scholarly journals 650 Target the activin receptor 1c on CD4+ T cells to achieve anti-tumor therapeutic effects

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A679-A679
Author(s):  
Ying Zheng ◽  
Andriana Lebid ◽  
Andrew Pardoll ◽  
Juan Fu ◽  
Chirag Patel ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Activin A ◽  
Pcr Analysis ◽  
Therapeutic Effects ◽  
Wild Type ◽  
Cd4 Cells ◽  
Activin Receptor ◽  
Tumor Bearing

BackgroundActivins, members of the transforming growth factor-ß (TGF-ß) superfamily, were isolated and identified in endocrine system, and have been widely studied in endocrine-related cancers,1 2 but not substantially in the context of immune system and endocrine-unrelated cancers.3–5 It has been reported that upon binding to the receptors, activins cause the intracellular recruitment and phosphorylation of smad proteins, which mediate the expression of Foxp3.6–9 Therefore, we hypothesized that the blockade of the interaction of activins and their receptors will inhibit the activins-mediated Foxp3 induction in CD4+ T cells, thus modify the immune suppressive tumor microenvironment and achieve the goal of cancer immunotherapy.MethodsELISA (enzyme-linked immunosorbent assay) has been performed to determine the plasma level of Activin A in tumor-bearing mice and cancer patients. In vitro iTreg (induced regulatory T cells) differentiation has been done to naïve CD4+ cells isolated from wild type mice in the presence or absence of Activin A, and the percentage of Foxp3+ cells was demonstrated by flow cytometric analysis. qRT-PCR analysis has been conducted to determine the mRNA level of activin receptor isotypes in the immune subpopulations sorted from Foxp3-YFP mice. In the end, in vivo subcutaneous transplanted tumor studies have been done to evaluate the anti-tumor therapeutic effects of activin-receptor 1c blockade.ResultsWe show here that tumor-bearing mice had elevated Activin A levels, which correlated directly with tumor burden. Likewise, cancer patients had elevated plasma Activin A compared to healthy controls. Importantly, our in vitro studies suggested that Activin A promoted differentiation of conventional CD4+ cells into Foxp3-expressing induced Tregs, especially when TGF-ß was limited. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1C (Acvr1c) was uniquely expressed on Tregs and was highly upregulated during iTreg differentiation. Mice deficient in Acvr1c were more resistant to cancer progression compared to wild type mice. This phenotype correlated with reduced expression of the FoxP3 transcription factor in CD4+ cells. Similar phenomena were observed when we treated the mice with anti-Acvr1c antibody after tumor inoculation. This anti-tumor therapeutic effect was more significant when anti-Acvr1c antibody was administrated in combination with anti-PD-1 antibody.ConclusionsBlocking Activin A signaling through its receptor 1c is a promising and disease-specific strategy for preventing the accumulation of immunosuppressive iTregs in cancer. Hence it represents a potential candidate for cancer immunotherapy.AcknowledgementsThis research is supported by the Bloomberg-Kimmel Institute (Immunometabolism Program & Immune Modulation Program), the Melanoma Research Alliance, the NIH (RO1AI099300, RO1AI089830, and R01AI137046), and The DoD (PC130767).ReferencesRisbridger GP, Schmitt JF, Robertson DM. Activins and inhibins in endocrine and other tumors. Endocr Rev 2001;22(6):836–858.Cui X, et al. Perspectives of small molecule inhibitors of activin receptor-like kinase in anti-tumor treatment and stem cell differentiation (Review). Mol Med Rep 2019;19(6):5053–5062.Michael IP, et al. ALK7 signaling manifests a homeostatic tissue barrier that is abrogated during tumorigenesis and metastasis. Dev Cell 2019;49(3):409–424.Wu B, et al. The TGF-ß superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation. Immunity 2021;54(2):308–323.Antsiferova M, et al. Activin promotes skin carcinogenesis by attraction and reprogramming of macrophages. MBO Mol Med 2017;9(1):27–45.Tsuchida K, et al. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol Cell Endocrinol 2004;220(1–2):59–65.Khalil AM, et al. Differential binding activity of TGF-ß family proteins to select TGF-ß receptors. J Pharmacol Exp Ther 2016;358(3):423–430.Huber S, et al. Activin a promotes the TGF-beta-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells. J Immunol 2009;182(8):4633–4640.Iizuka-Koga M, et al. Induction and maintenance of regulatory T cells by transcription factors and epigenetic modifications. J Autoimmun 2017;83:113–121.Ethics ApprovalAll animal experiments were performed under protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC).

