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.     

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.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.     

Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

Commensal A4 bacteria inhibit intestinal Th2‐cell responses through induction of dendritic cell TGF‐β production

It has been shown that while commensal bacteria promote Th1, Th17 and Treg cells in lamina propria (LP) in steady‐state conditions, they suppress mucosal Th2 cells. However, it is still unclear whether there are specific commensal organisms down‐regulating Th2 responses, and the mechanism involved. Here we demonstrate that commensal A4 bacteria, a member of the Lachnospiraceae family, which produce an immunodominant microbiota CBir1 antigen, inhibits LP Th2‐cell development. When transferred into the intestines of RAG^?/? mice, CBir1‐specific T cells developed predominately towards Th1 cells and Th17 cells, but to a lesser extent into Th2 cells. The addition of A4 bacterial lysates to CD4⁺ T‐cell cultures inhibited production of IL‐4. A4 bacteria stimulated dendritic cell production of TGF‐β, and blockade of TGF‐β abrogated A4 bacteria inhibition of Th2‐cell development in vitro and in vivo. Collectively, our data show that A4 bacteria inhibit Th2‐cell differentiation by inducing dendritic cell production of TGF‐β.     

Increased susceptibility of Fas ligand‐deficient gld mice to Trypanosoma cruzi infection due to a Th2‐biased host immune response

Infection of BALB / c mice with Trypanosoma cruzi resulted in up‐regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4⁺ T cells, activation‐induced CD4⁺ T cell death (AICD), and in Fas : FasL‐mediated cytotoxicity. When CD4⁺ T cells from infected mice were co‐cultured with T. cruzi‐infected macrophages, onset of AICD exacerbated parasite replication. CD4⁺ T cells from T. cruzi‐infected FasL‐deficient BALB gld / gld mice had no detectable AICD in vitro and their activation with anti‐TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld / gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild‐type mice. Secretion of Th2 cytokines IL‐10 and IL‐4 by CD4⁺ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti‐IL‐4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild‐type mice. These results indicate that, besides controlling CD4⁺ T cell AICD and parasite replication in vitro, an intact Fas : FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2‐type immune response to the parasite.     

T cell receptor specificities of Toxoplasma gondii-reactive mouse CD4+ T lymphocytes and Th1 clones

The Toxoplasma gondii-directed CD4⁺ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains Vβ4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4⁺ splenocytes. This repertoire was also detected among T. gondii-specific CD4⁺ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-γ and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4⁺ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates. Received: 3 February 1997     

Inhibition of transforming growth factor-�� signalling attenuates interleukin (IL)-18 plus IL-2-induced interstitial lung disease in mice

Interstitial lung disease (ILD) is an intractable disease induced by various factors in humans. However, there is no universally effective treatment for ILD. In this study, we investigated the role of transforming growth factor (TGF)‐β signalling in the pathogenesis of ILD by using model mice. Injection of interleukin (IL)‐18 plus IL‐2 in C57BL6 (B6) mice resulted in acute ILD by infiltration of natural killer (NK) cells and a significant increase of TGF‐β mRNA in the lung. To examine the pathogenetic role of TGF‐β in ILD mice, we used SB‐431542 (4‐[4‐(1,3‐benzodioxol‐5‐yl)‐5‐(2‐pyridinyl)‐1H‐imidazol‐2‐yl]‐benzamide), which is a potent and selective inhibitor of TGF‐β receptor I (TβRI), also known as activin receptor‐like kinase 5 (ALK5). Treatment of B6‐ILD mice with SB‐431542 resulted in improvement of ILD, delay in mortality, reduction of the expression of interferon (IFN)‐γ and IL‐6 in the lungs. The same treatment also decreased significantly the percentage of natural killer (NK) cells in the lungs (P < 0·05) and mRNA expression levels of certain chemokines such as CCL2, CCL3, CCL4, CCL5 and CXCL10 in B6‐ILD. These findings were confirmed by IL‐18 plus IL‐2 treatment of Smad3‐deficient (Smad3^–/–) mice (P < 0·05). Our results showed that inhibition of TGF‐β signalling reduced the percentage of NK cells and the expression of certain chemokines in the lungs, resulting in improvement of ILD. The findings suggest that TGF‐β signalling may play an important role in the pathogenesis of IL‐18 plus IL‐2‐induced ILD in mice.     

