Treg Production Research Articles

Sunitinib Mediates Reversal of Myeloid-Derived Suppressor Cell Accumulation in Renal Cell Carcinoma Patients

Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-gamma were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-gamma were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. Metastatic RCC patients had elevated levels of CD33(+)HLA-DR(-) and CD15(+)CD14(-) MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3(+)CD4(+)CD25(hi)Foxp3(+) Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-gamma. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at >/=1.0 microg/mL. Sunitinib did not induce MDSC maturation in vitro. Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

Pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance and protects against experimental autoimmune encephalomyelitis

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a widely expressed neuropeptide originally discovered in the hypothalamus. It closely resembles vasoactive intestinal peptide (VIP), a neuropeptide well known to inhibit macrophage activity, promote Th2-type responses, and enhance regulatory T cell (Treg) production. Recent studies have shown that administration of PACAP, like VIP, can attenuate dramatically the clinical and pathological features of murine models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However, specific roles (if any) of endogenous VIP and PACAP in the protection against autoimmune diseases have not been explored. Here, we subjected PACAP-deficient mice to myelin oligodendrocyte glycoprotein (MOG(35-55))-induced EAE. MOG immunization of PACAP-deficient mice triggered heightened clinical and pathological manifestations of EAE compared to wild-type mice. The increased sensitivity was accompanied by enhanced mRNA expression of proinflammatory cytokines (TNFalpha, IL-6, IFN-gamma, IL-12p35, IL-23p19, and IL-17), chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, and RANTES/CCL5), and chemotactic factor receptors (CCR1, CCR2, and CCR5), but downregulation of the anti-inflammatory cytokines (IL-4, IL-10, and TGF-beta) in the spinal cord. Moreover, the abundance of CD4(+)CD25(+)FoxP3(+) Tregs in lymph nodes and levels of FoxP3 mRNA in the spinal cord were also diminished. The reduction in Tregs was associated with increased proliferation and decreased TGF-beta secretion in lymph node cultures stimulated with MOG. These results demonstrate that endogenous PACAP provides protection in EAE and identify PACAP as an intrinsic regulator of Treg abundance after inflammation.

Distinct Roles of IL-10 in Regulation of Peanut Allergy in Humans

RATIONALE: Enhanced T cell, Treg and/or APC-derived IL-10 production might underlie clinical tolerance to many non-self antigens. Elevated plasma IL-10 was recently proposed as a biomarker of clinical tolerance in tree nut allergy. Here, we evaluate IL-10's contributions to regulation of peanut-driven cytokine responses.

Low frequency of CD4+CD25+ Treg in SLE patients: a heritable trait associated with CTLA4 and TGFβ gene variants

BackgroundCD4+CD25+ regulatory T cells play an essential role in maintaining immune homeostasis and preventing autoimmunity. Therefore, defects in Treg development, maintenance or function have been associated with several human autoimmune diseases including Systemic Lupus Erythematosus (SLE), a systemic autoimmune disease characterized by loss of tolerance to nuclear components and significantly more frequent in females.ResultsTo investigate the involvement of Treg in SLE pathogenesis, we determined the frequency of CD4+CD25+CD45RO+ T cells, which encompass the majority of Treg activity, in the PBMC of 148 SLE patients (76 patients were part of 54 families), 166 relatives and 117 controls. SLE patients and their relatives were recruited in several Portuguese hospitals and through the Portuguese Lupus Association. Control individuals were blood donors recruited from several regional blood donor centers. Treg frequency was significantly lower in SLE patients than healthy controls (z = -6.161, P < 0.00001) and intermediate in the relatives' group. Remarkably, this T cell subset was also lower in females, most strikingly in the control population (z = 4.121, P < 0.001). We further ascertained that the decreased frequency of Treg in SLE patients resulted from the specific reduction of bona fide FOXP3+CD4+CD25+ Treg. Treg frequency was negatively correlated with SLE activity index (SLEDAI) and titers of serum anti-dsDNA antibodies. Both Treg frequency and disease activity were modulated by IVIg treatment in a documented SLE case. The segregation of Treg frequency within the SLE families was indicative of a genetic trait. Candidate gene analysis revealed that specific variants of CTLA4 and TGFβ were associated with the decreased frequency of Treg in PBMC, while FOXP3 gene variants were associated with affection status, but not with Treg frequency.ConclusionSLE patients have impaired Treg production or maintenance, a trait strongly associated with SLE disease activity and autoantibody titers, and possibly resulting from the inability to convert FOXP3+CD25- into FOXP3+CD25+ T cells. Treg frequency is highly heritable within SLE families, with specific variants of the CTLA4 and TGFβ genes contributing to this trait, while FOXP3 contributes to SLE through mechanisms not involving a modulation of Treg frequency. These findings establish that the genetic components in SLE pathogenesis include genes related to Treg generation or maintenance.

