Immature dendritic cells convert anergic nonregulatory T cells into Foxp3−IL‐10+ regulatory T cells by engaging CD28 and CTLA‐4

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide‐specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL‐10^?Foxp3^?CTLA‐4⁺CD25^lowEgr2⁺ T cells could be converted into suppressive IL‐10⁺Foxp3^?CTLA‐4⁺CD25^highEgr2⁺ cells resembling type‐1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF‐β during anergy induction favored Foxp3⁺ Treg‐cell induction, while TGF‐β had little effect when added to the second stimulation. Expression of both CD28 and CTLA‐4 molecules on anergic T cells was required to allow their conversion into Tr1‐like cells. Suppressor activity was enabled via CD28‐mediated CD25 upregulation, acting as an IL‐2 sink, together with a CTLA‐4‐mediated inhibition of NFATc1/α activation to shut down IL‐2‐mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1‐like cells.     

Synergistic effect of TGF‐β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3⁺ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4⁺CD25^? cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3⁺ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3⁺ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.     

Interleukin‐10‐secreting type 1 regulatory T cells in rodents and humans

Summary: Interleukin‐10 (IL‐10)‐secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL‐10 and transforming growth factor‐β(TGF‐β), and by their ability to suppress antigen‐specific effector T‐cell responses via a cytokine‐dependent mechanism. In contrast to the naturally occurring CD4⁺CD25⁺ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL‐10‐dependent process in vitro and in vivo. Specialized IL‐10‐producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL‐10‐producing T‐cell populations that are induced by IL‐10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4⁺CD25⁺ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Loss of regulatory characteristics in CD4+CD25+/hi T cells induced by impaired transforming growth factor beta secretion in pneumoconiosis

Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4⁺CD25⁺ T cells compared to controls, whereas the CD4⁺CD25⁺ and CD4⁺CD25^hi T cells were enriched with Th1‐ and Th17‐like cells but not Foxp3‐expressing Treg cells and evidenced by significantly higher T‐bet, interferon (IFN)‐γ, and interleukin (IL)‐17 expression but lower Foxp3 and transforming growth factor (TGF)‐β expression. Regarding the CD4⁺CD25^hi T‐cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF‐β and an enrichment in T‐bet, RORγt, IFN‐γ, and IL‐17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14⁺ monocytes and CD19⁺ B cells, the levels of IFN‐γ and IL‐17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF‐β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF‐β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3⁺ Treg cells associated with impaired TGF‐β secretion.     

Integrated T-cell receptor and costimulatory signals determine TGF-β-dependent differentiation and maintenance of Foxp3+ regulatory T cells

Foxp3‐expressing Tregs play a non‐redundant role in protecting against immune pathologies. Foxp3⁺ Tregs can arise intra‐ and extra‐thymically, however, the signals directing their differentiation and maintenance in the periphery are not well understood. We show that stimulation of mouse naïve CD4⁺ T cells in vitro with optimal doses of anti‐CD3/anti‐CD28 resulted in high frequencies of Foxp3⁺ T cells via a TGF‐β‐dependent mechanism. Addition of TGF‐β and retinoic acid overcame the inhibition of Foxp3 expression observed during high‐strength anti‐CD3/anti‐CD28 stimulation. Reducing the strength of TCR or costimulatory signals with inhibitors of mammalian target of rapamycin (mTOR) or MEK/ERK signalling also enhanced expression of Foxp3 in a TGF‐β‐dependent manner. Addition of TGF‐β was further required to maintain Foxp3 expression in ex vivo derived Foxp3⁺ Tregs upon prolonged anti‐CD3/anti‐CD28 signalling. Thus, induction/maintenance of Foxp3 expression by TGF‐β is modulated by the integrated strength of TCR/costimulatory signals.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

Intestinal inflammation abrogates the tolerogenic properties of MLN CD103+ dendritic cells

Intestinal CD103⁺ DC promote the differentiation of Foxp3⁺ Treg from naïve CD4⁺ T cells through mechanisms involving TGF‐β and the dietary metabolite, retinoic acid (RA). In this study, we have analysed whether the specialised features of CD103⁺ DC are conserved in colitis. Our results show that inflammation dampens the tolerogenic properties of MLN CD103⁺ DC, which is associated with lower expression of tgfβ2 and aldh1a2. Accordingly, CD103⁺ DC taken from colitic mice are impaired in their ability to induce Foxp3⁺ Treg and instead favour the emergence of IFN‐γ‐producing CD4⁺ T cells compared with their steady‐state counterparts. BrdU‐labelling studies and analysis of ontogeny markers show that CD103⁺ DC from steady‐state and colitic settings retain similar subset composition and developmental pathways. These results indicate that MLN CD103⁺ DC are not hard‐wired to promote tolerance but can adapt to environmental conditions. The inflammatory properties of MLN CD103⁺ DC in colitic mice may reflect defective gut tolerogenic conditioning or altered migratory pathways and raise the possibility that migratory DC populations contribute to the pathogenesis of inflammatory bowel disease.     

