Critical role for the immunoproteasome subunit LMP7 in the resistance of mice to Toxoplasma gondii infection

Proteasome‐mediated proteolysis is responsible for the generation of immunogenic epitopes presented by MHC class I molecules, which activate antigen‐specific CD8⁺ T cells. Immunoproteasomes, defined by the presence of the three catalytic subunits LMP2, MECL‐1, and LMP7, have been hypothesized to optimize MHC class I antigen processing. In this study, we demonstrate that the infection of mice with a protozoan parasite, Toxoplasma gondii, induced the expression of LMP7 mRNA in APC and increased the capacity of APC to induce the production of IFN‐γ by antigen‐specific CD8⁺ T cells. In vitro infection of a DC cell line with T. gondii also induced the expression of LMP7 and resulted in enhanced proteasome proteolytic activity. Finally, mice lacking LMP7 were highly susceptible to infection with T. gondii and showed a reduced number of functional CD8⁺ T cells. These results demonstrate that proteasomes containing LMP7 play an indispensable role in the survival of mice infected with T. gondii, presumably due to the efficient generation of CTL epitopes required for the functional development of CD8⁺ T cells.     

Characterization of regulatory T cells in obese omental adipose tissue in humans

Obesity‐associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti‐inflammatory cells, notably regulatory T cells (Tregs) expressing the IL‐33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity‐associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non‐T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.     

Influenza matrix 1‐specific human CD4+FOXP3+ and FOXP3− regulatory T cells can be detected long after viral clearance

Control and termination of infection with Influenza A virus is associated with increased IL‐10 production in mouse models. Notably, IL‐10 can be produced by Treg. Therefore, we investigated whether the population of IL‐10‐producing influenza‐specific CD4⁺ T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza‐specific IL‐10‐producing T cells were detected in all human donors displaying influenza‐specific immunity. Isolation of Matrix 1 protein‐specific IL‐10‐producing T‐cell clones revealed that a substantial proportion of these T‐cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3⁺ and FOXP3^? CD4⁺ Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus‐infected cells. In vitro suppression was not mediated by IL‐10 but involved interference with the IL‐2 axis. The isolated Treg suppressed amongst others the IL‐2 production of influenza‐specific T‐helper cells as well as partially prevented the upregulation of the high‐affinity IL‐2 receptor on CD8 effector cells. So far the induction of virus‐specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus‐specific Treg can also be induced by viruses that are rapidly cleared in humans.     

An Optimized CFSE‐based T‐cell Suppression Assay to Evaluate the Suppressive Capacity of Regulatory T‐Cells Induced by Human Tolerogenic Dendritic Cells

In autoimmune diseases or transplant graft rejection, a therapy that will prevent or reduce the present immune activation is highly desired. Ex vivo generated tolerogenic dendritic cells (DC) are considered to have a strong potential as cellular therapy for these diseases. One of the mechanisms of immune suppression mediated by tolerogenic DC is the induction of regulatory T‐cells (Treg). Consequently, the efficacy of such DC to induce Treg will reflect their tolerogenic capacity. Because no specific markers have been described for human induced (i)Treg yet, the Treg can only be appreciated by functionality. Therefore, we have optimized an in vitro suppression assay to screen for human DC‐induced‐Treg activity. IL‐10‐generated tolerogenic DC were used to induce Treg that were previously shown to effectively suppress the proliferation of responder T‐cells stimulated with allogeneic mature DC (mDC). Our results show that the suppressive capacity of IL‐10 DC‐induced Treg measured in the suppression assay increases with the iTreg dose and decreases with higher numbers of antigen‐presenting cells (APC) as T‐cell stimulation. Lowering the ratio between responder T‐cells and stimulator mDC present in the coculture clearly improved the read‐out of the suppression assay. Furthermore, mDC‐primed T‐cells in the suppression assay were shown to be an essential control condition. In conclusion, we recommend titrations of both APC and iTreg in the suppression assay and to include a negative control condition with T‐cells primed by mDC, to distinguish specific and functional suppression by iTreg from possible generalized suppressive activity.     

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.     

Role of CD4+ Foxp3+ Regulatory T Cells in Protection Induced by a Live Attenuated,Replicating Type I Vaccine Strain of Toxoplasma gondii

Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii type I and type II strains. The involvement of regulatory T cells (Tregs) in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4⁺ Foxp3⁺ T regulatory cells followed by recruitment/expansion of CD4⁺ Foxp3⁻ CD25⁺ effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Tregs were of the TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4⁺ Foxp3⁺ cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of the TH1/TH17-like type. In addition, in vivo Treg induction in RH-infected mice with interleukin-2 (IL-2)/anti-IL-2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Tregs may contribute to the protective response after vaccination with Mic1.3KO.     