Intensive Extra Corporeal Phototherapy (ECP) Courses In Acute Graft Versus Host Disease (aGVHD) Patients

AGVHD may occur in a high proportion of allo-transplanted patients and is one the main causes of death. ECP treatment may successfully be employed in patients refractory or not eligible to high doses steroid therapy. The ECP intensification, as well as the timely start of the courses, are probably fundamental to enhance the probability and the rate of clinical response. Ten aGVHD III-IV patients, 7 males and 3 females, were enrolled. Four were adults and six were children with liver, gut or skin localization. The mean time from aGVHD diagnosis to ECP starting was seven days. Three ECP were performed weekly for three consecutive weeks then two procedures every two weeks were carried out in six months. A mean of 16 (6–29) procedures per patient was carried out, being 4 (1–6) months the mean duration per treatment. Phenotype studies disclosed Treg cells and circulating oligoclonal TCRVB subsets in in two and five patients, respectively. Cyclosporine A was used as primary immunosuppressive drug in six patients or associated to MMF or tacrolimus in two and one patients. One patient received MMF alone and four, Infliximab (3–6 doses). Two patients died for progressive GVHD and in one ECP was stopped for severe CMV reactivation. Seven patients (78%) had fast and significant clinical recovery after the first six, intensified ECP and the mean follow-up observed is of 29 (6–50) months. One child responsive after seven ECP courses died for acute liver failure, one month after the ECP had been concluded One child relapsed and died for NB in the first year post transplantation, two adults are alive with relapsed HL, the others are alive without clinical or molecular signs of relapse. As regards phenotype studies, in two responsive patients, not treated with Cyclosporine A, Treg absolute counts tripled during ECP treatment while oligoclonal TCRVB families disappeared in all responsive patients. Despite the limited number of the subjects treated, the rapid clinical recovery observed in these highly compromised patients enforces the indication to employ intensive ECP courses to accelerate the clinical remission and to reduce the immunosuppression. As regards the possible use of the Treg or the TCRVB oligoclonal subsets modifications as prognostic factors during ECP treatment, the true involvement of these subpopulations is up to date, unclear. In particular Cyclosporine A might inhibit the production of Tregs and probably, other biological markers must be individuated. AGVHD may occur in a high proportion of allo-transplanted patients and is one the main causes of death. ECP treatment may successfully be employed in patients refractory or not eligible to high doses steroid therapy. The ECP intensification, as well as the timely start of the courses, are probably fundamental to enhance the probability and the rate of clinical response. Ten aGVHD III-IV patients, 7 males and 3 females, were enrolled. Four were adults and six were children with liver, gut or skin localization. The mean time from aGVHD diagnosis to ECP starting was seven days. Three ECP were performed weekly for three consecutive weeks then two procedures every two weeks were carried out in six months. A mean of 16 (6–29) procedures per patient was carried out, being 4 (1–6) months the mean duration per treatment. Phenotype studies disclosed Treg cells and circulating oligoclonal TCRVB subsets in in two and five patients, respectively. Cyclosporine A was used as primary immunosuppressive drug in six patients or associated to MMF or tacrolimus in two and one patients. One patient received MMF alone and four, Infliximab (3–6 doses). Two patients died for progressive GVHD and in one ECP was stopped for severe CMV reactivation. Seven patients (78%) had fast and significant clinical recovery after the first six, intensified ECP and the mean follow-up observed is of 29 (6–50) months. One child responsive after seven ECP courses died for acute liver failure, one month after the ECP had been concluded One child relapsed and died for NB in the first year post transplantation, two adults are alive with relapsed HL, the others are alive without clinical or molecular signs of relapse. As regards phenotype studies, in two responsive patients, not treated with Cyclosporine A, Treg absolute counts tripled during ECP treatment while oligoclonal TCRVB families disappeared in all responsive patients. Despite the limited number of the subjects treated, the rapid clinical recovery observed in these highly compromised patients enforces the indication to employ intensive ECP courses to accelerate the clinical remission and to reduce the immunosuppression. As regards the possible use of the Treg or the TCRVB oligoclonal subsets modifications as prognostic factors during ECP treatment, the true involvement of these subpopulations is up to date, unclear. In particular Cyclosporine A might inhibit the production of Tregs and probably, other biological markers must be individuated.

