Suppressive Activity Of Tregs Research Articles

IL-10 Both Inhibits Treg Suppressive Activity and is a Survival Factor for Human Natural Tregs

ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) supports automated NMR data collection and backbone and side chain assignment for [U–13C, U–15N]-labeled proteins. Given the sequence of the protein and data for the orthogonal 2D 1H–15N and 1H–13C planes, the algorithm automatically directs the collection of tilted plane data from a variety of triple-resonance experiments so as to follow an efficient pathway toward the probabilistic assignment of 1H, 13C, and 15N signals to specific atoms in the covalent structure of the protein. Data collection and assignment calculations continue until the addition of new data no longer improves the assignment score. ADAPT-NMR was first implemented on Varian (Agilent) spectrometers [A. Bahrami, M. Tonelli, S.C. Sahu, K.K. Singarapu, H.R. Eghbalnia, J.L. Markley, PLoS One 7 (2012) e33173]. Because of broader interest in the approach, we present here a version of ADAPT-NMR for Bruker spectrometers. We have developed two AU console programs (ADAPT_ORTHO_run and ADAPT_NMR_run) that run under TOPSPIN Versions 3.0 and higher. To illustrate the performance of the algorithm on a Bruker spectrometer, we tested one protein, chlorella ubiquitin (76 amino acid residues), that had been used with the Varian version: the Bruker and Varian versions achieved the same level of assignment completeness (98% in 20 h). As a more rigorous evaluation of the Bruker version, we tested a larger protein, BRPF1 bromodomain (114 amino acid residues), which yielded an automated assignment completeness of 86% in 55 h. Both experiments were carried out on a 500 MHz Bruker AVANCE III spectrometer equipped with a z-gradient 5 mm TCI probe. ADAPT-NMR is available at http://pine.nmrfam.wisc.edu/ADAPT-NMR in the form of pulse programs, the two AU programs, and instructions for installation and use.

IL-6 Produced by Dendritic Cells from Lupus-Prone Mice Inhibits CD4+CD25+ T Cell Regulatory Functions

The B6.Sle1.Sle2.Sle3 triple congenic mouse (B6.TC) is a model of lupus coexpressing the three major NZM2410-derived susceptibility loci on a C57BL/6 background. B6.TC mice produce high titers of antinuclear nephrogenic autoantibodies and a highly penetrant glomerulonephritis. Previous studies have shown the Sle1 locus is associated with a reduced number of regulatory T cells (Treg) and that Sle3 results in intrinsic defects of myeloid cells that hyperactivate T cells. In this report, we show that B6.TC dendritic cells (DCs) accumulate in lymphoid organs and present a defective maturation process, in which bone marrow-derived, plasmacytoid, and myeloid DCs express a significantly lower level of CD80, CD86, and MHC class II. B6.TC DCs also induce a higher level of proliferation in CD4(+) T cells than B6 DCs, and B6.TC DCs block the suppressive activity of Treg. B6.TC DCs overproduce IL-6, which is necessary for the blockade of Treg activity, as shown by the effect of anti-IL-6 neutralizing Ab in the suppression assays. The overproduction of IL-6 by DCs and the blockade of Treg activity maps to Sle1, which therefore not only confers a reduced number of Treg but also blocks their ability to regulate autoreactive T cells. Taken together, these results provide a genetic and mechanistic evidence for systemic autoimmunity resulting from an impaired regulatory T cell compartment in both number and function and for Sle1-expressing DCs playing a major role in the latter defect though their production of IL-6.

Enhanced IL-6 Production by Bone Marrow Derived Dendritic Cells from Lupus-Prone Mice Inhibits CD4+CD25+ T Cell Mediated Suppression.