scholarly journals Conversion of CD4+ CD25− cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus

2005 ◽  
Vol 201 (1) ◽  
pp. 127-137 ◽  
Author(s):  
Shuang Liang ◽  
Pascale Alard ◽  
Yuan Zhao ◽  
Sarah Parnell ◽  
Sherry L. Clark ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Critical Role ◽  
Cd4 Cells ◽  
Natural Conditions ◽  
Foxp3 Mrna ◽  
Proliferation In Vitro ◽  
Regulatory Phenotype

The CD4+ CD25+ regulatory T cells play a critical role in controlling autoimmunity, but little is known about their development and maintenance. In this study, we investigated whether CD4+ CD25− cells can convert to CD4+ CD25+ regulatory T cells in vivo under natural conditions. CD4+ CD25− cells from CD45.1+ mice were sorted and transferred into congenic CD45.2+ mice. Converted CD4+ CD25+ cells could be detected in lymphoid organs as early as 1 wk after transfer and by 6 wk after transfer, 5–12% of transferred CD4+ cells expressed CD25. Converted CD4+ CD25+ cells themselves failed to proliferate after stimulation, but could suppress proliferation of responder cells in vitro, and also expressed high levels of Foxp3 mRNA. In addition, CD4+ CD25− cells transferred into thymectomized congenic mice converted to CD4+ CD25+ cells that also suppressed responder cell proliferation in vitro, and expressed high levels of Foxp3 mRNA. Finally, CD4+ CD25− cells transferred into B7−/− mice failed to convert into CD4+ CD25+ cells that exhibit the regulatory phenotype. These data indicate that CD4+ CD25− cells convert into CD4+ CD25+ regulatory T cells spontaneously in vivo and suggest that this conversion process could contribute significantly to the maintenance of the peripheral CD4+ CD25+ regulatory T cell population.

scholarly journals Efficient Therapeutic Function and Mechanisms of Human Polyclonal CD8+CD103+Foxp3+ Regulatory T Cells on Collagen-Induced Arthritis in Mice

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Juan Sun ◽  
Yiming Yang ◽  
Xiaona Huo ◽  
Beibei Zhu ◽  
Zhenhua Li ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
Regulatory T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Therapeutic Effects ◽  
Igg Antibody ◽  
Th1 Cell ◽  
Inflammatory Microenvironment

Objective. To investigate the potential therapeutic effect in a rheumatoid arthritis model of stable human CD8+ regulatory T cells (hCD8+Tregs) induced by TGF-β1 and rapamycin (RAPA) in vitro. Methods. Human CD8+T cells were isolated from human peripheral blood mononuclear cells and induced/expanded with TGF-β1 and RAPA along with anti-CD3/28 beads and IL-2 in vitro and harvested as hCD8+Tregs. The phenotypes, suppressive characteristics, and stability of the hCD8+Tregs in an inflammatory microenvironment were examined in vitro. Human CD8+Tregs were transfused into an acollagen-induced arthritis (CIA) mouse model, and their therapeutic effects and related mechanisms were investigated. Results. Human CD8+Tregs induced by TGF-β1/RAPA showed high expression of Foxp3 and CD103, exhibited vigorous suppression ability, and were stable in inflammatory microenvironments. In CIA mice, the clinical scores, levels of anti-collagen IgG antibody, and cartilage destruction were significantly reduced after adoptive transfusion with hCD8+Tregs. Moreover, hCD8+Treg treatment significantly reduced the number of Th17 cells, increased the number of CD4+IFN-γ+T cells, and produced self CD4+Foxp3+Tregs in vivo. In an in vitro cell coculture assay, hCD8+Tregs significantly inhibited mouse CD4+ effector T cell proliferation, induced mouse CD4+Foxp3+Treg and CD4+IFN-γ+Th1 cell production, reduced Th17 cell development, and downregulated CD80/86 expression on mature DCs (mDCs). Conclusion. TGF-β1/RAPA can induce hCD8+Tregs with stable suppressive characteristics, which could significantly alleviate the severity of CIA based on their stable suppressive ability in an inflammatory microenvironment and further influence the function of other downstream cell subtypes. Human CD8+Tregs might be a therapeutic strategy for rheumatoid arthritis.

