IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells

Interleukin-2 (IL-2) treatment is currently used to enhance T cell-mediated immune responses against tumors or in viral infections. At the same time, IL-2 is essential for the peripheral homeostasis of CD4(+)CD25(+)Foxp3(+ )regulatory T cells (Treg). In our study, we show that IL-2 is also an important activator of Treg suppressive activity in vivo. IL-2 treatment induces Treg expansion as well as IL-10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL-2 via gene-gun vaccination using IL-2 encoding DNA plasmids (pIL-2) inhibited naive antigen-specific T cell proliferation as well as a Th1-induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL-2-treated mice and the suppression depends on the synergistic action of IL-10 and TGF-beta. These data highlight that during therapeutic treatment with IL-2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.     

Localization of IL-17+Foxp3+ T cells in esophageal cancer

The etiology of cancer is unclear. Recent studies indicate that some cytokines, such as interleukin (IL)-17, and regulatory T cells are involved in the development of cancer. This study aims to detect a subset of T cell, IL17+Foxp3+ T cell, in the pathogenesis of esophageal cancer (Eca). Twelve patients with squamous Eca were recruited in this study. The surgically removed Eca tissue was collected. Cells isolated from Eca tissue were analyzed by flow cytometry. The results showed that 2-10% Eca tissue-derived CD4(+) T cells expressed Foxp3; only 0.2-0.8% non-ca tissue-derived CD4(+) T cells expressed Foxp3. Further analysis showed that 3-15% Eca-isolated CD4(+) T cells were also IL-17 positive whereas only 0.4-1.5% non-ca tissue-isolated CD4(+) T cells were IL-17 positive. We also found that about 4.8-11.2% Foxp3(+) IL-17(+) T cells in isolated CD4(+) T cells from Eca tissue that were significantly less than in non-ca tissue derived CD4(+) T cells. Less than 1% Foxp3(+) IL-17(+) T cells in isolated CD4(+) T cells in both Eca patients and healthy controls. Treatment with hypoxia markedly increased the expression of IL-6 in peripheral CD68+ cells. Coculturing CD68+ cells and Foxp3+ T cells under hypoxic environment resulted in abundant expression of IL-17 in Foxp3+ T cells that could be blocked by pretreatment with either anti-IL-17 or anti-transforming growth factor beta antibodies. We conclude that IL-17+Foxp3+ T cells may contribute to the development of Eca.     

Foxp3+IL-17+ T cells promote development of cancer-initiating cells in colorectal cancer

The pathogenesis of CRC remains to be further understood. This study was designed to elucidate the role of Foxp3+IL-17+ T cells in the pathogenesis of CRC. Surgically removed CRC tissue was collected from 12 patients with CRC. The frequency and cytokine profile of Foxp3+IL-17+ T cells in CRC were examined by flow cytometry. Chemokine CXCL11 was examined in CRC tissue by Western blotting. Treg chemotaxis was examined in a transwell system. The effect of Foxp3+IL-17+ T cells on induction of cancer-initiating cells was examined; the latter's Akt and MAPK activities and colony formation were examined afterward. Abundant Foxp3+IL-17+ T cells were detected in CRC tissue that expresses high levels of TGF-β, CXCR3, CCR6, and RORγt. High levels of CXCL11 were detected in CRC tissue-derived CD68+ cells, which had a strong chemotactic effect on Foxp3+ Tregs. Hypoxia induced the expression of IL-17 in Foxp3+ Tregs; Foxp3+IL-17+ T cells were capable of inducing CRC-associated cell markers in BMMo and drove the cells to be cancer-initiating cells. High levels of phosphorylated Akt and MAPK were detected in the induced cancer-initiation cells; the latter has the capability to form a colony. CRC tissue-derived Foxp3+IL-17+ cells have the capacity to induce cancer-initiating cells.     

CD8+ Foxp3+ T cells share developmental and phenotypic features with classical CD4+ Foxp3+ regulatory T cells but lack potent suppressive activity

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.     

