Treg Cell Population Research Articles

Epigallocatechin-3-Gallate Ameliorates Experimental Autoimmune Encephalomyelitis by Altering Balance among CD4 + T-Cell Subsets

The green tea component epigallocatechin-3-gallate (EGCG) may be beneficial in autoimmune diseases; however, the underlying mechanisms are not well understood. In this study, we determined the effect of EGCG on the development of experimental autoimmune encephalomyelitis, an animal model for human multiple sclerosis, and the underlying mechanisms. Female C57BL/6 mice were fed EGCG (0%, 0.15%, 0.3%, and 0.6% in diet) for 30 days and then immunized with specific antigen myelin oligodendrocyte glycoprotein 35-55. EGCG dose dependently attenuated clinical symptoms and pathological features (leukocyte infiltration and demyelination) in the central nervous system and inhibited antigen-specific T-cell proliferation and delayed-type hypersensitivity skin response. We further showed that EGCG reduced production of interferon-γ, IL-17, IL-6, IL-1β, and tumor necrosis factor-α; decreased types 1 and 17 helper T cells (Th1 and Th17, respectively); and increased regulatory T-cell populations in lymph nodes, the spleen, and the central nervous system. Moreover, EGCG inhibited expression of transcription factors T-box expressed in T cells and retinoid-related orphan receptor-γt, the specific transcription factor for Th1 and Th17 differentiation, respectively; the plasma levels of intercellular adhesion molecule 1; and CCR6 expression in CD4(+) T cells. These results indicate that EGCG may attenuate experimental autoimmune encephalomyelitis autoimmune response by inhibiting immune cell infiltration and modulating the balance among pro- and anti-autoimmune CD4(+) T-cell subsets. Thus, we identified a novel mechanism that underlies EGCG's beneficial effect in autoimmune disease.

B Cell Depletion Enhances T Regulatory Cell Activity Essential in the Suppression of Arthritis

The efficacy of B cell-depletion therapy in rheumatoid arthritis has driven interest in understanding the mechanism. Because the decrease in autoantibodies in rheumatoid arthritis does not necessarily correlate with clinical outcome, other mechanisms may be operative. We previously reported that in proteoglycan-induced arthritis (PGIA), B cell-depletion inhibits autoreactive T cell responses. Recent studies in B cell-depletion therapy also indicate a role for B cells in suppressing regulatory mechanisms. In this study, we demonstrate that B cells inhibited both the expansion and function of T regulatory (Treg) cells in PGIA. Using an anti-CD20 mAb, we depleted B cells from mice with PGIA and assessed the Treg cell population. Compared to control Ab-treated mice, Treg cell percentages were elevated in B cell-depleted mice, with a higher proportion of CD4(+) T cells expressing Foxp3 and CD25. On a per-cell basis, CD4(+)CD25(+) cells from B cell-depleted mice expressed increased amounts of Foxp3 and were significantly more suppressive than those from control Ab-treated mice. The depletion of Treg cells with an anti-CD25 mAb concurrent with B cell-depletion therapy restored the severity of PGIA to levels equal to untreated mice. Although titers of autoantibodies did not recover to untreated levels, CD4(+) T cell recall responses to the immunizing Ag returned as measured by T cell proliferation and cytokine production. Thus, B cells have the capacity to regulate inflammatory responses by enhancing effector T cells along with suppressing Treg cells.

Prolonged renal allograft survival by donor interleukin-6 deficiency: association with decreased alloantibodies and increased intragraft T regulatory cells

Both humoral and cellular immune responses are involved in renal allograft rejection. Interleukin (IL)-6 is a regulatory cytokine for both B and Foxp3 (forkhead box P3)-expressing regulatory T (Treg) cells. This study was designed to investigate the impact of donor IL-6 production on renal allograft survival. Donor kidneys from IL-6 knockout (KO) vs. wild-type (WT) C57BL/6 mice (H-2(b)) were orthotopically transplanted to nephrotomized BALB/c mice (H-2(d)). Alloantibodies and Treg cells were examined by fluorescence-activated cell sorting analysis. Graft survival was determined by the time to graft failure. Here, we showed that a deficiency in IL-6 expression in donor kidneys significantly prolonged renal allograft survival compared with WT controls. IL-6 protein was upregulated in renal tubules and endothelium of renal allografts following rejection, which correlated with an increase in serum IL-6 compared with that in those receiving KO grafts or naive controls. The absence of graft-producing IL-6 or lower levels of serum IL-6 in the recipients receiving IL-6 KO allografts was associated with decreased circulating anti-graft alloantibodies and increased the percentage of intragraft CD4(+)CD25(+)Foxp3(+) Treg cells compared with those with WT allografts. In conclusion, the lack of graft-producing IL-6 significantly prolongs renal allograft survival, which is associated with reduced alloantibody production and/or increased intragraft Treg cell population, implying that targeting donor IL-6 may effectively prevent both humoral and cellular rejection of kidney transplants.