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive Tregs

This study determines levels of regulatory T cells (T(regs)), naive T(regs), immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)-infected patients receiving prolonged highly active anti-retroviral therapy (HAART) who have known thymic output, and explores if naive T(regs) may represent recent thymic emigrant T(regs). HIV-infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T(regs) (CD3(+)CD4(+)CD25(+)CD127(low)), naive T(regs) (CD3(+)CD4(+)CD25(+)CD45RA(+)) and activation markers (CD38(+)human leucocyte antigen D-related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T(REC)) content in CD4(+) cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T(regs) were significantly higher in HIV-infected patients compared with controls, both after 1 and 5 years of HAART (P<0.001), despite fully suppressed HIV-RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T(regs) were elevated significantly in HIV-infected patients (P<0.001) and were associated with thymic output measured as the T(REC) frequency in CD4(+) cells (P=0.038). In summary, T(reg) levels in HIV-infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T(regs) may contribute to higher T(reg) levels in HIV-infection.

W1020 Outcome of Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis Is Regulated By CD4+Cd25+ Treg Production of Fgl2

W1020 Outcome of Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis Is Regulated By CD4+Cd25+ Treg Production of Fgl2

Expanded murine regulatory T cells: Analysis of phenotype and function in contact hypersensitivity reactions

Regulatory T cells (Treg) exert suppressive functions in the periphery of the body for the maintenance of tolerance. The functional analysis of Treg is hampered by the fact that only small numbers (5%–10% among the CD4 + T cells) of Treg exist in peripheral blood and tedious isolation methods further reduce the yield of high-purity Treg. We therefore set out to expand isolated murine Treg in ex vivo cultures with help of anti-CD3/anti-CD28 antibodies in the presence of IL-2. Our expansion-protocol described herein resulted in a 200-fold expansion of Treg and does not involve feeder cells, beads or other cellular compounds that would interfere with further in vivo use of the expanded cells. Expanded Treg could even be stored in liquid nitrogen and thawed without loss of function. Functional analysis revealed that expanded Treg are superior suppressors of T cell functions in vitro and in vivo and when applied in a model for contact hypersensitivity reactions, Treg were able to suppress the ear swelling reaction significantly. Thereby the expanded Treg home to secondary lymphoid organs in similar manner as observed for freshly isolated Treg. Accordingly, the expansion procedure does not effect the expression of specific homing markers. Thus, this protocol will facilitate the production of Treg as an “off-the-shelf reagent” and offers the possibility to explore the application of Treg as a cellular therapeutic in several allergic and autoimmune diseases.