Localization of IL-17+Foxp3+ T Cells in Esophageal Cancer

The etiology of cancer is unclear. Recent studies indicate that some cytokines, such as interleukin (IL)-17, and regulatory T cells are involved in the development of cancer. This study aims to detect a subset of T cell, IL17+Foxp3+ T cell, in the pathogenesis of esophageal cancer (Eca). Twelve patients with squamous Eca were recruited in this study. The surgically removed Eca tissue was collected. Cells isolated from Eca tissue were analyzed by flow cytometry. The results showed that 2–10% Eca tissue-derived CD4⁺ T cells expressed Foxp3; only 0.2–0.8% non-ca tissue-derived CD4⁺ T cells expressed Foxp3. Further analysis showed that 3–15% Eca-isolated CD4⁺ T cells were also IL-17 positive whereas only 0.4–1.5% non-ca tissue-isolated CD4⁺ T cells were IL-17 positive. We also found that about 4.8–11.2% Foxp3⁺ IL-17⁺ T cells in isolated CD4⁺ T cells from Eca tissue that were significantly less than in non-ca tissue derived CD4⁺ T cells. Less than 1% Foxp3⁺ IL-17⁺ T cells in isolated CD4⁺ T cells in both Eca patients and healthy controls. Treatment with hypoxia markedly increased the expression of IL-6 in peripheral CD68+ cells. Coculturing CD68+ cells and Foxp3+ T cells under hypoxic environment resulted in abundant expression of IL-17 in Foxp3+ T cells that could be blocked by pretreatment with either anti-IL-17 or anti-transforming growth factor beta antibodies. We conclude that IL-17+Foxp3+ T cells may contribute to the development of Eca.     

Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro

Cross-talk has been shown to occur between the immune system and bone metabolism pathways. In the present study, we investigated the impact of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells on osteoclastogenesis and bone resorption. Treg cells that were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy adults inhibited both the differentiation of osteoclasts (OCs) from human embryo bone marrow cells (BMCs) and the pit formation in a dose-dependent manner. In cell cocultures, the production levels of both interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) were proportionally upregulated as the ratio of Treg cells to BMCs was increased, and the inhibition of OC differentiation and bone resorption by Treg cells was completely reversed by anti-IL-10 and anti-TGF-β1 antibodies. Treatment of BMC and Treg cell cocultures with 17β-estradiol (E2) at concentrations between 10⁻⁷ and 10⁻⁹ mol/l suppressed OC differentiation and bone resorption more efficiently than it did in cultures of BMCs alone; this enhanced suppression occurred via the stimulation of Treg cell IL-10 and TGF-β1 expression. These data suggest that Treg cells suppress OC differentiation and bone resorption by secreting IL-10 and TGF-β1. E2 enhances the suppressive effects of Treg cells on OC differentiation and bone resorption by stimulating IL-10 and TGF-β1 secretion from these cells. Therefore, Treg cell-derived IL-10 and TGF-β1 are likely involved in the regulation of E2 on bone metabolism and represent potential therapeutic targets for the treatment of postmenopausal osteoporosis (PMO).     

Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells

CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3(-) T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3(-) T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy.     

Resident CD8+ T cells suppress CD4+ T cell-dependent late allergic airway responses

BACKGROUND: The role of CD8+ T cells in the immune response to airway challenge with an allergen is poorly understood. OBJECTIVE: The aim of this study was to test the hypothesis that resident naive CD8+ T cells modulate the magnitude of CD4+ T cell-dependent allergic airway responses. METHODS: Cervical lymph node CD4+ T cells (2 x 10(6)) were harvested from ovalbumin (OVA)- or sham-sensitized rats and injected intraperitoneally into naive Brown Norway recipients. The recipients were treated with a CD8alpha mAb (OX-8) to deplete the resident CD8+ T cells (n = 12) or mouse ascites (n = 12). Two days after adoptive transfer, the recipient animals were OVA challenged, lung resistance was measured for 8 hours, and bronchoalveolar lavage (BAL) was performed. RESULTS: After OVA challenge, primed CD4-transferred CD8-depleted rats had larger early airway responses and late airway responses compared with primed CD4-transferred CD8-nondepleted rats (early airway responses: 158.6% +/- 19.2% vs 115.7% +/- 5.9%, P < .05; late airway responses: 8.5% +/- 1.7% vs 4.4% +/- 0.9%, P < .05). BAL eosinophilia was also greater (4.67% +/- 0.45% vs 2.34 +/- 0.26%, P < .01). The cells in BAL fluid expressing IL-4 mRNA were not significantly changed by CD8 depletion, but IL-5 mRNA+ cells were higher in number, and IFN-gamma mRNA+ cells were fewer in the CD8-depleted group. CONCLUSIONS: Resident CD8+ T cells downregulate the late allergic response and airway inflammation evoked by CD4+ T-cell transfers in Brown Norway rats. This downregulation does not require antigen priming.     

CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model

Th17 cells and CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are thought to promote and suppress inflammatory responses, respectively. Here we explore why under Th17 cell polarizing conditions, Treg cells did not suppress, but rather upregulated, the expression of interleukin-17A (IL-17A), IL-17F, and IL-22 from responding CD4(+) T?cells (Tresp cells). Upregulation of IL-17 cytokines in Tresp cells was dependent on?consumption of IL-2 by Treg cells, especially at early time points both in?vitro and in?vivo. During an oral Candida albicans infection in mice, Treg cells induced IL-17 cytokines in Tresp cells, which markedly enhanced fungal clearance and recovery from infection. These findings show how Treg cells can promote acute Th17 cell responses to suppress mucosal fungus infections and reveal that Treg cells?have a powerful capability to fight infections besides their role in maintaining tolerance or immune homeostasis.     

Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells

Studies in both animal models and humans have shown a subset of B cells behaving as immuno-regulatory cells, being a source of inhibitory cytokines such as IL-10 and TGF-β. Our aims were to establish the presence of human B regulatory (Breg) cells and to assess their ability to suppress proliferation of CD4(+) T cells and to mediate T regulatory (Treg) cells' properties. For this purpose, human Breg, CD4(+) T and Treg cells were purified using magnetic microbeads. CFSE-labeled CD4(+) T cells were stimulated and cultured alone or with Breg cells. Their proliferative response was determined 72 hours later based on the CFSE staining. In parallel, Treg cells were cultured alone or with Breg cells in different conditions for 24 hours, and then stained and analyzed for Foxp3 and CTLA-4 expression. We found that, the co-culture of Breg cells (defined as CD25(high) CD27(high) CD86(high) CD1d(high) IL-10(high) TGF-β(high)) with autologous stimulated CD4(+) T cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4(+) T cells. Furthermore, Foxp3 and CTLA-4 expression in Treg cells were enhanced by non-stimulated and further by ODN-CD40L stimulated Breg cells. The regulatory function of Breg cells on Treg cells was mainly dependent on a direct contact between Breg and Treg cells, but was also TGF-β but not IL-10 dependent. In conclusion, human Breg cells decrease the proliferation of CD4(+) T cells and also enhance the expression of Foxp3 and CTLA-4 in Treg cells by cell-to-cell contact.     

Total glucosides of paeony induces regulatory CD4(+)CD25(+) T cells by increasing Foxp3 demethylation in lupus CD4(+) T cells

Total glucosides of paeony (TGP), an active compound extracted from Paeony root, has been used in therapy for autoimmune diseases. However the molecular mechanism of TGP in the prevention of autoimmune response remains unclear. In this study, we found that TGP treatment significantly increased the percentage and number of Treg cells in lupus CD4(+) T cells. Further investigation revealed that treatment with TGP increased the expression of Foxp3 in lupus CD4(+) T cells by down-regulating Foxp3 promoter methylation levels. However, we couldn't observe similar results in healthy control CD4(+) T cells treated by TGP. Moreover, our results also showed that IFN-γ and IL-2 expression was enhanced in TGP-treated lupus CD4(+) T cells. These findings indicate that TGP inhibits autoimmunity in SLE patients possibly by inducing Treg cell differentiation, which may in turn be due to its ability to regulate the methylation status of the Foxp3 promoter and activate IFN-γ and IL-2 signaling.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

