1140918040Prevention of abortion in the CBAxDBA/2 model by intravaginal TGF-β is associated with local recruitment of predominantly CD8+ Foxp3+ T cells to the genital tract

Problem: Spontaneous abortions in DBA/2‐mated CBA/J mice can be prevented by an immune response to BALB/c, and CD4⁺25⁺ Treg cells as well as CD8⁺ T cells have been proposed to confer protection. Recently a 2 ng dose of intravaginal TGF‐β3 at the time of exposure to DBA/2 semen was shown to be effective. TGF‐β is known to facilitate development of Treg cells. Is there evidence for local Treg induction? Methods: The phenotype of cellular recruitment to the vaginal wall and uterus was established by immunostaining tissue sections from CBA/J females following intravaginal TGF‐β treatment. The phenotype of cells in vaginal washings 48 hr after TGF‐β was determined by flow cytometry. Results: Increased numbers of CD3⁺, CD25⁺, and CD11c⁺ cells were found in vaginal mucosa with increasing doses of TGF‐β. A 2 ng TGF‐β3 treatment at the time of estrus recruited Foxp3⁺ cells to the vaginal lumen, and the majority of these were CD8⁺; CD4⁺ cells were also present, but expressed only low levels of CD25 and CTLA4. A 20 ng dose recruited predominantly CD4⁺8⁺ Foxp3⁺ cells. Conclusion: Induction of Tregs to semen‐associated DBA/2 antigens may prevent pregnancy loss in the CBAxDBA/2 model without the need for BALB/c as an immunogen. The Treg phenotype in the genital tract is compatible with additional members of the Treg family that recognize Class I MHC and associated paternal peptides and prevent abortions.     

Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine‐protective regulatory T cells

The gut is home to a large number of Treg, with both CD4⁺ CD25⁺ Treg and bacterial antigen‐specific Tr1 cells present in normal mouse intestinal lamina propria. It has been shown recently that intestinal mucosal DC are able to induce Foxp3⁺ Treg through production of TGF‐β plus retinoic acid (RA). However, the factors instructing DC toward this mucosal phenotype are currently unknown. Curcumin has been shown to possess a number of biologic activities including the inhibition of NF‐κB signaling. We asked whether curcumin could modulate DC to be tolerogenic whose function could mimic mucosal DC. We report here that curcumin modulated BM‐derived DC to express ALDH1a and IL‐10. These curcumin‐treated DC induced differentiation of naïve CD4⁺ T cells into Treg resembling Treg in the intestine, including both CD4⁺CD25⁺ Foxp3⁺ Treg and IL‐10‐producing Tr1 cells. Such Treg induction required IL‐10, TGF‐β and retinoic acid produced by curcumin‐modulated DC. Cell contact as well as IL‐10 and TGF‐β production were involved in the function of such induced Treg. More importantly, these Treg inhibited antigen‐specific T‐cell activation in vitro and inhibited colitis due to antigen‐specific pathogenic T cells in vivo.     

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.     

HCV‐infected hepatocytes drive CD4+CD25+Foxp3+ regulatory T‐cell development through the Tim‐3/Gal‐9 pathway

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T‐cell Ig and mucin domain protein‐3 (Tim‐3) and galectin‐9 (Gal‐9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim‐3/Gal‐9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim‐3/Gal‐9 interactions regulate HCV‐mediated Treg‐cell development, here we provide pilot data showing that HCV‐infected human hepatocytes express higher levels of Gal‐9 and TGF‐β, and upregulate Tim‐3 expression and regulatory cytokines TGF‐β/IL‐10 in co‐cultured human CD4⁺ T cells, driving conventional CD4⁺ T cells into CD25⁺Foxp3⁺ Treg cells. Additionally, recombinant Gal‐9 protein can transform TCR‐activated CD4⁺ T cells into Foxp3⁺ Treg cells in a dose‐dependent manner. Importantly, blocking Tim‐3/Gal‐9 ligations abrogates HCV‐mediated Treg‐cell induction by HCV‐infected hepatocytes, suggesting that Tim‐3/Gal‐9 interactions may regulate human Foxp3⁺ Treg‐cell development and function during HCV infection.     