The B6.Sle1.Sle2.Sle3 triple congenic mouse (B6.TC) is a model of lupus due to the co-expression of the three major NZM2410-derived susceptibility loci on a C57BL/6 background. B6.TC mice produce high titers of anti-nuclear nephrogenic autoantibodies and a highly penetrant glomerulonephritis. Previous studies have shown the Sle1 locus is associated with a reduced number of regulatory T cells (Treg), and that Sle3 results in intrinsic defects in myeloid cells that hyperactivate T cells. Here, we show that B6.TC dendritic cells (DCs) accumulate in lymphoid organs and present a defective maturation process, in which bone-marrow derived DCs, plasmacytoid and myeloid DCs express a significantly lower level of CD80, CD86 and class II MHC than B6 controls. B6.TC DCs also induce a higher level of proliferation in CD4+ T cells than B6 DCs, and B6.TC DCs block the suppressive activity of Treg. B6.TC DCs over-produce IL-6, which is necessary for the blockade of Treg activity, as shown by anti-IL-6 neutralizing antibody in the suppression assays. The over-production of IL-6 by DCs and the blockade of Treg activity maps to Sle1, which therefore not only confers a reduced number of Treg, but also blocks their ability to regulate autoreactive T cells. Taken together, these results provide a genetic and mechanistic evidence for systemic autoimmunity resulting from an impaired regulatory T cell compartment both in number and function, and for Sle1-expressing DCs playing a major role in the latter defect though their production of IL-6.

Lack of Suppressive CD4+CD25+FOXP3+ T Cells in Advanced Stages of Primary Cutaneous T-Cell Lymphoma

Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapable of suppressing autologous CD4+CD25- T-cell proliferation, whereas suppressive function was intact in the other six patients. Suppressive activity of Tregs inversely correlated with the peripheral blood tumor burden. T-plastin gene expression, used as a Sezary cell marker, confirmed that Sezary cells were heterogeneous for CD25 expression. Mixed lymphocyte reactions demonstrated that CD4+CD25- T cells from patients who lacked functional Tregs were susceptible to suppression by Tregs from healthy controls, and had not become suppressive themselves. Furthermore, we found reduced expression of Foxp3 in the CD4+CD25+ Tregs of these patients relative to the other six CTCL patients and controls. Our findings thus indicate a dysfunction of peripheral Tregs in certain CTCL patients, which correlates with tumor burden.

Toll-like receptor 2 signaling modulates the functions of CD4+ CD25+ regulatory T cells.

Toll-like receptors (TLRs) are primary sensors of both innate and adaptive immune systems and play a pivotal role in response against structurally conserved components of pathogens. Synthetic bacterial lipoprotein (BLP) Pam3Cys-SK4 is a TLR2 agonist that is capable of modulating T cell immune responses. We show here that BLP, together with anti-CD3 antibody [T cell receptor (TcR) activation], induced proliferation of both CD4+ CD25+ regulatory T cells (Tregs) and CD4+ CD25- (effector) T cells in the absence of antigen-presenting cells. The expanded Tregs showed a transient loss of suppressive activity. Moreover, BLP rendered effectors resistant to the suppression of Tregs by increasing IL-2 secretion. BLP also transiently suppressed the induction of Foxp3 (X-linked forkhead/winged helix transcription factor) mRNA in Tregs at the first 8-15 h after T cell receptor activation. Consistent with this observation, BLP-stimulated Tregs regained their inhibitory activity and prevented spontaneous colitis induced by effectors in severe combined immunodeficient mice. Our results demonstrate a previously unrecognized pathway by which TLR expressed on T cells may directly modulate the immune response. Thus, during an acute bacterial infection, BLP may rapidly increase the host's adaptive immunity by expanding effectors and also by attenuating the suppressive activity of Tregs. In the process, BLP also expands the Tregs, which recover their suppressive activity when the infection has subsided, in time to limit potential autoimmunity that might result from the overactivated effectors.

Low circulating regulatory T-cell levels after acute rejection in liver transplantation

