Parasitic Nematode-Induced CD4+Foxp3+T Cells Can Ameliorate Allergic Airway Inflammation

Background

The recruitment of CD4⁺CD25⁺Foxp3⁺T (T_reg) cells is one of the most important mechanisms by which parasites down-regulate the immune system.

Methodology/Principal Findings

We compared the effects of T_reg cells from Trichinella spiralis-infected mice and uninfected mice on experimental allergic airway inflammation in order to understand the functions of parasite-induced T_reg cells. After four weeks of T. spiralis infection, we isolated Foxp3-GFP-expressing cells from transgenic mice using a cell sorter. We injected CD4⁺Foxp3⁺ cells from T. spiralis-infected [Inf(+)Foxp3⁺] or uninfected [Inf(-)Foxp3⁺] mice into the tail veins of C57BL/6 mice before the induction of inflammation or during inflammation. Inflammation was induced by ovalbumin (OVA)-alum sensitization and OVA challenge. The concentrations of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and the levels of OVA-specific IgE and IgG1 in the serum were lower in mice that received intravenous application of Inf(+)Foxp3⁺ cells [IV(inf):+(+) group] than in control mice. Some features of allergic airway inflammation were ameliorated by the intravenous application of Inf(-)Foxp3⁺ cells [IV(inf):+(-) group], but the effects were less distinct than those observed in the IV(inf):+(+) group. We found that Inf(+)Foxp3⁺ cells migrated to inflammation sites in the lung and expressed higher levels of T_reg-cell homing receptors (CCR5 and CCR9) and activation markers (Klrg1, Capg, GARP, Gzmb, OX40) than did Inf(-)Foxp3⁺ cells.

Conclusion/Significance

T. spiralis infection promotes the proliferation and functional activation of T_reg cells. Parasite-induced T_reg cells migrate to the inflammation site and suppress immune responses more effectively than non-parasite-induced T_reg cells. The adoptive transfer of Inf(+)Foxp3⁺ cells is an effective method for the treatment and prevention of allergic airway diseases in mice and is a promising therapeutic approach for the treatment of allergic airway diseases.     

Defective CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis,counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4⁺CD25⁺ T_reg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T_reg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4⁺CD25⁺ T cells in the central and peripheral lymphoid tissues. In addition, CD4⁺CD25⁺ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4⁺CD25⁺ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T_reg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T_reg cells and accessory cells. Our results demonstrate that the decrease in T_reg cell activity in CIA is counter-regulated by endogenous IFN-γ.     

Commitment to the Regulatory T Cell Lineage Requires CARMA1 in the Thymus but Not in the Periphery

Regulatory T (T_reg) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4⁺ T cells in a cytokine dependent manner. T_reg cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4⁺Foxp3⁺ T_reg cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T_reg cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFβ) and interleukin-2 (IL-2). In vivo, a small peripheral T_reg pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T_reg cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.     

CD44 expression positively correlates with Foxp3 expression and suppressive function of CD4+ Treg cells

Background  

CD4⁺CD25⁺ regulatory T (T_reg) cells develop in the thymus and can suppress T cell proliferation, modulated by Foxp3 and cytokines; however, the relevance of CD44 in T_reg cell development is less clear. To address this issue, we analyzed Foxp3 expression in CD44⁺ T_reg cells by using multiple parameters, measured the levels of the immunoregulatory cytokine interleukin (IL)-10 in various thymocyte subsets, and determined the suppressor activity in different splenic T_reg cell populations.     

The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>T cells

It is now generally accepted that CD4⁺CD25⁺ T_reg cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4⁺CD25⁺ cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4⁺CD25⁺ cells in the inflamed joint as compared with CD4⁺CD25^- cells. These data imply a significant role for CD4⁺CD25⁺ T_reg cells in the control of chronic arthritis. However, transferred T_reg cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of T_reg cell transfer in potential therapeutic applications.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Activation and Recruitment of Regulatory T Cells via Chemokine Receptor Activation in Trichinella spiralis-Infected Mice

As most infections by the helminth parasite elicit the recruitment of CD4⁺CD25⁺Foxp3⁺ T (T_reg) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated T_reg cells, we compared the expression levels of T_reg cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of T_reg cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated T_reg cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of T_reg cells in the muscle tissue.     