The GARP/Latent TGF‐β1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance

Treg cells can secrete latent TGF‐β1 (LTGF‐β1), but can also utilize an alternative pathway for transport and expression of LTGF‐β1 on the cell surface in which LTGF‐β1 is coupled to a distinct LTGF‐β binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF‐β1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF‐β1 in the induction of oral tolerance. When Foxp3^? OT‐II T cells were transferred to wild‐type recipient mice followed by OVA feeding, the conversion of Foxp3^? to Foxp3⁺ OT‐II cells was dependent on recipient Treg cells. Neutralization of IL‐2 in the recipient mice also abrogated this conversion. The GARP/LTGF‐β1 complex on recipient Treg cells, but not dendritic cell‐derived TGF‐β1, was required for efficient induction of Foxp3⁺ T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvβ8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF‐β1 complex.     

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.     

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.     

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.     

TGF‐β regulation of encephalitogenic and regulatory T cells in multiple sclerosis

Transforming growth factor beta (TGF‐β) is a pleiotropic cytokine that has been shown to influence the differentiation and function of T cells. The role that TGF‐β plays in immune‐mediated disease, such as multiple sclerosis (MS), has become a major area of investigation since CD4⁺ T cells appear to be a major mediator of autoimmunity. This review provides an analysis of the literature on the role that TGF‐β plays in the generation and regulation of encephalitogenic and regulatory T cells (Treg) in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as well as in T cells of MS patients. Since TGF‐β plays a major role in the development and function of both CD4⁺ effector and Treg, which are defective in MS patients, recent studies have found potential mechanisms to explain the basis for these T‐cell defects to establish a foundation for potentially modulating TGF‐β signaling to restore normal T‐cell function in MS patients.     

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.     

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.     

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.     

Novel CD8+ Treg suppress EAE by TGF‐β‐ and IFN‐γ‐dependent mechanisms

Although CD8⁺ Treg‐mediated suppression has been described, CD8⁺ Treg remain poorly characterized. Here we identify a novel subset of CD8⁺ Treg that express latency‐associated peptide (LAP) on their cell surface (CD8⁺LAP⁺ cells) and exhibit regulatory activity in vitro and in vivo. Only a small fraction of CD8⁺LAP⁺ cells express Foxp3 or CD25, although the expression levels of Foxp3 for these cells are higher than their LAP^? counterparts. In addition to TGF‐β, CD8⁺LAP⁺ cells produce IFN‐γ, and these cells suppress EAE that is dependent on both TGF‐β and IFN‐γ. In an adoptive co‐transfer model, CD8⁺LAP⁺ cells suppress myelin oligodendrocyte glycoprotein (MOG)‐specific immune responses by inducing or expanding Foxp3⁺ cells and by inhibiting proliferation and IFN‐γ production in vivo. Furthermore, in vivo neutralization of IFN‐γ and studies with IFN‐γ‐deficient mice demonstrate an important role for IFN‐γ production in the function of CD8⁺LAP⁺ cells. Our findings identify the underlying mechanisms that account for the immunoregulatory activity of CD8⁺ T cells and suggest that induction or amplification of CD8⁺LAP⁺ cells may be a therapeutic strategy to help control autoimmune processes.     

Beta2‐adrenergic receptor signaling in CD4+ Foxp3+ regulatory T cells enhances their suppressive function in a PKA‐dependent manner

Beta2‐adrenergic receptor (B2AR) signaling is known to impair Th1‐cell differentiation and function in a cAMP‐dependent way, leading to inhibition of cell proliferation and decreased production of IL‐2 and IFN‐γ. CD4⁺ Foxp3⁺ Treg cells play a key role in the regulation of immune responses and are essential for maintenance of self‐tolerance. Nevertheless, very little is known about adrenergic receptor expression in Treg cells or the influence of noradrenaline on their function. Here we show that Foxp3⁺ Treg cells express functional B2AR. B2AR activation in Treg cells leads to increased intracellular cAMP levels and to protein kinase A (PKA)‐dependent CREB phosphorylation. We also found that signaling via B2AR enhances the in vitro suppressive activity of Treg cells. B2AR‐mediated increase in Treg‐cell suppressive function was associated with decreased IL‐2 mRNA levels in responder CD4⁺ T cells and improved Treg‐cell‐induced conversion of CD4⁺ Foxp3^? cells into Foxp3⁺ induced Treg cells. Moreover, B2AR signaling increased CTLA‐4 expression in Treg cells in a PKA‐dependent way. Finally, we found that PKA inhibition totally prevented the B2AR‐mediated increase in Treg‐cell suppressive function. Our data suggest that sympathetic fibers are able to regulate Treg‐cell suppressive activity in a positive manner through B2AR signaling.