Immune regulatory CD4+CD25+ T cells play a crucial role in inducing and maintaining allograft tolerance in experimental models of transplantation (Tx). In humans, the effect of Tx and immunosuppression on the function and homeostasis of CD4+CD25+ regulatory T cells (Tregs) is not well characterized. In this study, the frequency of Tregs in liver transplant recipients was determined based on flow cytometric analysis of CD4, CD25, CD45RO, and cytotoxic T lymphocyte antigen (CTLA)-4 markers, and the suppressor activity of Tregs was assessed in a mixed-leukocyte reaction. A link between Tregs, acute rejection, and immune-suppressive treatment was investigated. Liver transplant recipients had significantly higher Treg levels in peripheral blood pre-Tx than healthy controls. After Tx, a significant drop in the Treg fraction was observed. This reduction of circulating Tregs was transient and was associated with immunosuppression. In recipients who did not develop rejection, a relative recovery of Treg levels was seen within the first year after Tx. Recipients who experienced an episode of steroid-treated acute rejection, however, had sustained low Treg levels. The suppressive activities of CD4+CD25+ Tregs from rejectors, nonrejectors, and healthy controls on proliferation and interferon (IFN)-gamma production were indistinguishable. In conclusion, the percentage of CD4+CD25+CD45RO+CTLA-4+ quadruple-positive Tregs in peripheral blood decreases significantly after liver Tx. Treatment with methylprednisolone during Tx and for acute rejection is associated with low circulating Tregs. Despite these quantitative differences between rejectors and nonrejectors, the suppressive quality of CD4+CD25+ Tregs is identical in both groups.

Effective Depletion of Alloreactive T Cells by CD95l-Transduced Cells with Preservation of FoxP3 Regulatory T-Cells.

Adoptive transfer of donor T-cells after stem cell transplantation across HLA barriers could bridge the period of prolonged immunodeficiency until new thymic derived T-cells develop. In such an approach, however, donor T-cells reactive to alloantigens of the recipient induce GvHD, necessitating their selective elimination, inactivation or modulation. Depletion of alloreactive cells has been attempted by monoclonal antibodies, but prevention of GvHD was insufficient in clinical application so far. We developed a system allowing efficient selective depletion of alloreactive cells by CD95L-mediated apoptosis, which in addition has the advantage of preserving FoxP3 regulatory T-cells. An apoptosis-resistant B-cell line (C1RA1) was transduced with lentiviral CD95L (C1RA1-CD95L). CD95L was stably expressed by 65–80% of the cells and was fully functional to induce apoptosis in Jurkat cells. MNC of donor A were stimulated with APC of donor B, and, after 5 days of culture, viable responder cells were enriched and cultured with C1RA1-CD95L for another 3 days. T cells obtained by this culture system had lost proliferative activity towards cells of donor B, while proliferation against 3rd-party cells was fully preserved. Likewise, cytotoxic activity against donor B was markedly reduced. Since CD4+CD25+ T-cells were still detectable at levels of 7 to 10% of MNC at the end of the culture despite effective depletion of alloactivated cells, we analyzed for the presence of FoxP3+ regulatory T-cells (Treg). While gene expression of FoxP3 was almost undetectable in MNC before culture, it was present after activation of MNC and was strongly expressed after induction of apoptosis with C1RA1-CD95L at the end of our culture system. Suppression of proliferation of about 50% by these cells was noted in a suppressor assay using fresh MNC from donor A stimulated with cells of donor B. When suppression was analyzed in a stimulation system with 3rd-party stimulatory cells, reduction of proliferation was less prominent, indicating the possibility that the suppressive activity of Treg preserved in our culture system could be alloantigen-specific. In summary, we have developed a culture system, allowing alloantigen-specific depletion of T cells while preserving FoxP3+ regulatory T-cells. This sequential system of alloantigen-specific stimulation followed by CD95L-mediated apoptosis of activated cells by an unrelated, modified cell line offers the advantage of broad applicability in clinical transplantation settings.

Role of glucocorticoid‐induced TNF receptor family gene (GITR) in collagen‐induced arthritis