Adoptive transfer of T<Subscript>reg</Subscript> depleted autologous T cells in advanced renal cell carcinoma

Purpose

CD4⁺CD25⁺ regulatory T (T_reg) cells are present in increased numbers in patients with advanced cancer and CD25⁺ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25⁺ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets.

Patients and methods

Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25⁺ cells using CliniMACS^® System then re-infused into the patient.

Results

Efficient CD25⁺ depletion from all leukapheresis products was achieved and 0.55–5.87 × 10⁷/kg CD3⁺ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25⁺ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of T_reg cells.

Conclusions

Given the transient nature of the reduction in CD25⁺ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when T_reg cell levels are depleted.

     

Mechanisms of Cyclic Nucleotide Phosphodiesterases in Modulating T Cell Responses in Murine Graft-versus-Host Disease

Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3⁺ CD4⁺ T cells (T_reg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of T_reg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for T_reg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor T_reg cells. Towards the underlying mechanisms, in vitro studies revealed that T_reg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing T_reg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon T_reg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of T_reg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.     

Early increases in superantigen-specific Foxp3+ regulatory T cells during mouse mammary tumor virus infection

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3⁺ regulatory T cells (T_reg) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4⁺ CD25⁺ T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25⁺ cells at different stages of the infection. When CD25⁺ cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3⁻ T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that T_reg cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25⁺ cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages T_reg cells may favor viral persistence. Thus, our results indicated that T_reg cells play an important and complex role during MMTV infection.     

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4⁺CD25⁺FOXP3⁺ circulating cells (CD4 T_reg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4⁺ T_reg cells rapidly decreased after primary tumour debulking, while CD8⁺CD25⁺FOXP3⁺ (CD8 T_reg) cells are not detectable in peripheral blood. Similar results on CD4 T_reg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 T_reg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β₁ and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8⁺ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating T_reg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.     

Analysis of the Dynamics of Infiltrating CD4+ T Cell Subsets in the Heart during Experimental Trypanosoma cruzi Infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed during both the acute and chronic phases of the disease, is characterized by an inflammatory mononuclear cell infiltrate that includes CD4⁺ T cells. It is known that Th1 cytokines help to control infection. The role that T_reg and Th17 cells may play in disease outcome, however, has not been completely elucidated. We performed a comparative study of the dynamics of CD4⁺ T cell subsets after infection with the T. cruzi Y strain during both the acute and chronic phases of the disease using susceptible BALB/c and non-susceptible C57BL/6 mice infected with high or low parasite inocula. During the acute phase, infected C57BL/6 mice showed high levels of CD4⁺ T cell infiltration and expression of Th1 cytokines in the heart associated with the presence of T_reg cells. In contrast, infected BALB/c mice had a high heart parasite burden, low heart CD4⁺ T cell infiltration and low levels of Th1 and inflammatory cytokines, but with an increased presence of Th17 cells. Moreover, an increase in the expression of IL-6 in susceptible mice was associated with lethality upon infection with a high parasite load. Chronically infected BALB/c mice continued to present higher parasite burdens than C57BL/6 mice and also higher levels of IFN-γ, TNF, IL-10 and TGF-β. Thus, the regulation of the Th1 response by T_reg cells in the acute phase may play a protective role in non-susceptible mice irrespective of parasite numbers. On the other hand, Th17 cells may protect susceptible mice at low levels of infection, but could, in association with IL-6, be pathogenic at high parasite loads.     

