe au maintien autocrine de la sous-population immunorégulatrice dans la LLC. / B cells produce pro-inflammatory or immunosuppressive factors to modulate the immuneresponse. In Chronic lymphocytic leukemia (CLL), a subset of the tumor lymphocytes produces IL10 and share immunoregulatory functions with regulatory B cells. CLL cell ssurvival is driven by antigenic response and pro-survival cytokines such as IL6. This project aimed at deciphering the cytokines profile of CLL subsets and analyzing their functional relevance. We identified immunoregulatory subsets producing IL-10, TGFβ1 and for the firsttime FOXP3. In patients, the increased proportion of cells expressing IL10 was correlated with decrease in IL6⁺ cells. Importantly we described an autocrine survival loop driven by IL10 in these cells. IL10 triggering led to STAT3 activation, induction of active pro-survival factors altogether with IL10 self-induction. Interrupting this loop with a blocking ab against IL10R prevented survival of the cells. IL6 did not manage such mechanisms. In conclusion,this work demonstrates that IL10 is an important mediator in CLL; the cytokine alters immune recognition of the tumor cells and sustains leukemic cells survival via the autocrine loop.

Zöliakie ist durch eine dysregulierte Immunreaktion auf die in Getreiden enthaltene Proteinfraktion Gluten charakterisiert. Die Assoziation der Erkrankung mit Polymorphismen in immunregulatorischen Genen weist auf eine Rolle von regulatorischen T-Zellen im Krankheitsgeschehen hin. Foxp3+ regulatorische T-Zellen haben eine essentielle Bedeutung für die Aufrechterhaltung der intestinalen Immunhomöostase und die Limitierung von Autoimmunität. In der vorliegenden Arbeit wurde eine 2005 bis 2010 diagnostizierte Gruppe von 51 Kindern und Jugendlichen mit Zöliakie untersucht. Diese Gruppe wurde mit 51 geschlechts- und altersadaptierten Kontrollen ohne Zöliakie verglichen. Es wurden anamnestische, paraklinische und histologische Daten mit der Verteilung von CD3+Foxp3+ regulatorischen T-Zellen in der Dünndarmschleimhaut untersucht. Patienten mit Zöliakie wiesen eine leichte Anämie, jedoch keine signifikante Wachstums- und Gewichtsentwicklung auf, was die oligosymptomatische Verlaufsform in der Gesamtkohorte unterstreicht. Es konnte gezeigt werden, dass CD3+Foxp3+ regulatorische T-Zellen bei Zöliakie-Patienten vermehrt in der Dünndarmschleimhaut akkumulieren. Weiterhin korreliert die Häufigkeit CD3+Foxp3+ regulatorischer T-Zellen sowohl mit dem Schweregrad der Schleimhaut-schädigung (gemessen an der Marsh-Oberhuber-Klassifikation, dem Zotten-Krypten Verhältnis oder der Zahl der intraepithelialen Lymphozyten) als auch mit den Titern Zöliakie-spezifischer Antikörper. Die Akkumulation CD3+Foxp3+ regulatorischer T Zellen lässt sich partiell als Folge einer Anreicherung von CD4+ T-Zellen auf Kosten CD8+ T-Zellen erklären. Die Daten weisen darauf hin, dass Foxp3+ regulatorische T Zellen sekundär als Folge des gluteninduzierten Entzündungsprozesses in der Schleimhaut akkumulieren, diesen offensichtlich aber nicht effektiv begrenzen. Die mögliche Assoziation der Immundysregulation der Zöliakie mit Foxp3+ regulatorischen T-Zellen ist damit nicht durch eine numerische Reduktion sondern wahrscheinlich durch partielle funktionelle Defekte bedingt.:Bibliographische Beschreibung 2 Inhaltsverzeichnis 3 Abkürzungsverzeichnis 5 1. Einleitung 6 1.1 Zöliakie 6 1.1.1 Epidemiologie 6 1.1.2 Äthiopathogenese 6 1.1.3 Diagnostik 10 1.1.4 Therapie 11 1.2 Regulatorische T-Zellen 