In rheumatoid arthritis (RA), a widespread autoimmune/inflammatory joint disease, early activation of effector CD4+ T lymphocytes, and cytokine production is followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, and disease. GITR (glucocorticoid-induced TNFR family-related gene), a costimulatory molecule for T lymphocytes, increases CD4+CD25- effector T cell activation while inhibiting suppressor activity of CD4+CD25+ T regulatory (Treg) cells. We analyzed the role of GITR in type II collagen (CII) -induced arthritis (CIA) using GITR-/- and GITR+/+ mice. Results indicate significantly less CIA induction in GITR-/- mice than in GITR+/+ mice, with marked differences in erythema, edema, neutrophil infiltration, joint injury, and bone erosion. Production of IFNgamma, IL-6, TNFalpha, MIP-1alpha, and MIP-2, inducible NOS (iNOS), COX-2, and nitrotyrosine poly-ADP-ribose (PAR) were also less in CII-treated GITR-/- mice. Although CD4+CD25+ Treg cells from GITR+/+ and GITR-/- CII-challenged mice exerted similar suppressor activity in vitro, GITR triggering abrogated GITR+/+ Treg suppressor activity and costimulated CD4+CD25- GITR+/+ effector cells. Furthermore, Treg cells from GITR-/- protected more than Treg cells from GITR+/+ mice against CIA when cotransferred with Treg-depleted splenocytes from arthritic GITR+/+ animals into severe combined immunodeficient (SCID) mice. In conclusion, GITR plays a critical role in the immunological response against CII and in the development of CIA.

Regulation of CD4+CD25+ regulatory T cell activity: it takes (IL‐)two to tango

Although CD4(+)CD25(+) regulatory T cells (Treg) represent a well-characterized population of T cells with in vitro and in vivo suppressive capacity, the basic mechanisms of suppression are still not understood. The constitutive expression of the high-affinity receptor for IL-2 has raised the question about the role of IL-2 in Treg function. Here, we review recent data indicating that IL-2 is not only necessary for the homeostasis of Treg but is also critical for the activation of Treg function. Since Treg do not produce IL-2 by themselves, their capacity to utilize IL-2 secreted by other T cells appears to be an essential component of Treg biology. This indicates that Treg suppressive activity is controlled by interaction with activated target cells via the soluble mediator IL-2. In Treg, IL-2 has been identified as a potent inducer of the immunosuppressive cytokine IL-10, an important mediator of Treg suppression in vivo. The efficient capture of IL-2 by Treg may, under conditions of limited IL-2 supply, cause IL-2 deprivation of responder T cells. This competition can explain some of the currently discussed discrepancies between in vivo and in vitro activity of Treg.

The negative regulatory function of the lymphocyte‐activation gene‐3 co‐receptor (CD223) on human T cells

Accumulating evidence indicates that the CD4 homologue lymphocyte activation gene-3 (LAG-3) plays a down-regulatory role on T-cell responses. However, the role of LAG-3/major histocompatibility complex (MHC) class II interactions on primary human T-cell responses, as well as the mechanism by which down-regulation occurs, are not clear. Here, we show that LAG-3 colocalized with CD3, CD4 or CD8 in areas of cholesterol-rich raft aggregation during this primary response, as well as in the clustered raft region formed between T cells and antibody-coated beads. Addition of a blocking LAG-3-specific monoclonal antibody to both CD4 and CD8 primary resting T cells activated under conditions of antigen-presenting cell-driven stimulation and low antigen concentrations augments CD69 activation antigen expression, T-cell expansion and T helper 1 (Th1, but not Th2) cytokine production. Blocking LAG-3/MHC class II interactions leads to an increase in the number of cells entering division at these low concentrations of antigen and to more rounds of divisions with an accumulation of cells in the S-phase of the cell cycle. These results indicate that LAG-3 signalling inhibits early events in primary activation of human CD4 and CD8 T cells and further support a role for LAG-3 signalling in regulating the expansion of activated effector or memory T cells, either directly or indirectly through Treg suppressor activity.

Inhibition of CD4+25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide

AbstractRegulatory T cells (TREGs) control the key aspects of tolerance and play a role in the lack of antitumor immune responses. Cyclophosphamide (CY) is a chemotherapeutic agent with a dose-dependent, bimodal effect on the immune system. Although a previous study demonstrated that CY reduces the number of TREGs, the mechanism involved in this process has yet to be defined. In this report, it is established that low-dose CY not only decreases cell number but leads to decreased functionality of TREGs. CY treatment enhances apoptosis and decreases homeostatic proliferation of these cells. Expression of GITR and FoxP3, which are involved in the suppressive activity of TREGs, is down-regulated after CY administration, though the level of expression varies depending on the time studied. This is the first report demonstrating that CY, in addition to decreasing cell number, inhibits the suppressive capability of TREGs. The relevance of the loss of suppressor functionality and the changes in gene expression are further discussed.