Mesenchymal Stem Cell-Mediated Immature Dendritic Cells Induce Regulatory T Cell-Based Immunosuppressive Effect

Immature dendritic cells (imDCs) are increasingly viewed as mediators of T-cell tolerance. We investigated factors enabling induction of regulatory T (Treg) cells through syngeneic imDC/mesenchymal stem cell (MSC) co-cultures in vitro and immunosuppressive effects of MSC-mediated imDCs (MSCs were excluded after 72 h co-culture) in vivo. In these experiments, we found that Foxp3+ Treg cell population remarkably increased after the T cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naïve T cells into Treg cells by MSCs and imDCs. In parallel, TGF-β secretion was markedly induced from the imDC+MSC+splenocyte culture supernatant to a significant level at 72-h co-culture, compared to the MSC or imDC+splenocyte co-culture. Based on these results, using a murine melanoma tumor model, we confirmed that the subcutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Consequently, these results suggested that immune tolerance with MSC-mediated DCs leads to immunosuppression induced by at least Foxp3-specific Treg cells. This tool may be useful in clinical trials due to the yet unknown side effects of stem cell therapy.

Down-regulation of Treg cells and up-regulation of TH1/TH2 cytokine ratio were induced by polysaccharide from Radix Glycyrrhizae in H22 hepatocarcinoma bearing mice.

Radix Glycyrrhizae polysaccharide (GP) possesses multiple pharmacological activities. However, the effect of GP on CD4+CD25+ regulatory T (Treg) cells has not been elucidated. This study aimed to investigate the effects of GP on Treg cells and Th1/Th2 cytokines in H22 hepatocarcinoma tumor-bearing mice. The results demonstrated that GP inhibits tumor progression. In the lymph nodes of the tumor microenvironment and spleen, the proportion of Treg cells was significantly higher in the tumor-bearing mice. GP administration down-regulated the population of Treg cells (P < 0.01) and decreased lymph node Foxp3 and IL-10 mRNA expression (P < 0.01). In addition, GP treatment decreased IL-10 and TGF-β level (P < 0.01) and increased IL-2 and IL-12p70 level in serum (P < 0.01). In conclusion, GP reduced the proportion of Treg cells and Foxp3 lowered expression in Treg cells, and up-regulated Th1/Th2 cytokine ratio in serum in the tumor bearing mice, which might partially cause the inhibition of tumor growth.

Idiopathic retroperitoneal fibrosis mimicking malignant lymphoma

We present a case of atypical idiopathic retroperitoneal fibrosis (iRPF) presenting as a large pelvic tumor, for which it proved difficult to exclude T-cell malignant lymphoma. Histopathological examination of biopsy material showed collagenous tissue and fat with an exuberant and predominant T-cell infiltrate, largely consisting of CD4(+) cells expressing the IL-2 receptor-α chain (CD25). Focal plasma cells were negative for the immunoglobulin G4 (IgG4) isotype. T-cell receptor gene rearrangement (TRGR) pattern showed a Gaussian distribution, in keeping with a polyclonal T-cell population. Awareness of the sometimes exuberant and predominant T-cell infiltrate in iRPF should lead to earlier consideration of this disorder. This is particularly the case where there is an atypically localized and/or extensive mass, for which early exclusion of monoclonality with TRGR may provide helpful. Immunohistochemical findings suggest that CD4(+) CD25(+) cells, which are part of a naturally occurring population of regulatory T-cells, may be involved in the pathogenesis of iRPF.

