Guarding the immune system: suppression of autoimmunity by CD4+CD25+ immunoregulatory T cells

CD4+CD25+Foxp3+ T cells (CD25+ T regulatory [Treg] cells) are a naturally occurring suppressor T-cell population that regulates a wide variety of immune responses. A major function of CD25+ Treg cells is to inhibit the activity of self-reactive T cells that can potentially cause autoimmune disease. This review examines the recent advances in CD25+ Treg cell biology, with particular focus on the thymic and peripheral development of CD25+ Treg cells, the signals that promote their expansion and maintenance in the periphery and the mechanism by which they mediate their suppressor activity in peripheral lymphoid tissues. An understanding of these issues is likely to facilitate the development of CD25+ Treg-cell-based therapies for the treatment of autoimmune disease.     

Midkine inhibits inducible regulatory T cell differentiation by suppressing the development of tolerogenic dendritic cells

Midkine (MK), a heparin-binding growth factor, reportedly contributes to inflammatory diseases, including Crohn's disease and rheumatoid arthritis. We previously showed that MK aggravates experimental autoimmune encephalomyelitis (EAE) by decreasing regulatory CD4(+)CD25(+)Foxp3(+) T cells (Tregs), a population that regulates the development of autoimmune responses, although the precise mechanism remains uncertain. In this article, we show that MK produced in inflammatory conditions suppresses the development of tolerogenic dendritic cells (DCregs), which drive the development of inducible Treg. MK suppressed DCreg-mediated expansion of the CD4(+)CD25(+)Foxp3(+) Treg population. DCregs expressed significantly higher levels of CD45RB and produced significantly less IL-12 compared with conventional dendritic cells. However, MK downregulated CD45RB expression and induced IL-12 production by reducing phosphorylated STAT3 levels via src homology region 2 domain-containing phosphatase-2 in DCreg. Inhibiting MK activity with anti-MK RNA aptamers, which bind to the targeted protein to suppress the function of the protein, increased the numbers of CD11c(low)CD45RB(+) dendritic cells and Tregs in the draining lymph nodes and suppressed the severity of EAE, an animal model of multiple sclerosis. Our results also demonstrated that MK was produced by inflammatory cells, in particular, CD4(+) T cells under inflammatory conditions. Taken together, these results suggest that MK aggravates EAE by suppressing DCreg development, thereby impairing the Treg population. Thus, MK is a promising therapeutic target for various autoimmune diseases.     

Thymus exosomes-like particles induce regulatory T cells

Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3(+) regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4(+)CD25(-) T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4(+)CD25(-) T cells in vitro and in vivo. We further show that neutralization of TGF-beta in ELPs partially reverses thymic ELP-mediated induction of CD4(+)Foxp3(+) T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3(+) Tregs. Also, TGF-beta of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.     

Kinetics and activation requirements of contact-dependent immune suppression by human regulatory T cells

Naturally occurring regulatory T cells (Tregs) maintain self tolerance by dominant suppression of potentially self-reactive T cells in peripheral tissues. However, the activation requirements, the temporal aspects of the suppressive activity, and mode of action of human Tregs are subjects of controversy. In this study, we show that Tregs display significant variability in the suppressive activity ex vivo as 54% of healthy blood donors examined had fully suppressive Tregs spontaneously, whereas in the remaining donors, anti-CD3/CD2/CD28 stimulation was required for Treg suppressive activity. Furthermore, anti-CD3/CD2/CD28 stimulation for 6 h and subsequent fixation in paraformaldehyde rendered the Tregs fully suppressive in all donors. The fixation-resistant suppressive activity of Tregs operated in a contact-dependent manner that was not dependent on APCs, but could be fully obliterated by trypsin treatment, indicating that a cell surface protein is directly involved. By add-back of active, fixed Tregs at different time points after activation of responding T cells, the responder cells were susceptible to Treg-mediated immune suppression up to 24 h after stimulation. This defines a time window in which effector T cells are susceptible to Treg-mediated immune suppression. Lastly, we examined the effect of a set of signaling inhibitors that perturb effector T cell activation and found that none of the examined inhibitors affected Treg activation, indicating pathway redundancy or that Treg activation proceeds by signaling mechanisms distinct from those of effector T cells.     

Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.     

Cutting edge: CD4+CD25+ regulatory T cells impaired for intestinal homing can prevent colitis

Transfer of CD4(+)CD45RB(high) T cells into RAG(-/-) mice causes colitis, which can be prevented by CD4(+)CD25(+) regulatory T cells (Treg). Colitis induction by CD4(+)CD45RB(high) T cells requires beta(7) integrin-dependent intestinal localization, but the importance of beta(7) integrins for Treg function is unknown. In this study, we show that beta(7)(-/-) Treg were effective in preventing colitis. Treg expanded in vivo to the same extent as CD4(+)CD45RB(high) T cells after transfer and they did not inhibit CD4(+)CD45RB(high) T cell expansion in lymphoid tissues, although they prevented the accumulation of Th1 effector cells in the intestine. beta(7)(-/-) Treg were significantly reduced in the large intestine, however, compared with wild-type Treg, and regulatory activity could not be recovered from the intestine of recipients of beta(7)(-/-) Treg. These data demonstrate that Treg can prevent colitis by inhibiting the accumulation of tissue-seeking effector cells and that Treg accumulation in the intestine is dispensable for colitis suppression.     

Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model

CD4(+) regulatory T cells (Tregs) control adaptive immune responses and promote self-tolerance. Various humanized mouse models have been developed in efforts to reproduce and study a human immune system. However, in models that require T cell differentiation in the recipient murine thymus, only low numbers of T cells populate the peripheral immune systems. T cells are positively selected by mouse MHC and therefore do not function well in an HLA-restricted manner. In contrast, cotransplantation of human fetal thymus/liver and i.v. injection of CD34(+) cells from the same donor achieves multilineage human lymphohematopoietic reconstitution, including dendritic cells and formation of secondary lymphoid organs, in NOD/SCID mice. Strong Ag-specific immune responses and homeostatic expansion of human T cells that are dependent on peripheral human APCs occur. We now demonstrate that FOXP3(+)Helios(+) "natural" Tregs develop normally in human fetal thymic grafts and are present in peripheral blood, spleen, and lymph nodes of these humanized mice. Humanized mice exhibit normal reversal of CD45 isoform expression in association with thymic egress, postthymic "naive" to "activated" phenotypic conversion, and suppressive function. These studies demonstrate the utility of this humanized mouse model for the study of human Treg ontogeny, immunobiology and therapy.     

Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis

Regulatory CD4(+)CD25(+) T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-gamma production by CD4(+)CD25(-) and CD8(+) T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-beta, suggesting a mechanism of linked immunosuppression. Tregs/CD4(+)CD25(-) T cell cocultures lead to similar changes in IFN-gamma and IL-10 secretion in patients and controls, whereas increased TGF-beta secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8(+) T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4(+)CD25(-) T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4(+)CD25(-) T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4(+)CD25(-) T cell spontaneous apoptosis may contribute to the development of autoimmunity.     

Increased regulatory versus effector T cell development is associated with thymus atrophy in mouse models of multiple myeloma

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play a central role in cancer tolerance. However, mechanisms leading to their accumulation in cancer remain unknown. Although the thymus is the main site of Treg development, thymic contribution to Treg expansion in cancer has not been directly examined. Herein, we used two murine models of multiple myeloma (MM), 5T2 MM and 5T33 MM, to examine Treg accumulation in peripheral lymphoid organs, including spleen, lymph nodes, bone marrow, and blood, and to explore thymic Treg development during malignancy. We found that peripheral ratios of suppressive-functional Tregs increased in both models of MM-inflicted mice. We found that thymic ratios of Treg development in MM increased, in strong association with thymus atrophy and altered developmental processes in the thymus. The CD4(+)CD8(+) double-positive population, normally the largest thymocyte subset, is significantly decreased, whereas the CD4(-)CD8(-) double-negative population is increased. Administration of thymocytes from MM-inflicted mice compared with control thymocytes resulted in increased progression of the disease, and this effect was shown to be mediated by Tregs in the thymus of MM-inflicted mice. Our data suggest that increased ratios of Treg development in the thymus may contribute to disease progression in MM-inflicted mice.     

Natural naive CD4+CD25+CD127low regulatory T cell (Treg) development and function are disturbed in multiple sclerosis patients: recovery of memory Treg homeostasis during disease progression

Patients with relapsing-remitting multiple sclerosis (RR-MS) show a suboptimal CD4(+)CD25(+) regulatory T cell (Treg) function, whereas no Treg alterations are observed in secondary progressive MS (SP-MS) patients. To clarify the difference in Treg activity between early and chronic disease stages in MS, we analyzed the functional capacity and homeostatic parameters of naive CD4(+)CD25(+)CD127(low)CD45RA(+) Tregs (nTregs) and their memory counterparts CD4(+)CD25(+)CD127(low)CD45RO(+) Tregs (mTregs) in untreated MS patients and healthy controls. Interestingly, whereas the suppressive capacity of FACS-sorted nTregs was impaired in both early and chronic MS patients, only the latter group showed a restored mTreg function. Consistent with this observation, chronic MS patients had increased numbers of mTregs as compared with age-matched early MS patients, whereas nTreg frequencies did not differ significantly. TCR excision circle numbers were reduced in nTregs of early MS patients, suggestive of a diminished nTreg thymic output. Moreover, a decreased number of CD31(+) mTregs were observed in early vs chronic MS patients, indicating that inflammatory processes drive the homeostatic turnover of mTregs during the early disease stage. Additionally, early MS patients showed a more restricted nTreg and mTreg TCR BV gene profile as compared with healthy controls and chronic MS patients. Finally, analysis of IFN-beta and glatiramer acetate-treated MS patients showed that these immunomodulatory drugs modify nTreg homeostasis. Taken together, this study provides strong evidence for a disturbed thymic nTreg development and function in MS patients. Moreover, memory Treg but not naive Treg homeostasis recovers during disease progression.     