Attenuation of Immune-Mediated Influenza Pneumonia by Targeting the Inducible Co-Stimulator (ICOS) Molecule on T Cells

Inducible Co-stimulator (ICOS) plays a critical role in mediating T cell differentiation and function and is considered a key player in balancing T effector and T regulatory (T_reg) cell responses. Here we show that activation of the ICOS signalling pathway during acute influenza A virus (IAV) infection by application of an agonistic ICOS antibody reduced the frequency of CD8⁺ T cells in the respiratory tract of IAV infected animals and delayed pathogen elimination. In line with this, immune-mediated influenza pneumonia was significantly ameliorated in mice that received ICOS agonist as indicated by significantly reduced alveolar infiltrations and bronchointerstitial pneumonia, while at the same time virus-related pathology remained unaffected. Importantly, ICOS agonist treatment resulted in expansion of CD4⁺Foxp3⁺ T_regs in IAV infected mice, which was associated with elevated levels of the immunosuppressive cytokine IL-10 in the alveolar space. Together, our findings suggest a prominent role of ICOS signaling during acute IAV infection by increasing the T_reg/CD8⁺ T cell ratio with beneficial outcome on immune-mediated pneumonia and underline the suitability of ICOS as potential therapeutic target for immune intervention in those infectious conditions characterized by strong immunopathology rather than virus-mediated cytopathic effects.     

TGF-β Signalling Is Required for CD4+ T Cell Homeostasis But Dispensable for Regulatory T Cell Function

TGF-β is widely held to be critical for the maintenance and function of regulatory T (T_reg) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-β receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-β–driven peripheral tolerance is not regulated by TGF-β signalling on mature CD4⁺ T cells. Inducible TR2 ablation specifically on CD4⁺ T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4⁺ T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4⁺ T cells does not result in the collapse of the T_reg cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-β signalling and the TR2–deficient T_reg cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-β signalling on mature CD4⁺ T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice.     

Th1-Like ICOS+ Foxp3+ Treg Cells Preferentially Express CXCR3 and Home to β-Islets during Pre-Diabetes in BDC2.5 NOD Mice

Type 1 diabetes (T1D) occurs through a breakdown of self-tolerance resulting in the autoimmune destruction of the insulin producing β-islets of the pancreas. A numerical and functional waning of CD4⁺Foxp3⁺ regulatory T (T_reg) cells, prompted by a pancreatic IL-2 deficiency, accompanies Th1 autoimmunity and T1D progression in non-obese diabetic (NOD) mice. Recently, we identified a dominant subset of intra-islet T_reg cells that expresses the ICOS costimulatory receptor and promotes self-tolerance delaying the onset of T1D. ICOS co-stimulation potently enhances IL-2 induced survival and proliferation, and suppressive activity of T_reg cells in situ. Here, we propose an ICOS-dependent mechanism of T_reg cell homing to the β-islets during pre-diabetes in the NOD model via upregulation of the CXCR3 chemokine receptor. The islet-specific ICOS⁺ T_reg cell subset preferentially expresses CXCR3 in the pancreatic lymph nodes (pLN) in response to T_eff cell-mediated pancreatic inflammation, an expression correlating with the onset and magnitude of IFN-γ production by T_eff cells in pancreatic sites. We also reveal that intra-pancreatic APC populations and insulin-producing β, but not α nor δ, islet cells secrete the CXCR3 chemokines, CXCL9, 10 and 11, and selectively promote ICOS⁺CXCR3⁺ T_reg cell chemotaxis in vitro. Strikingly, islet-derived T_reg cells also produce these chemokines suggesting an auto-regulation of homing by this subset. Unlike ICOS^- cells, ICOS⁺ T_reg cells adopt a Th1-like T_reg phenotype while maintaining their suppressive capacity, characterized by expression of T-bet and CXCR3 and production of IFN-γ in the draining pLNs. Finally, in vivo neutralization of IFN-γ blocked T_reg cell CXCR3 upregulation evincing its role in regulating expression of this chemokine receptor by T_reg cells. Thus, CXCR3-mediated trafficking of T_reg cells could represent a mechanism of homeostatic immunoregulation during diabetogeneesis.     