Interleukin‐17 Facilitates the Immune Suppressor Capacity of High‐Grade Glioma‐Derived CD4 (+) CD25 (+) Foxp3 (+) T Cells Via Releasing Transforming Growth Factor Beta

High‐grade glioma is a malignant tumour; the pathogenesis is to be further investigated. Interleukin (IL)‐17 is an inflammatory cytokine. Chronic inflammation is a pathological feature of cancer. This study aimed to characterize the glioma‐derived IL‐17⁺ regulatory T cells (Treg). In this study, single cells were isolated from surgically removed high‐grade glioma tissue and examined by flow cytometry. The immune suppressor effect of IL‐17⁺ Tregs on CD8⁺ T cells was assessed in vitro. The results showed that abundant IL‐17⁺ Tregs were found in high‐grade glioma tissue. The immune suppressor molecule, transforming growth factor (TGF)‐beta, was detected in the IL‐17⁺ Tregs. The proliferation of CD8⁺ T cells was suppressed by culturing with the IL‐17⁺ Tregs, which was partially abrogated by neutralizing antibodies of either TGF‐beta or IL‐17 and completely abrogated by neutralizing antibodies against both TGF‐beta and IL‐17. In conclusion, IL‐17⁺ Tregs exist in the high‐grade glioma tissue; this subset of T cells can suppress CD8⁺ T cell activities via releasing TGF‐beta and IL‐17.     

Inhibitory activity of anti-interleukin-4 and anti-interleukin-10 antibodies on Toxoplasma gondii proliferation in mouse peritoneal macrophages cocultured with splenocytes from infected mice

Cocultures of splenocytes from Toxoplasma gondii-immunized mice or from naive mice, separated by a transwell membrane from naive macrophage layers, induced a decrease in T. gondii proliferation in macrophages in comparison with cultures without splenocytes or cocultures with splenocytes from infected mice. Interleukin 4 (IL-4) and IL-10 levels increased in cocultures of splenocytes from infected mice with naive macrophages. In contrast, the levels of these cytokines decreased in cocultures with splenocytes from immunized mice. No correlation was found between the release of interferon-gamma (IFN-γ) and the inhibition of parasite multiplication. Cocultures with splenocytes from immunized mice induced an increase in tumor necrosis factor-alpha (TNF-α) levels. In contrast, in cocultures with splenocytes from infected mice, TNF-α production decreased. In cocultures with splenocytes from infected mice, T. gondii proliferation in macrophages was neutralized by anti-IL4 or anti-IL10 antibodies and was associated with increased TNF-α production. Moreover, this study demonstrates the significant combined effect of IL-4 and IL-10 on the down-regulation of macrophage-effector functions. A soluble positive signal was given by splenocytes to induce the production of TNF-α by macrophages. This signal was inhibited by IL4 and IL10. This process is biologically relevant in the regulation of T. gondii proliferation. Received: 2 August 1999 / Accepted: 30 August 1999     

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.     

T regulatory cells control T-cell proliferation partly by the release of soluble CD25 in patients with B-cell malignancies

Interleukin‐2 (IL‐2) is one of the most studied cytokines driving T‐cell proliferation, activation and survival. It binds to the IL‐2 receptor consisting of three chains, the α (CD25), β and common γ (γc). The binding of the CD25 chain to IL‐2 is necessary to expose high‐affinity binding sites for the β and γc chains, which, in turn, are responsible for downstream signalling. A high level of soluble CD25 (sCD25) has been associated with a poor prognosis in patients with non‐Hodgkin’s lymphoma. The function and source of origin of this soluble receptor is not well investigated. In the present study we hypothesized that T regulatory (Treg) cells may release CD25 to act as a decoy receptor for IL‐2, thereby depriving T‐effector cells of IL‐2. Peripheral blood from patients with B‐cell malignancies (n = 26) and healthy controls (n = 27) was investigated for the presence and function of FoxP3⁺ Treg cells and sCD25 by multi‐colour flow cytometry and enzyme‐linked immunosorbent assay. Further, the proliferative capacity of T cells was evaluated with or without the presence of recombinant sCD25. The results demonstrate that Treg cells from patients had lower CD25 expression intensity and that they released CD25 in vitro. Further, high levels of Treg cells correlated with sCD25 plasma concentration. Recombinant sCD25 could suppress T‐cell proliferation in vitro. In conclusion, the release of sCD25 by Treg cells may be a mechanism to deprive IL‐2 and thereby inhibit anti‐tumour T‐cell responses.     

CD4+FoxP3+ regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection

The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4⁺FoxP3⁺ Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild‐type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4⁺FoxP3⁺ Treg cells by application of diphtheria toxin. In Treg cell‐depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA‐3⁺ T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)‐4, IL‐5, and IL‐13. In contrast, only a mild increase in the Th1‐associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.     