Immunosuppressive therapy influences the accelerated age-dependent T-helper cell differentiation in systemic lupus erythematosus remission patients

Background

CD4⁺ T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4⁺ regulatory T cells (Tregs) and CD4⁺ responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality.

Methods

To determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA⁺CD31⁺ recent thymic emigrant (RTE) Tregs/Tresps and CD45RA⁺CD31^? mature naive (MN) Tregs/Tresps, as well as CD45RA^?CD31⁺ and CD45RA^?CD31^? memory Tregs/Tresps (CD31⁺ and CD31^? memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays.

Results

With age, we found an increased differentiation of RTE Tregs via CD31⁺ memory Tregs and of RTE Tresps via MN Tresps into CD31^? memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31⁺ memory Tresps, whereby CD31^? memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels.

Conclusions

Our data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable.

     

Immune tolerance: Are regulatory T cell subsets needed to explain suppression of autoimmunity?

The potential for self-reactive T cells to cause autoimmune disease is held in check by Foxp3(+) regulatory T cells (Tregs), essential mediators of peripheral immunological tolerance. Tregs have the capacity to suppress multiple branches of the immune system, tightly controlling the different subsets of effector T cells across multiple different tissue environments. Recent genetic experiments have found mutations that disrupt specific Treg: effector T cell relationships, leading to the possibility that subsets of Tregs are required to suppress each subset of effector T cells. Here we review the environmental factors and mechanisms that allow Tregs to suppress specific subsets of effector T cells, and find that a parsimonious explanation of the genetic data can be made without invoking Treg subsets. Instead, Tregs show a functional and chemotactic plasticity based on microenvironmental influences that allows the common pool of cells to suppress multiple distinct immune responses.     

Impaired Thymic Export and Increased Apoptosis Account for Regulatory T Cell Defects in Patients with Non-ST Segment Elevation Acute Coronary Syndrome

Regulatory T (Treg) cells play a protective role against the development of atherosclerosis. Previous studies have revealed Treg cell defects in patients with non-ST elevation acute coronary syndrome (NSTACS), but the mechanisms underlying these defects remain unclear. In this study, we found that the numbers of peripheral blood CD4⁺CD25⁺CD127^low Treg cells and CD4⁺CD25⁺CD127^lowCD45RA⁺CD45RO⁻ naive Treg cells were lower in the NSTACS patients than in the chronic stable angina (CSA) and the chest pain syndrome (CPS) patients. However, the number of CD4⁺CD25⁺CD127^lowCD45RA⁻CD45RO⁺ memory Treg cells was comparable in all of the groups. The frequency of CD4⁺CD25⁺CD127^lowCD45RO⁻CD45RA⁺CD31⁺ recent thymic emigrant Treg cells and the T cell receptor excision circle content of purified Treg cells were lower in the NSTACS patients than in the CSA patients and the CPS controls. The spontaneous apoptosis of Treg cells (defined as CD4⁺CD25⁺CD127^lowannexin V⁺7-AAD⁻) was increased in the NSTACS patients compared with the CSA and CPS groups. Furthermore, oxidized LDL could induce Treg cell apoptosis, and the oxidized LDL levels were significantly higher in the NSTACS patients than in the CSA and CPS groups. In accordance with the altered Treg cell levels, the concentration of TNF-α was increased in the NSTACS patients, resulting in a decreased IL-10/TNF-α ratio. These findings indicate that the impaired thymic output of Treg cells and their enhanced susceptibility to apoptosis in the periphery were responsible for Treg cell defects observed in the NSTACS patients.     

Virus-Specific Regulatory T Cells Ameliorate Encephalitis by Repressing Effector T Cell Functions from Priming to Effector Stages

Several studies have demonstrated the presence of pathogen-specific Foxp3⁺ CD4 regulatory T cells (Treg) in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg) and conventional CD4 T cells (M133 Tconv) in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN). Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.     

Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim(-/-), Bim(-/-)Puma(-/-), Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3(-/-)) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.     

Shaping of the autoreactive regulatory T cell repertoire by thymic cortical positive selection

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.     

Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells

CD28/B7 blockade leads to exacerbated autoimmune disease in the nonobese diabetic mouse strain as a result of a marked reduction in the number of CD4(+)CD25(+) regulatory T cells (Tregs). Herein, we demonstrate that CD28 controls both thymic development and peripheral homeostasis of Tregs. CD28 maintains a stable pool of peripheral Tregs by both supporting their survival and promoting their self-renewal. CD28 engagement promotes survival by regulating IL-2 production by conventional T cells and CD25 expression on Tregs.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.