12 1.2.1 Typen regulatorischer T-Zellen 13 1.2.2 Suppressionsmechanismen von CD4+CD25+Foxp3+ regulatorischen T-Zellen 14 1.3 Zielstellung der Arbeit 17 2. Material und Methoden 19 2.1 Ethikvotum 19 2.2 Ablauf der Studie 19 2.3 Rekrutierung der Studienpopulation 20 2.3.1 Gruppe der Zöliakie-Patienten 22 2.3.2 Kontrollgruppe 22 2.4 Telefoninterview – Erfassung anamnestischer Daten 23 2.5 Anthropometrische Daten 23 2.6 Klinische Chemie 24 2.7 Zöliakie - spezifische Antikörper 24 2.8 Färbungen der histologischen Schnitte 25 2.8.1 Vorbereitung der Gewebeproben 25 2.8.2 Hämatoxylin-Eosin (HE)-Färbung 25 2.8.3 Immunhistochemische Färbungen 26 2.8.4 Immunfluoreszenzfärbungen 27 2.8.5 Mikroskopie und Fotographie 28 2.9 Morphometrische Messung der histologischen Schnitte 28 2.9.1 Zotten- und Kryptenmessung 28 2.9.2 Bestimmung der Anzahl intraepithelialer Lymphozyten 29 2.9.3 Ermittlung von Zelldichten 29 2.10 Statistische Auswertung 30 3. Ergebnisse 31 3.1 Charakterisierung der Studienpopulation 31 3.1.1 Der Schweregrad der Schleimhautschädigung korreliert mit dem Geschlecht 31 3.1.2 Die Einführung glutenhaltiger Nahrung erfolgt bei Kindern mit Zöliakie früher 32 3.1.3 Keine Unterschiede in Körpergröße und Körpergewicht zwischen den Studiengruppen 36 3.1.4 Klinische Chemie - Leichte Anämie bei Kindern und Jugendlichen mit Zöliakie 39 3.1.5 Serologische Charakterisierung der Studienpopulation 41 3.2 Histologische Charakterisierung der Dünndarmschleimhaut 42 3.2.1 Das Zotten-Kryptenverhältnis sinkt mit zunehmendem Marsh-Stadium 42 3.2.2 Zunahme von intraepithelialen Lymphozyten bei Patienten mit Zöliakie 44 3.2.3 Erhöhte Infiltrationsdichte in der Lamina propria von Zöliakie-Patienten 46 3.3 Die Anzahl von CD3+Foxp3+ T-Zellen ist in der Dünndarmschleimhaut von Zöliakie- Patienten erhöht 48 3.4. Erhöhte CD4/CD8-Ratio in der Dünndarmschleimhaut von Zöliakie-Patienten 50 3.5 Dichte von regulatorischen Foxp3+ T-Zellen korreliert mit histologischen, hämatologischen und serologischen Parametern 52 4. Diskussion 56 4.1 Zöliakie – Umweltfaktoren und Immunregulation 56 4.2 Rolle von Foxp3+ T-Zellen bei Zöliakie 58 4.3 Immungenetik bei Zöliakie 63 4.4 Stärken und Schwächen der Studie 68 5. Zusammenfassung 69 Literaturverzeichnis 71 Appendix 84 Lebenslauf 85 Persönliche Daten 85 Danksagung 86 Erklärung über die eigenständige Abfassung der Arbeit 88

Depending on the duration and severity, psychological tension and physical stress can enhance or suppress the immune system in both humans and animals. Although it has been established that chronic stress exerts a significant suppressive effect on immune function, the mechanisms by which affects immune responses remain elusive. By employing an in vivo murine system, we revealed that TGF-β1/Smad2/3/Foxp3 axis was remarkably activated following chronic stress. Furthermore, TLR9 and p38 MAPK played a critical role in the activation of TGF-β1/Smad2/3/Foxp3 signaling cascade. Moreover, inhibition of TGF-β1/Smad2/3/Foxp3 or p38 significantly attenuated chronic stress-induced lymphocyte apoptosis and apoptosis-related proteins, as well as the differentiation of T regulatory cells in spleen. Interestingly, disequilibrium of pro-inflammatory and anti-inflammatory cytokines balance caused by chronic stress was also rescued by blocking TGF-β1/Smad2/3/Foxp3 axis. These findings yield insight into a novel mechanism by which chronic stress modulates immune functions and identifies new targets for the development of novel anti-immune suppressant medications.