DC expressing transgene Foxp3 are regulatory APC

Tolerogenic DC and suppressive Foxp3⁺ Treg play important roles in preventing autoimmunity and allograft rejection. We report that (adenovirus mediated) ectopic expression of Foxp3 in human DC (i.e. DC.Foxp3) yields an APC that severely limits T‐cell proliferation and type‐1 immune responses from the naïve, but not memory, pool of responder T cells in vitro. In marked contrast, the frequencies of type‐2 and Treg responses were dramatically increased after stimulation of naïve T cells with DC.Foxp3 versus control DC. DC.Foxp3‐induced CD4⁺CD25⁺ Treg cells potently suppressed the proliferation of, and IFN‐γ production from, CD4⁺ and CD8⁺ responder T cells. Notably, the immunosuppressive biology of DC.Foxp3 was effectively normalized by addition of 1‐methyl‐tryptophan or neutralizing anti‐TGF‐β1 Ab during the period of T‐cell priming. These data suggest the potential utility of regulatory DC.Foxp3 and/or DC.Foxp3‐induced CD4⁺CD25⁺ Treg as translational agents for the amelioration or prevention of pathology in the setting of allograft transplantation and/or autoimmunity.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

Schistosoma mansoni egg antigens induce Treg that participate in diabetes prevention in NOD mice

Schistosoma mansoni soluble egg antigens (SEA) profoundly regulate the infected host's immune system. We previously showed that SEA prevents type 1 diabetes in NOD mice and that splenocytes from SEA‐treated mice have reduced ability to transfer diabetes to NOD.scid recipients. To further characterize the mechanism of diabetes prevention we examined the cell types involved and showed that CD25⁺ T‐cell depletion of splenocytes from SEA‐treated donors restored their ability to transfer diabetes. Furthermore, SEA treatment increased the number and proportional representation of Foxp3⁺ T cells in the pancreas of NOD mice. We have used in vitro systems to analyze the effect of SEA on the development of NOD Foxp3⁺ T cells. We find that SEA can induce Foxp3 expression in naïve T cells in a TGF‐β‐dependent manner. Foxp3 induction requires the presence of DC, which we also show are modified by SEA to upregulate C‐type lectins, IL‐10 and IL‐2. Our studies show that SEA can have a direct effect on CD4⁺ T cells increasing expression of TGF‐β, integrin β8 and galectins. These effects of SEA on DC and T cells may act in synergy to induce Foxp3⁺ Treg in the NOD mouse.     

Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells

Summary: Th3 CD4⁺ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T‐cell subset which primarily secretes transforming growth factor (TGF)‐β, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4⁺ TGF‐β‐secreting cells with suppressive properties have been generated from interleukin (IL)‐4‐deficient animals. In vitro differentiation of Th3 cells from Th precursors from T‐cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF‐β, IL‐4, IL‐10, and anti‐IL‐12. Th3 CD4⁺ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF‐β with various amounts of IL‐4 and IL‐10. Because Th3 regulatory cells are triggered in an antigen‐specific fashion but suppress in an antigen‐non‐specific fashion, they mediate “bystander suppression” when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL‐4. Anti‐CD86 but not anti‐CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF‐β and has a unique class of dendritic cells. CD4⁺CD25⁺ regulatory T‐cell function also appears related to TGF‐β.     

Regulatory T cells in the periphery

Summary: Recognition of a systemic antigen by CD4⁺ T cells in a lymphopenic host leads to the sequential generation of pathogenic effector cells and protective CD25⁺ forkhead box protein (Foxp3⁺) regulatory T cells (Tregs) in the periphery. Such an experimental model is potentially valuable for defining the stimuli that determine the balance of effector and regulatory T cells. Our studies have shown that interleukin‐2 (IL‐2) enhances the development of effector cells and is essential for the peripheral generation of regulatory cells. Other models of peripheral Treg generation suggest that the concentration of antigen, the nature of the antigen‐presenting cells, and cytokines such as transforming growth factor‐β and IL‐10 may all influence the peripheral generation of Tregs.     

Induced regulatory T cells are phenotypically unstable and do not protect mice from rapidly progressive glomerulonephritis

Regulatory T (Treg) cells are a suppressive CD4⁺ T‐cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4⁺ T cells were cultured in vitro with interleukin‐2 (IL‐2), transforming growth factor‐β1, all‐trans‐retinoic acid and monoclonal antibodies against interferon‐γ and IL‐4, generating Foxp3⁺ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL‐12p40 or by using RORγt^–/– CD4⁺ T cells. Induced Treg cells were transferred into models of delayed‐type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen‐specific delayed‐type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti‐IL‐12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed‐type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of RORγt^–/– CD4⁺ T cells or iTreg cells generated from sensitized CD4⁺ Foxp3^– cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T‐cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.     

TGF‐β‐producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti‐T cell immunoglobulin domain and mucin domain‐1 and anti‐CD45RB antibody treatment results in tolerance to full MHC‐mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen‐specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4⁺CD25^? T cells than with naive B cells. We also show that Breg cells express the TGF‐β associated latency‐associated peptide and that Breg‐cell mediated graft prolongation post‐adoptive transfer is abrogated by neutralization of TGF‐β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C‐C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody‐induced transplantation tolerance, Breg cells promote graft survival by promoting Treg‐cell development, possibly via TGF‐β production.     

The regulation of regulation: interleukin‐10 increases CD4+ CD25+ regulatory T cells but impairs their immunosuppressive activity in murine models with schistosomiasis japonica or asthma

CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells play an important role in maintaining immune homeostasis. Interleukin‐10 (IL‐10), a cytokine with anti‐inflammatory capacities, also has a critical role in controlling immune responses. In addition, it is well known that production of IL‐10 is one of the suppression mechanisms of Treg cells. However, the action of IL‐10 on Treg cells themselves remains insufficiently understood. In this study, by using a Schistosoma japonicum‐infected murine model, we show that the elevated IL‐10 contributed to Treg cell induction but impaired their immunosuppressive function. Our investigations further suggest that this may relate to the up‐regulation of serum transforming growth factor (TGF‐β) level but the decrease in membrane‐bound TGF‐β of Treg cells by IL‐10 during S. japonicum infection. In addition, similar IL‐10‐mediated regulation on Treg cells was also confirmed in the murine model of asthma. In general, our findings identify a previously unrecognized opposing regulation of IL‐10 on Treg cells and provide a deep insight into the precise regulation in immune responses.     

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

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