Isolation strategies of regulatory T cells for clinical trials: Phenotype, function, stability, and expansion capacity

Recent clinical results demonstrate the highly effective potency of regulatory T cells (Tregs) to control graft-versus-host disease (GvHD). In this presented study, we directly compared different Treg subpopulations in order to define the most promising Treg target cell population for cellular intervention studies with respect to their phenotype, functional properties, stability, and expansion capacity. Different Treg cell populations have been isolated from healthy donors and characterized by fluorescence activated cell sorting (FACS) analysis for their phenotypic marker and purity, functional properties by suppression assay, stability by Treg-specific demethylated region (TSDR) of the Foxp3 promoter and their invitro expansion capacity. The direct comparison of the respective Treg target cell populations identified CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs as the most promising Treg population for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function, and stability. The CD4(+)CD25(hi) Tregs qualified as the best candidate for invitro expansion combining a highly stable phenotype with strong suppressive potential and attractive cell yield after repetitive stimulation. The suppressive capacity of freshly isolated CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs is comparable to freshly isolated CD4(+)CD25(hi), but inferior to CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs. Invitro expansion of CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs resulted in cell populations with less suppressive potency compared with CD4(+)CD25(hi) expanded Tregs correlating well with a higher TSDR demethylation level. In conclusion, future clinical trials should favor CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs for direct Treg cell transfer, whereas CD4(+)CD25(hi) Tregs qualify as best candidate for invitro expansion.

Mechanisms of immune-tolerance to allergens on the mucosal surfaces

The immune system is a highly interactive network, which makes it’s decisions on the basis of all body tissues, infections, normal flora bacteria and almost any environmental agents. In recent years, regulatory T cells (TReg) cells have become a prime target for strategies aimed at inducing tolerance to ffod antigens. Immune tolerance in the context of allergy can be defined as persistence of efficacy following discontinuation of treatment, implying an altered allergen-specific memory T and B cell response. Various populations of TReg cells have been shown to play a central role and their identification as key regulators of immunological processes in peripheral tolerance to food- and aero-allergens has opened an important era in the prevention and treatment of allergic diseases. Both naturally occurring CD4+ CD25+ TReg cells and inducible populations of allergen-specific interleukin-10 (IL-10)-secreting T regulatory type 1 (Tr1) cells inhibit allergen-specific effector cells in experimental models. Skewing of allergen-specific effector T cells to a regulatory phenotype appears as a key event in the development of healthy immune response to allergens and successful outcome in allergen-specific immunotherapy. FoxP3+ CD4+CD25+ TReg cells and Tr1 cells contribute to the control of allergen-specific immune responses in several major ways, which can be summarized as suppression of dendritic cells that support the generation of effector T cells; suppression of effector Th1, Th2 and Th17 cells; suppression of allergen-specific IgE, induction of IgG4; suppression of mast cells, basophils and eosinophils; interaction with resident tissue cells and remodeling, suppression of effector T cell migration to tissues. Current strategies for drug development exploit these observations with the potential for preventive therapies and cure for allergic diseases.

Seminal Fluid Regulates Accumulation of FOXP3+ Regulatory T Cells in the Preimplantation Mouse Uterus Through Expanding the FOXP3+ Cell Pool and CCL19-Mediated Recruitment1

Regulatory T (Treg) cells facilitate maternal immune tolerance of the semiallogeneic conceptus in early pregnancy, but the origin and regulation of these cells at embryo implantation is unclear. During the preimplantation period, factors in the seminal fluid delivered at coitus cause expansion of a CD4(+)CD25(+) putative Treg cell population in the para-aortic lymph nodes draining the uterus. Using flow cytometry, immunohistochemistry, and real-time quantitative PCR (qPCR) for the signature Treg cell transcription factor FOXP3, we confirmed the identity of the expanded lymph node population as FOXP3(+) Treg cells and showed that this is accompanied by a comparable increase in the uterus of FOXP3(+) Treg cells and expression of Foxp3 mRNA by Day 3.5 postcoitum. Seminal plasma was necessary for uterine Treg cell accumulation, as mating with seminal vesicle-deficient males failed to elicit an increase in uterine Treg cells. Furthermore seminal fluid induced expression of mRNA encoding the Treg chemokine CCL19 (MIP3beta), which acts through the CCR7 receptor to regulate Treg cell recruitment and retention in peripheral tissues. Glandular and luminal epithelial cells were identified as the major cellular origins of uterine CCL19, and exposure to both seminal plasma and sperm was required for maximum expression. Together, these results indicate that Treg cells accumulate in the uterus prior to embryo implantation and that seminal fluid is a key regulator of the uterine Treg cell population, operating by both increasing the pool of available Treg cells and promoting their CCL19-mediated recruitment from the circulation into the implantation site.