Murine liver antigen presenting cells control suppressor activity of CD4+CD25+ regulatory T cells

CD4(+)CD25(+) regulatory T cells (Treg) are important mediators of peripheral immune tolerance; however, whether Treg participate also in hepatic immune tolerance is not clear. Therefore, we tested the potential of Treg to suppress stimulation of CD4(+) T cells by liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), or hepatocytes. In the absence of Treg, all 3 types of liver cells could stimulate CD4(+) T cell proliferation; in the presence of Treg, however, CD4(+) T cell proliferation was suppressed. Interaction with KC even stimulated the expansion of the Treg population; LSEC or hepatocytes, in contrast, could not induce proliferation of Treg. Because liver inflammation can be induced by infection, we tested the potential of liver cells to modify Treg suppressor activity in the presence of microbial signals. In the presence of immune-stimulatory CpG-oligonucleotides, LSEC, KC, and hepatocytes could indeed overcome Treg-mediated suppression; in the presence of lipopolysaccharide (LPS), however, only KC and hepatocytes, but not LSEC, could overcome Treg suppressor activity. Hepatocytes from mice with deficient toll-like receptor-4 signaling failed to abrogate Treg suppression in response to LPS, indicating that overcoming Treg suppressor activity was indeed a response of the liver cell and not of the Treg. In conclusion, Treg can suppress CD4(+) T cell stimulation by liver cells. However, in response to microbial signals, the liver cells can overcome the suppressive activity of Treg. Thus, liver cells may facilitate the transition from hepatic immune tolerance to hepatic inflammation by controlling Treg suppressor activity.

Regulatory T Cell Suppression and Anergy Are Differentially Regulated by Proinflammatory Cytokines Produced by TLR-Activated Dendritic Cells

CD25(+)CD4(+) regulatory T cells (Tregs) are required for the maintenance of peripheral tolerance to certain self Ags. In this study, the requirements for murine Treg-suppressive activity and proliferation were examined in the context of the maturation of myeloid dendritic cells (DCs). We find that the suppressive function of Tregs is critically dependent on immature DCs and is readily reversed by the maturation of DCs induced by GM-CSF, but does not require TLR activation of either DCs or Tregs. In contrast, reversal of Treg anergy is dependent on TLR activation of DCs, and involves the potentiation of Treg responsiveness to IL-2 by cooperative effects of IL-6 and IL-1, both of which are produced by TLR-activated, mature DCs. Thus, proinflammatory cytokines produced by TLR-activated, mature DCs are required for reversal of Treg anergy, but are not required to overcome Treg suppression.

Distinct roles for the OX40-OX40 ligand interaction in regulatory and nonregulatory T cells.

The OX40 (CD134) molecule is induced primarily during T cell activation and, as we show in this study, is also expressed on CD25+CD4+ regulatory T (Treg) cells. A necessary role for OX40 in the development and homeostasis of Treg cells can be inferred from the reduced numbers of the cells present in the spleens of OX40-deficient mice, and their elevated numbers in the spleens of mice that overexpress the OX40 ligand (OX40L). The homeostatic proliferation of Treg cells following transfer into lymphopenic mice was also found to be potentiated by the OX40-OX40L interaction. Suppression of T cell responses by Treg cells was significantly impaired in the absence of OX40, indicating that, in addition to its homeostatic functions, OX40 contributes to efficient Treg-mediated suppression. However, despite this, we found that CD25-CD4+ T cells became insensitive to Treg-mediated suppression when they were exposed to OX40L-expressing cells, or when they were treated with an agonistic OX40-specific mAb. OX40 signaling could also abrogate the disease-preventing activity of Treg cells in an experimental model of inflammatory bowel disease. Thus, although the data reveal important roles for OX40 signaling in Treg cell development, homeostasis, and suppressive activity, they also show that OX40 signals can oppose Treg-mediated suppression when they are delivered directly to Ag-engaged naive T cells.