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.     

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.     

CD4+CD25+ Treg induction by an HSP60‐derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent

Chronic schistosome infection results in the suppression of host immune responses, allowing long‐term schistosome survival and restricting pathology. Current theories suggest that Treg play an important role in this regulation. However, the mechanism of Treg induction during schistosome infection is still unknown. The aim of this study was to determine the mechanism behind the induction of CD4⁺CD25⁺ T cells by Schistosoma japonicum HSP60 (SjHSP60)‐derived peptide SJMHE1 as well as to elucidate the cellular and molecular basis for the induction of CD4⁺CD25⁺ T cells during S. japonicum infection. Mice immunized with SJMHE1 or spleen and LN cells from naïve mice pretreated with SJMHE1 in vitro all displayed an increase in CD4⁺CD25⁺ T‐cell populations. Release of IL‐10 and TGF‐β by SJMHE1 stimulation may contribute to suppression. Adoptively transferred SJMHE1‐induced CD4⁺CD25⁺ T cells inhibited delayed‐type hypersensitivity in BALB/c mice. Additionally, SJMHE1‐treated APC were tolerogenic and induced CD4⁺ cells to differentiate into suppressive CD4⁺CD25⁺ Treg. Furthermore, our data support a role for TLR2 in SJMHE1‐mediated CD4⁺CD25⁺ Treg induction. These findings provide the basis for a more complete understanding of the S. japonicum–host interactions that contribute to host homeostatic mechanisms, preventing an excessive immune response.     

Interleukin-9 as a T helper type 17 cytokine

The production of interleukin‐9 (IL‐9) by CD4 T cells has gathered renewed interest as the result of the observation that its expression is broader than originally thought. This includes the production of IL‐9 by a recently characterized subset of CD4 helper T (Th) cells that are termed Th9 as well as production by additional T‐cell subsets including Th17 cells. There is an incomplete understanding as to which IL‐9‐producing T‐cell subsets develop under physiological conditions. We describe the conditions used to generate IL‐9 in Th17 cells in vitro. We also summarize conditions where both IL‐9 and IL‐17 are found in vivo and propose that Th17 cells producing IL‐9 may co‐exist and interact with Th9 cells during conditions of autoimmunity, allergy and infection.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

In vitro‐induced Th17 cells fail to induce inflammation in vivo and show an impaired migration into inflamed sites

Recently, IL‐17 produced by Th17 cells was described as pro‐inflammatory cytokine with an eminent role in autoimmune diseases, e.g. rheumatoid arthritis. A lack of IL‐17 leads to amelioration of collagen‐induced arthritis. IL‐17 induction in naïve CD4⁺ T cells depends on IL‐6 and TGF‐β and is enhanced by IL‐23. The in vivo inflammatory potential of in vitro‐primed Th17 cells however, remains unclear. Here, we show that, although IL‐17 neutralisation results in amelioration of murine OVA‐induced arthritis, in vitro‐primed Th17 cells cannot exacerbate arthritic symptoms after adoptive transfer. Furthermore, Th17 cells cannot induce an inflammatory delayed type hypersensitivity reaction because they fail to migrate into inflamed sites, possibly due to the lack of CXCR3 expression. Also, re‐isolated Th17 cells acquired IFN‐γ expression, indicating instability of the Th17 phenotype. Taken together, the data show that IL‐6, TGF‐β and IL‐23 might not provide sufficient signals to induce “fully qualified” Th17 cells.