The Role of Mouse Mesenchymal Stem Cells in Differentiation of Naive T-Cells into Anti-Inflammatory Regulatory T-Cell or Proinflammatory Helper T-Cell 17 Population

Bone marrow-derived mesenchymal stem cells (MSCs) modulate immune response and can produce significant levels of transforming growth factor-β (TGF-β) and interleukin-6 (IL-6). These 2 cytokines represent the key factors that reciprocally regulate the development and polarization of naive T-cells into regulatory T-cell (Treg) population or proinflammatory T helper 17 (Th17) cells. In the present study we demonstrate that MSCs and their products effectively regulate expression of transcription factors Foxp3 and RORγt and control the development of Tregs and Th17 cells in a population of alloantigen-activated mouse spleen cells or purified CD4(+)CD25(-) T-cells. The immunomodulatory effects of MSCs were more pronounced when these cells were stimulated to secrete TGF-β alone or TGF-β together with IL-6. Unstimulated MSCs produce TGF-β, but not IL-6, and the production of TGF-β can be further enhanced by the anti-inflammatory cytokines IL-10 or TGF-β. In the presence of proinflammatory cytokines, MSCs secrete significant levels of IL-6, in addition to a spontaneous production of TGF-β. MSCs producing TGF-β induced preferentially expression of Foxp3 and activation of Tregs, whereas MSC supernatants containing TGF-β together with IL-6 supported RORγt expression and development of Th17 cells. The effects of MSC supernatants were blocked by the inclusion of neutralization monoclonal antibody anti-TGF-β or anti-IL-6 into the culture system. The results showed that MSCs represent important players that reciprocally regulate the development and differentiation of uncommitted naive T-cells into anti-inflammatory Foxp3(+) Tregs or proinflammatory RORγt(+) Th17 cell population and thereby can modulate autoimmune, immunopathological, and transplantation reactions.

Autoimmune regulator (AIRE)-deficient CD8+CD28lowregulatory T lymphocytes fail to control experimental colitis

Mutations in the gene encoding the transcription factor autoimmune regulator (AIRE) are responsible for autoimmune polyendocrinopathy candidiasis ectodermal dystrophy syndrome. AIRE directs expression of tissue-restricted antigens in the thymic medulla and in lymph node stromal cells and thereby substantially contributes to induction of immunological tolerance to self-antigens. Data from experimental mouse models showed that AIRE deficiency leads to impaired deletion of autospecific T-cell precursors. However, a potential role for AIRE in the function of regulatory T-cell populations, which are known to play a central role in prevention of immunopathology, has remained elusive. Regulatory T cells of CD8(+)CD28(low) phenotype efficiently control immune responses in experimental autoimmune and colitis models in mice. Here we show that CD8(+)CD28(low) regulatory T lymphocytes from AIRE-deficient mice are transcriptionally and phenotypically normal and exert efficient suppression of in vitro immune responses, but completely fail to prevent experimental colitis in vivo. Our data therefore demonstrate that AIRE plays an important role in the in vivo function of a naturally occurring regulatory T-cell population.

Repeated Seminal Fluid Exposure Expands Regulatory T Cell Populations in Mice.

Regulatory T cells (Treg cells) are required to achieve successful allogeneic pregnancy through their actions in mediating maternal immune tolerance towards conceptus antigens at the time of embryo implantation. In mice, we have shown that seminal fluid exposure is a key factor in maximising the Treg cell pool available systemically and present in the implantation site. Both the seminal plasma and sperm components of seminal fluid contribute to Treg cell activation and proliferation through provision of alloantigens and the immune deviating cytokine transforming growth factor-beta (TGFB), as well as potentially other signalling agents. Additionally, seminal fluid induces uterine expression of chemokines such as CCL19 that facilitate Treg cell recruitment into the uterine endometrium in the peri-implantation period. In women, clinical studies have shown that regular coitus in the absence of barrier methods of contraception over at least a three month period can diminish the incidence of preeclampsia and improve reproductive outcomes. We thus hypothesised that in mice, repeated mating events expand the Treg cell pool beyond that present after a single mating event. To examine the effect of repeated seminal fluid exposure on the maternal Treg cell population, C57BL/6 (B6) female mice were mated once or four times (over a 4-6 week period) to vasectomised (VAS) BALB/c males, then killed on day 3.5 post coitum (pc). Treg cells were quantified by flow cytometry as CD3+CD4+CD25+FoxP3+ cells in the uterus-draining para-aortic lymph nodes, peripheral lymph nodes, spleen and blood. Four matings resulted in an approximately 2-fold increase in the size of the Treg cell population in para-aortic lymph nodes compared with no mating or one mating event. Increases were also seen in the cervical lymph nodes but not the spleen or blood. Surgical removal of the seminal vesicle gland to generate SVX Balb/c males abolished their capacity to elicit expansion of the Treg cell pool. Repeated syngeneic matings of B6 females with VAS B6 males also failed to elicit any further expansion compared with one mating. In conclusion, this study demonstrates that repeated exposure to seminal fluid can expand the Treg cell pool, and that seminal plasma and male alloantigens are involved in eliciting this response. Our findings begin to provide a mechanistic explanation for observations in humans that pregnancy complications are more prevalent after a short duration of sexual relationship or use of barrier methods of contraception. This research was supported by NHMRC Program ID453556 and Fellowship ID627017 awarded to SAR. (platform)