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4⁺CD25⁺Foxp3⁺ T (regulatory T; T_reg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of T_reg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and T_reg cells recruitment.     

Regulatory T cells and IL-10 independently counterregulate cytotoxic T lymphocyte responses induced by transcutaneous immunization

Background

The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T_reg) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.

Methodology/Principal Findings

TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3⁺ regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10^-/-) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T_reg in IL-10^-/- mice and the use of B cell deficient JHT^-/- mice, we can exclude T_reg and B cells as source of IL-10 in the setting of TCI.

Conclusion/Significance

We identify T_reg and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T_reg function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors.     

In Vivo Induction of Functionally Suppressive Induced Regulatory T Cells from CD4+CD25- T Cells Using an Hsp70 Peptide

Therapeutic peptides that target antigen-specific regulatory T cells (Tregs) can suppress experimental autoimmune diseases. The heat shock protein (Hsp) 70, with its expression elevated in inflamed tissue, is a suitable candidate antigen because administration of both bacterial and mouse Hsp70 peptides has been shown to induce strong immune responses and to reduce inflammation via the activation or induction of Hsp specific Tregs. Although two subsets of Tregs exist, little is known about which subset of Tregs are activated by Hsp70 epitopes. Therefore, we set out to determine whether natural nTregs (derived from the thymus), or induced iTregs (formed in the periphery from CD4⁺CD25^- naïve T cells) were targeted after Hsp70-peptide immunization. We immunized mice with the previously identified Hsp70 T cell epitope B29 and investigated the formation of functional iTregs by using an in vitro suppression assay and adoptive transfer therapy in mice with experimental arthritis. To study the in vivo induction of Tregs after peptide immunization, we depleted CD25⁺ cells prior to immunization, allowing the in vivo formation of Tregs from CD4⁺CD25^- precursors. This approach allowed us to study in vivo B29-induced Tregs and to compare these cells with Tregs from non-depleted immunized mice. Our results show that using this approach, immunization induced CD4⁺CD25⁺ T cells in the periphery, and that these cells were suppressive in vitro. Additionally, adoptive transfer of B29-specific iTregs suppressed disease in a mouse model of arthritis. This study shows that immunization of mice with Hsp70 epitope B29 induces functionally suppressive iTregs from CD4⁺CD25^- T cells.     

The Retinoic Acid-Metabolizing Enzyme Cyp26b1 Regulates CD4 T Cell Differentiation and Function

The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of Cyp26b1, the T cell-specific family member, in CD4⁺ T cells. Mice with a conditional knockout of Cyp26b1 in T cells (Cyp26b1 ^−/− mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iT_reg) cell- and T_H17 cell-polarizing conditions in vitro. Further, Cyp26b1 expression was differentially regulated in iT_reg and T_H17 cells. Transfer of naïve Cyp26b1 ^−/− CD4⁺ T cells into Rag1 ^−/− mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of Cyp26b1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target Cyp26b1 for the treatment of inflammatory bowel disease.     

Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (T_regs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. T_reg abnormalities have been associated with several autoimmune diseases and there is interest in the role of T_regs in SLE. We previously demonstrated that transfer of expanded CD4⁺CD25⁺CD62L^HI T_regs slows the development of lupus in (NZBxNZW)F₁ (B/W) mice. However in the absence of T_reg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3⁻ effector T cells (T_effs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure T_reg subset defined by CD4⁺CD25⁺ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4⁺CD25⁺ cells produced a population containing 70–85% CD4⁺Foxp3⁺T_regs. Expanded T_regs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4⁺CD25⁺ T_regs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of T_eff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred T_regs or transferred T_effs expanded in the absence of T_regs. These studies demonstrate that adoptive transfer of expanded CD4⁺CD25⁺Foxp3⁺T_regs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured T_eff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated T_effs from autoimmune patients may not pose a significant risk of promoting disease.