Effects of different immunosuppressive regimens on regulatory T-cells in noninflamed colon of liver transplant recipients

Regulatory T-cells (Treg) are natural suppressors of autoimmunity. Previous studies indicate that immunosuppressive drugs, especially calcineurin-inhibitors, may interfere with Treg homeostasis. Inflammatory bowel disease (IBD) can relapse or develop de novo after liver transplantation. IBD is associated with a relative deficiency of Treg. The aim of this study was to determine the effect of long-term immunosuppression on the presence of Treg in the noninflamed colonic mucosa of liver transplant recipients. Colonic biopsies of normal mucosa of 36 liver transplant recipients on different types of immunosuppression and 11 controls were studied. Treg marker Foxp3 and Treg products transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) were studied by quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry. TGF-beta-induced Smad-protein 3 and 7 were studied by Q-PCR. No significant differences between controls and patients were observed in IL-10, TGF-beta, and Smad expression. Mucosal Foxp3 mRNA levels and Foxp3+CD3+ cells were significantly reduced in transplant recipients using prednisone/azathioprine/tacrolimus compared with controls but no direct relationship between Foxp3 expression and 1 specific drug was detected. These results challenge the hypothesis that calcineurin-induced reduction of Treg or TGF-beta expression predisposes nontransplanted tissue to inflammation, but indicate that combined immunosuppression hampers Treg development in the intestine.

The role of CTLA-4 in Treg-mediated suppression of T cell LIP (92.6)

Abstract Regulatory T cells are known to express CTLA-4 constitutively, however the role this inhibitory signaling molecule plays in Treg homeostasis and suppression of T cell responses is not well defined. Published reports suggest CTLA-4 expression is associated with Treg production of immunosuppressive cytokines TGF-β and IL-10 and is essential for cell-to-cell-suppression in vitro. Determining what role CTLA-4 expression on Tregs plays in vivo is complicated, however, due to differential expression on regulatory and non-regulatory T cells and development of fatal lymphoproliferative disease in CTLA-4-deficient mice. Recently published reports suggest blockade of CTLA-4 on Tregs abrogates their in vivo function, allowing the generation of effective anti-viral and anti-tumor T cell responses and/or emergence of autoimmune disease. Potential Treg suppressive mechanisms include CTLA-4-to-DC outside-in signaling through B7 and resultant upregulation/activation of tryptophan catabolism and suppression of T cell function/viability. It is thought naturally occurring (thymus-derived) Tregs may function this way to prevent activation and expansion of potentially pathogenic T cell clones in response to infectious stimuli. Our preliminary data suggests outside-in signaling plays a role in suppression of spontaneous T cell LIP by CD4+CD25+Foxp3+ cells (TR). Follow-up experiments will explore this suppressive mechanism in the context of T cell LIP and CD4 T cell LIP-induced autoimmunity.

Cytokine-induced IL-10–secreting CD8 T cells represent a phenotypically distinct suppressor T-cell lineage

AbstractPopulations of regulatory T cells (Tregs) control autoimmune and allergic immunopathology induced by self or foreign antigens. Several types of CD4+ MHC class II–restricted Treg populations have been characterized, but the biology of CD8+, MHC class I–restricted Tregs is less understood. We show here that CD8+ Tregs are rapidly generated in the presence of IL-4 and IL-12, produce IL-10, and exhibit a unique cell-surface phenotype with coexpression of activation and naive cell-associated markers. They block activation of naive or effector T cells and suppress IgG/IgE antibody responses and graft-versus-host disease in vivo. Suppression is dependent on cell contact and mediated by direct T-cell–T-cell interaction that antagonizes T-cell–receptor (TCR) signals. The data establish the existence of a CD8 T-cell suppressor effector subset distinct in both phenotype and function from T cytotoxic 1 (Tc1) and Tc2 cells. Production of such CD8 Tregs has potential for cell-based therapy of CD4 or CD8 T-cell–mediated disease.

CD4 +CD25 + T regulatory cells inhibit cytotoxic activity of CTL and NK cells in humans—impact of immunosenescence