α-1-Antitrypsin Gene Delivery Reduces Inflammation, Increases T-Regulatory Cell Population Size and Prevents Islet Allograft Rejection

Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmid-derived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated "EF") alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline-injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure-function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.

Raising a Treg cell army

Three independent teams have devised ways to expand populations of Treg cells up to therapeutically useful quantities. The techniques pave the way for clinical testing of Treg cells for preventing immunological complications of transplantation.

Analysis of Treg Cell Population Alterations in the Peripheral Blood of Patients Treated Surgically for Ovarian Cancer - A Preliminary Report

Treg cells constitute the main cell population that enables cancer cells to evade immune surveillance. An alteration in the Treg cell population might correspond to the diminishment of the tumour mass in patients with cancer and could therefore be a useful marker of the intensity of the selective suppression of the host immune system and also of the degree of radicalism of a procedure. Certainly, it is well known that in order for anti-cancer therapy to succeed the proper immune response against cancer cells must be restored. Furthermore, monitoring the level of selective immune system suppression during cancer therapy might yield information that would support a decision to supplement standard therapy by immunotherapy or to increase the degree of radicalism of the applied therapy. We examined the Treg cell populations in the peripheral blood of a group of patients treated surgically for ovarian cancer. In each patient, the peripheral blood samples were collected both prior to and 1 day after the surgical procedure, and then again 5 days after the procedure. The presence of regulatory T cells in the samples was analyzed by means of flow cytometry. In our study, the percentages of FOXP3(+) cells in the subpopulation of CD4(+) T lymphocytes found in the peripheral blood of the patients before the surgical intervention were statistically significantly higher than those observed in the peripheral blood of these same patients after the surgical procedure. It would seem that the alteration in the Treg cell subpopulation could be a key factor in determining the status of the tumour microenvironment. Most likely, it could provide information about whether the proper level of anti-cancer immune response could be restored. The possibility of restoring the immune response may directly correspond to the degree of radicalism of the surgical intervention.

Vitamin D3 targets epidermal and dermal dendritic cells for induction of distinct regulatory T cells

The vitamin D metabolite 1,25(OH)2D3 (VitD3) is a potent immunosuppressive drug and, among others, is used for topical treatment of psoriasis. Aproposed mechanism of VitD3-mediated suppression is priming of dendritic cells (DCs) to induce regulatory T (Treg) cells. Currently, there is confusion about the phenotype of VitD3-induced Treg cells and the DC-derived molecules driving their development. We investigated Treg cell induction after VitD3 priming of 2 distinct skin DC subsets: Langerhans cells (LCs) and dermal dendritic cells (DDCs). LCs and DDCs primed with VitD3 were cocultured with allogeneic naive T cells. The phenotype and function of the DCs and induced T cells were analyzed. Both VitD3-primed DC subtypes induced T cells with regulatory activity. Unexpectedly, whereas the Treg cell populations generated by VitD3-primed LCs were CD25(hi)CD127(lo) forkhead box protein 3 (Foxp3)-positive cells, which meet the criteria of classical inducible Treg cells, theTcells developing in response to VitD3-primed DDCs were Foxp3(-) T(R)1 cells expressing IL-10. Inhibition experiments revealed that LC-derived TGF-β is a key factorin the induction of Foxp3(+) Treg cells, whereas DDC-derived IL-10 is important for the induction of IL-10(+) T(R)1 cells. Thus we report the novel finding that distinct but closely related DC subsets are differentially programmed by VitD3 to support development of either TGF-β-dependent Foxp3(+) Treg cells or IL-10-dependent IL-10(+) Treg cells.