CD8+ cells suppress oil-induced arthritis

Oil-induced arthritis is a genetically restricted polyarthritis that develops in the DA rat after injection of the mineral oil Freund's incomplete adjuvant. Here, we investigated the role of the potentially disease-limiting cell populations CD8+ T cells, gammadelta T cells, natural killer (NK) cells and NK T cells in inguinal lymph nodes for the development of this adjuvant-induced arthritis. Flow cytometry analysis before and at disease onset revealed a higher proportion of lymph node T cells expressing NKR-P1 in the disease-resistant LEW.1AV1 compared with the disease-susceptible DA strain, suggesting that NK T cells might be disease protective. However, prophylactic in vivo administration of an anti-NKR-P1 MoAb (clone 10/78) did not consistently affect the disease course. The proportion of CD8+ T cells and the ratio CD4+/CD8+ T cells in inguinal lymph nodes did not differ significantly between DA and LEW.1AV1 rats before or at disease onset. Nevertheless, prophylactic in vivo depletion of CD8+ cells by the OX8 MoAb in the DA strain resulted in an earlier disease onset compared with the control group, demonstrating that CD8+ cells regulate arthritis development. In vivo depletion of gammadelta T cells by the V65 MoAb did not alter the disease course, indicating that the disease-suppressive CD8+ cells are alphabeta T cells or NK cells.     

CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells

A key issue in mammalian immunology is how CD4+CD25+Foxp3+ regulatory T cells (T(reg) cells) suppress immune responses. Here we show that T(reg) cells induced apoptosis of effector CD4+ T cells in vitro and in vivo in a mouse model of inflammatory bowel disease. T(reg) cells did not affect the early activation or proliferation of effector CD4+ T cells. Cytokines that signal through the common gamma-chain suppressed T(reg) cell-induced apoptosis. T(reg) cell-induced effector CD4+ T cell death required the proapoptotic protein Bim, and effector CD4+ T cells incubated with T(reg) cells showed less activation of the prosurvival kinase Akt and less phosphorylation of the proapoptotic protein Bad. Thus, cytokine deprivation-induced apoptosis is a prominent mechanism by which T(reg) cells inhibit effector T cell responses.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.     

IL-21R expression on CD8+ T cells promotes CD8+ T cell activation in coxsackievirus B3 induced myocarditis

IL-21 is a multi-functional cytokine which can promote survival, proliferation and activation of T and B lymphocytes including CD8 T cells. Previous studies have shown that autoimmune CD8+ T cells are the primary pathogenic effector cell in coxsackievirus B3 (CVB3) induced myocarditis in C57Bl/6 mice. To evaluate the role of IL-21 in promoting CD8+ T cell mediated cardiac injury in myocarditis, C57Bl/6 and IL-21RKO mice were infected with CVB3. IL-21RKO mice developed significantly less myocarditis than C57Bl/6 animals although cardiac virus titers were equivalent between the mouse strains. Numbers of CD8+IFNγ+ cells were decreased in IL-21RKO mice but numbers of either CD4+IFNγ+ or CD4+IL-4+ cells were not significantly different from C57Bl/6 animals indicating a selective effect of IL-21 signaling on the CD8+ T cell response. To confirm that IL-21 signaling exclusively functions at the level of the CD8+ T cell in CVB3 induced myocarditis, purified CD8+ cells were isolated from either C57Bl/6 or IL-21RKO donors and adoptively transferred into CD8KO recipients prior to CVB3 infection. CD8KO recipients given either C57Bl/6 or IL-21RKO CD8+ cells showed equivalent reconstitution of the CD8+ cells in the spleen but the recipients given C57Bl/6 CD8+ cells showed significantly greater myocarditis than recipients of IL-21RKO CD8+ cells. These data demonstrate that IL-21 signaling directly in the CD8+ cell population is required for CVB3-induced myocarditis.     

Heterogeneity of CD4(+) and CD8(+) T cells

There is extensive plasticity in the T-cell response to antigen. Helper CD4(+) T cells, cytotoxic CD8(+) T cells, the progression from na?ve to effector and memory T cells, and differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. More recently it has become apparent that T-cell populations display additional diversity in terms of phenotype, anatomical distribution and effector function.