We hypothesized that advanced age and medical conditions had an impact on the accumulation of CD4 +CD25 + T regulatory cells (Treg), which in turn could deteriorate cytotoxic activity of CD8 + T and NK cells. Volunteers were divided according to the Senieur Protocol into healthy young and elderly and non-healthy young and elderly subjects. The numbers of Treg cells in peripheral blood, their influence on CD8 + T and NK cells and production of IL2 as well as apoptosis intensity of Treg cells were measured. The number of Treg cells was higher in both elderly groups than in respective young ones. Compared to healthy subjects, those with medical conditions were revealed to have higher numbers of Treg cells. In addition, the highest accumulation of Treg cells in non-healthy elderly could be a result of their resistance to undergo apoptosis. The frequency of Treg cells correlated inversely with the activity of autologous cytotoxic cells in PBMC and production of IL2 by autologous CD4 +CD25 − Th cells. Thus, these parameters were the most highly decreased in non-healthy subjects, notably in the elderly. However, these parameters improved in the cultures of pure sorted cells. The only subset capable of decreasing them to the levels noted in PBMC when added back was Treg cells, which proved the link between the number of Treg cells, cytotoxic activity and production of IL2. Concluding, we found that Treg accumulated as a result of ageing and/or medical conditions were capable of decreasing cytotoxic activity of CD8 + T and NK cells and production of IL2.

Antibody-Induced Transplantation Tolerance That Is Dependent on Thymus-Derived Regulatory T Cells

Targeting of the CD45RB isoform by mAb (anti-CD45RB) effectively induces donor-specific tolerance to allografts. The immunological mechanisms underlying the tolerant state remain unclear although some studies have suggested the involvement of regulatory T cells (T-regs). Although their generative pathway remains undefined, tolerance promoting T-regs induced by systemic anti-CD45RB treatment have been assumed to originate in the peripheral immune system. We demonstrate herein that separable effects on the peripheral and central immune compartments mediate graft survival induced by anti-CD45RB administration. In the absence of the thymus, anti-CD45RB therapy is not tolerogenic though it retains peripheral immunosuppressive activity. The thymus is required for anti-CD45RB to produce indefinite graft survival and donor-specific tolerance, and this effect is accomplished through thymic production of donor-specific T-regs. These data reveal for the first time an Ab-based tolerance regimen that relies on the central tolerance pathway.

NFκB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation

CD4+CD25+ regulatory T cells (T(reg)) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on T(reg) has been shown to be a key player of regulating T(reg)-mediated suppression. A recent study reports that NF-kappaB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of T(reg) but not for its suppression capacity. In this report, we have shown that T(reg) from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive T(reg) proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type T(reg). Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62L(low) T(reg) both in thymus and periphery than their wild-type littermates. This CD62(low) subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.

Augmented Induction of CD4+CD25+ Treg using Monoclonal Antibodies to CD200R

CD200 is a transmembrane protein delivering immunoregulatory signals after engagement of CD200R. A family of CD200Rs exist (CD200R1-4) with different tissue expression and functional activity. In the presence of anti-CD200R2/3 monoclonal antibodies (mAbs), bone-marrow cells cultured in the presence of (interleukin [IL]-4+granulocyte-macrophage colony-stimulating factor) differentiate into dendritic cells (DCs), which induce CD4+CD25+ Treg. The effect of these mAbs on Treg induced in anti-CD3 activated thymocyte cultures is unknown. BL/6 bone-marrow cells were cultured with GMC-SF and IL-4 in the presence/absence of anti-CD200Rs to generate DCs that induced Treg in C3H lymph-node T cells. Treg were also induced in anti-CD3/CD28-activated C3H thymocytes. Treg activity was assayed by (1) suppression of mixed leukocyte culture (MLC) in cultures using C3H stimulator spleen cells and BL/6 stimulator cells, and (2) by expression of the transcription factor, Foxp3. Addition of anti-CD200R2/3 mAbs (but not anti-CD200R1) to bone-marrow cultures led to generation of DCs that induced a CD4+CD25+ (Treg) population inhibiting MLCs (C3H cells stimulated with C57BL/6 cells) in a cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and transforming growth factor (TGF)-beta-dependent manner. Anti-CD200R2, but not anti-R1/R3, augmented induction of Foxp3-expressing Treg from anti-CD3/CD28 activated thymocytes. Suppression in MLCs by anti-CD200R1 mAbs was dependent on IL-10 and TGF-beta. Unlike anti-CD200R1, anti-CD200R2 both promotes development of DCs with capacity to induce Treg and directly augments thymocyte production of Treg.