Abstract LB-157: Overcoming T-regulatory effects towards developing a more effective immunotherapy targeting multiple myeloma

Abstract Recent progress in dendritic cell (DC) and T cell therapies has offered a new avenue in treating multiple myeloma (MM). We have reported that MM patients' DCs cannot effectively present MM antigens and, instead, preferrentially activate T regulatory cells (Tregs). Furthermore, the MM cells actively suppress patients' DC and T cell functions (Han et al. 2008 Mol Ther. 16, 269–279). Therefore, an effective immunotherapy must overcome immune tolerance to the cancer cells and effectively expand functional MM-specific effector cells. To obtain critical data about MM patient's immune status, a longitudinal study of Treg cells before and after treatments of twenty MM patients was conducted. Patients' bone marrow (BM) and peripheral blood (PB) specimens were collected in the first visit and the follow up visits during treatment. Treg cell populations were examined using antibodies against CD4, CD25, CD127 and Foxp3. At the same time, naïve versus memory phenotype was assessed using antibodies against CD45RA, CCR7 and CD27. Besides the above markers, the surface molecule KLRG1 reflecting T cell senescence and longivity was also detected by flow cytometry. Analysis of Tregs at different time points showed a clear trend of gradual increase in Treg population both in the BM and PB of the MM patients; compared with healthy donors, MM patients' Tregs expand more than two to three folds. Surface marker analysis suggests that the expanded Tregs are mostly memory effector cells, which coincides with a decrease in the ratio of naïve Tregs. Analysis of MM-specific T cell responses by examining effector cytokines and cytolytic activities against autologous tumor cells revealed that BM contained more cytotoxic T cells than did PB. Increased expansion of MM-specific immune effector cells in culture was observed when Treg activities were suppressed using various immune modulators. Further understanding of Treg-mediated cancer tolerance will be essential to develop clinically more effective immunotherapy towards improved survival of MM patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2011-LB-157

Mechanisms of tolerance

Mechanisms of tolerance

Activation of Th17/Th1 and Th1, but not Th17, is associated with the acute cardiac event in patients with acute coronary syndrome

Objective Th1 activation and regulatory T (Treg) cell suppression have been observed in acute coronary syndrome (ACS), including unstable angina (UA) and acute myocardial infarction (AMI). However, the role of Th17 cell or IL-17A remains controversial in ACS patients, and little is known about the role of recently discovered Th17/Th1 cells, a subset of Th17 cells, in coronary artery disease (CAD). The purpose of this study is to investigate functional changes of Th17/Th1, Th17, Th1, Th2 and Treg cells in ACS patients. Methods The contents of Th17/Th1, Th17, Th1, Th2 and Treg cells, related gene expression, and plasma cytokines from CAD and control patients with normal coronary arteries (NCA) were measured by flow cytometry, real-time quantitative PCR and ELISA. Results Th17/Th1 and Th1 cell contents and related gene expression (T-bet, IFN-γ, STAT4, RORγt, STAT3 and IL-17) were significantly increased in ACS patients, whereas plasma IFN-γ only increased in CAD patients. In contrast, Treg cell population, Foxp3 levels, and plasma TGF-β1 were decreased in ACS patients compared with stable angina (SA) and NCA patients. Conclusion The study showed activation of Th17/Th1 and Th1 cell in ACS patients, which may provide insight into the mechanisms underlying culprit plaque relevant T-cell activation in ACS patients.

Sustained expression of circulating human alpha-1 antitrypsin reduces inflammation, increases CD4+FoxP3+ Treg cell population and prevents signs of experimental autoimmune encephalomyelitis in mice

Alpha-1-antitrypsin (AAT) is the primary circulating serine protease inhibitor, and is known to exert potent anti-inflammatory effects and to inhibit the progression of several autoimmune diseases. In this study, transgenic mice that over-express surfactant-driven human (h)AAT on the C57BL/6 background were evaluated for resistance to MOG-35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), compared to WT C57BL/6 control mice. According to the results, sustained levels of circulating hAAT profoundly inhibited induction of clinical signs, inflammatory lesions and demyelination observed in WT mice with EAE, concomitant with enhanced levels of CD4+FoxP3+ Treg cells, reduced secretion of MOG peptide-induced pro-inflammatory cytokines, IL-17, IL-1β & IL-6, diminished expression of caspase-1 and enhanced expression of CCR6. These results implicate hAAT as a potent immunoregulatory agent worthy of further investigation as a potential therapy in human autoimmune diseases including multiple sclerosis.