Mast cells are crucial in the resistance against Toxoplasma gondii oral infection

During oral infection, mucosal immunity assumes a predominant role. Here, we addressed the role of mast cells (MCs), which are mainly located in mucosa during oral infection with Toxoplasma gondii, using MC‐deficient (W/W^v) mice. We show that in the absence of MCs the resistance of W/W^v mice to oral infection was considerably reduced. W/W^v mice uniformly succumbed within 15 days of infection after administration of cysts of the ME49 strain of T. gondii. The rapid lethality of T. gondii in W/W^v mice correlated with a delayed Th1‐cell response, since IFN‐γ and IL‐12 levels peaked in the later phase of the infection. In vitro, BM‐derived MCs were able to recognize parasite lysate in a MyD88‐dependent way, reaffirming the role of this TLR adapter in immune responses to T. gondii. The importance of MCs in vivo was confirmed when W/W^v mice reconstituted with BM‐derived MCs from control mice retrieved an early strong Th1‐cell response and specially a significant IL‐12 production. In conclusion, MCs play an important role for the development of a protective immune response during oral infection with T. gondii.     

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.     

Changes in peripheral blood level of regulatory T cells in patients with malignant melanoma during treatment with dendritic cell vaccination and low-dose IL-2

In this study, changes in peripheral blood regulatory T cell (Treg) levels were evaluated in 46 progressive patients with melanoma treated with a dendritic cell‐based vaccine and concomitant low‐dose IFN‐α and IL‐2. The regulatory subset of CD4 T cells, characterized by CD25^high, was prospectively analysed in fresh blood, and treatment‐associated quantitative and qualitative changes were analysed. By the 4th vaccine, patients showed a marked increase in CD4⁺CD25^high T cell subset from 6% to 22% (P < 0.001). At the 6th vaccine, a general decline was observed and a significantly (P = 0.01) lower level of CD4⁺CD25^high Treg cells was reached in the group of patients who attained disease stabilization (9.5%) compared to patients with continued progressive disease (14.5%). However, when FoxP3 was employed for retrospective analysis of Tregs on frozen blood, this difference did not reach significance (P = 0.09). The vast majority of the Treg produced IL‐10 and, to a varying extent, TGF‐β. In addition, sorted CD4⁺CD25^highCD127^? Tregs were able to suppress proliferation of peripheral blood mononuclear cells in a dose‐dependent manner, thus suggesting a regulatory functionality. These findings emphasize the need for strategies to effectively eliminate Treg cells to optimize the clinical effectiveness of cancer immunotherapy.     

Functional changes in regulatory T cells during an experimental infection with sparganum (plerocercofid of Spirometra mansoni)

Regulatory T (Treg) cells are important in the regulation of immune response, but the exact regulation of Treg‐cell function in vivo is still not well known. In the present study, we investigated the functional activity of CD4⁺ CD25⁺ Treg cells as well as the frequency and number of CD4⁺ CD25⁺ FoxP3⁺ Treg cells in the spleens of experimentally infected mice with a tissue‐migrating parasite, sparganum (plerocercoid of Spirometra mansoni) for 3 weeks. The results demonstrated fluctuations in the Treg‐cell function during the parasite infection, being up‐regulated at day 3, down‐regulated until day 14, and thereafter up‐regulated again at day 21. We also investigated the cytokine‐producing capability of the splenocytes to study the pattern of immune response of the mice to the parasite. The results showed decreased capabilities of interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ) and IL‐17α production, whereas IL‐4‐producing and IL‐10‐producing capabilities were increased along with the parasitic infection. Meanwhile, IL‐6‐producing capability was increased to reach a peak at week 2, and thereafter was decreased to the baseline level. As a regulatory mechanism, we found that Treg‐cell function was attenuated in the presence of the crude extracts of sparganum, but was enhanced in the presence of the excretory–secretory products, suggesting that sparganum products were involved in the triggering and regulation of immune response in the acute and chronic phases, respectively. Results show that Treg cells are central in the immune homeostasis in vivo that is maintained by host–parasite interactions during the parasitic infection.     

Litomosoides sigmodontis induces TGF‐β receptor responsive,IL‐10‐producing T cells that suppress bystander T‐cell proliferation in mice

Helminth parasites suppress immune responses to prolong their survival within the mammalian host. Thereby not only helminth‐specific but also nonhelminth‐specific bystander immune responses are suppressed. Here, we use the murine model of Litomosoides sigmodontis infection to elucidate the underlying mechanisms leading to this bystander T‐cell suppression. When OT‐II T cells specific for the third‐party antigen ovalbumin are transferred into helminth‐infected mice, these cells respond to antigen‐specific stimulation with reduced proliferation compared to activation within non‐infected mice. Thus, the presence of parasitic worms in the thoracic cavity translates to suppression of T cells with a different specificity at a different site. By eliminating regulatory receptors, cytokines, and cell populations from this system, we provide evidence for a two‐staged process. Parasite products first engage the TGF‐β receptor on host‐derived T cells that are central to suppression. In a second step, host‐derived T cells produce IL‐10 and subsequently suppress the adoptively transferred OT‐II T cells. Terminal suppression was IL‐10‐dependant but independent of intrinsic TGF‐β receptor‐ or PD‐1‐mediated signaling in the suppressed OT‐II T cells. Blockade of the same key suppression mediators, i.e. TGF‐β‐ and IL‐10 receptor, also ameliorated the suppression of IgG response to bystander antigen vaccination in L. sigmodontis‐infected mice.