scholarly journals Rabbit ATG but not horse ATG promotes expansion of functional CD4+CD25highFOXP3+ regulatory T cells in vitro

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3675-3683 ◽  
Author(s):  
Xingmin Feng ◽  
Sachiko Kajigaya ◽  
Elena E. Solomou ◽  
Keyvan Keyvanfar ◽  
Xiuli Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Expression Patterns ◽  
Cell Number ◽  
Therapeutic Effects ◽  
Peripheral Blood Mononuclear ◽  
Activation Markers

Abstract Regulatory T cells (Treg) play important roles in suppressing immune responses and maintaining tolerance. Rabbit antithymocyte globulin (rATG) and horse ATG (hATG) are widely used in the treatment of immune-mediated syndromes, but their effects on Treg are unknown. We show here that in vitro culture of normal human peripheral blood mononuclear cells (PBMCs) with a low-dose rATG resulted in marked expansion of functional Treg by converting CD4+CD25− T cells to CD4+CD25+ T cells. hATG did not expand but rather decreased Treg. Immuno-blot showed increased expression of FOXP3 and NFAT1 in CD4+CD25− and CD4+CD25+ T cells exposed to rATG. PBMCs treated with rATG displayed increased interleukin-10 in culture supernatants than those treated with hATG. Furthermore, rATG and hATG showed differences in their potential to stimulate CD4+ T cells as examined using different activation markers. Microarray revealed that rATG induced markedly different gene-expression patterns in PBMCs, compared with hATG-treated or untreated PBMCs. Our findings indicate that rATG expanded Treg, probably through transcriptional regulation by enhanced NFAT1 expression, in turn conferring CD4+CD25− T cell FOXP3 expression and regulatory activity. The therapeutic effects of rATG may occur not only because of lymphocyte depletion but also enhanced Treg cell number and function.

scholarly journals Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue

2007 ◽  
Vol 204 (4) ◽  
pp. 723-734 ◽  
Author(s):  
Jessica R. Kocks ◽  
Ana Clara Marques Davalos-Misslitz ◽  
Gabriele Hintzen ◽  
Lars Ohl ◽  
Reinhold Förster
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Lymph Nodes ◽  
Lymphoid Tissue ◽  
Wild Type ◽  
Peripheral Lymph ◽  
Bronchial Lymph Node ◽  
Bone Marrow Chimeras ◽  
Deficient Mice

Presence and extent of bronchus-associated lymphoid tissue (BALT) is subject to considerable variations between species and is only occasionally observed in lungs of mice. Here we demonstrate that mice deficient for the chemokine receptor CCR7 regularly develop highly organized BALT. These structures were not present at birth but were detectable from day 5 onwards. Analyzing CCR7−/−/wild-type bone marrow chimeras, we demonstrate that the development of BALT is caused by alterations of the hematopoietic system in CCR7-deficient mice. These observations together with the finding that CCR7-deficient mice posses dramatically reduced numbers of regulatory T cells (T reg cells) in the lung-draining bronchial lymph node suggest that BALT formation might be caused by disabled in situ function of T reg cells. Indeed, although adoptive transfer of wild-type T reg cells to CCR7-deficient recipients resulted in a profound reduction of BALT formation, neither naive wild-type T cells nor T reg cells from CCR7−/− donors impair BALT generation. Furthermore, we provide evidence that CCR7-deficient T reg cells, although strongly impaired in homing to peripheral lymph nodes, are fully effective in vitro. Thus our data reveal a CCR7-dependent homing of T reg cells to peripheral lymph nodes in conjunction with a role for these cells in controlling BALT formation.

scholarly journals A secreted Echinococcus multilocularis activin A homologue promotes regulatory T cell expansion

2019 ◽  
Author(s):  
Justin Komguep Nono ◽  
Manfred B. Lutz ◽  
Klaus Brehm
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Larval Stage ◽  
Echinococcus Multilocularis ◽  
Alveolar Echinococcosis ◽  
Activin A ◽  
Dependent Manner ◽  
Cell Type

ABSTRACTBackgroundAlveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis remain elusive so far.Methodology/Principal findingsWe herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-β-dependent manner. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-β (TGF-β)/activin subfamily members. EmACT obtained from heterologous expression promoted host TGF-β-driven CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT in the in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment.Conclusions/SignificanceIn conclusion, we herein show that E. multilocularis larvae secrete a factor with clear structural and functional homologies to mammalian activin A. Like its mammalian homolog, this protein induces the secretion of IL-10 by T-cells and contributes to the expansion of TGF-β-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation.AUTHOR SUMMARYThe metacestode larval stage of the tapeworm E. multilocularis grows infiltratively, like a malignant tumor, within the organs of its human host, thus causing the lethal disease alveolar echinococcosis (AE). Immunosuppression plays an important role in both survival and proliferation of the metacestode, which mainly depends on factors that are released by the parasite. These parasite-derived molecules are potential targets for developing new anti-echinococcosis drugs and/or improving the effectiveness of current therapies. Additionally, an optimized use of such factors could help minimize pathologies resulting from over-reactive immune responses, like allergies and autoimmune diseases. The authors herein demonstrate that the E. multilocularis metacestode releases a protein, EmACT, with significant homology to activin A, a cytokine that might support host TGF-β in its ability to induce the generation of immunosuppressive regulatory T-cells (Treg) in mammals. Like its mammalian counterpart, EmACT was associated with the expansion of TGF-β-induced Treg and stimulated the release of elevated amounts of immunosuppressive IL-10 by CD4+ T-cells. The authors also demonstrate that Treg are locally expanded by the metacestode during an infection of mice. These data confirm an important role of Treg for parasite establishment and growth during AE and suggest a potential role of EmACT in the expansion of these immunosuppressive cells around the parasite.

scholarly journals Essential Roles of CD8+CD122+ Regulatory T Cells in the Maintenance of T Cell Homeostasis

2004 ◽  
Vol 200 (9) ◽  
pp. 1123-1134 ◽  
Author(s):  
Muhaimin Rifa'i ◽  
Yoshiyuki Kawamoto ◽  
Izumi Nakashima ◽  
Haruhiko Suzuki
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Wild Type ◽  
T Cell Homeostasis ◽  
Activated T Cells ◽  
Naturally Occurring ◽  
Recombination Activating Gene ◽  
Β Chain

Regulation of immune system is of paramount importance to prevent immune attacks against self-components. Mice deficient in the interleukin (IL)-2/IL-15 receptor β chain, CD122, are model animals of such immune attacks and characteristically have a high number of abnormally activated T cells. Here, we show that the transfer of CD8+CD122+ cells into CD122-deficient neonates totally prevented the development of abnormal T cells. Furthermore, recombination activating gene–2−/− mice that received wild-type mice–derived CD8+CD122− cells died within 10 wk after cell transfer, indicating that normal CD8+CD122− cells become dangerously activated T cells in the absence of CD8+CD122+ T cells. CD8+CD122+ cells could control activated CD8+ or CD4+ T cells both in vivo and in vitro. Our results indicate that the CD8+CD122+ population includes naturally occurring CD8+ regulatory T cells that control potentially dangerous T cells.

Rabbit ATG but Not Horse ATG Promotes Expansion of Functional CD4+CD25highFoxP3 Regulatory T Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2312-2312
Author(s):  
Xingmin Feng ◽  
Elena E. Solomou ◽  
Keyvan Keyvanfar ◽  
Thomas Herndon ◽  
Jichun Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Autoimmune Diseases ◽  
In Vitro Culture ◽  
Mononuclear Cells ◽  
Foxp3 Expression ◽  
Therapeutic Effects ◽  
Lymphocyte Depletion

Abstract CD4+CD25+ regulatory T cells (Treg) are believed to play important roles in suppressing immune responses and maintaining tolerance. Treg have the ability to prevent the development of autoimmune diseases, graft rejection and graft versus host disease (GVHD) in mice and perhaps also in humans. Immunosuppressive drugs such as rabbit ATG (rATG), horse ATG (hATG) and cyclosporine A (CsA) are widely used in conditioning for transplantation and for the treatment of autoimmune diseases and GVHD, but their effects on Treg remain to be fully elucidated. Lopez et al. (J Am Soc Nephrol. 2006;17:2844–2853.) first reported that in vitro culture of human peripheral blood mononuclear cells (PBMC) with rATG resulted in expansion of CD4+CD25+ T cells. In the current study, we show that in vitro culture of normal human PBMC with low dose rATG (10 μg/ml) resulted in marked expansion of CD4+CD25high T cells (rATG 8.00±0.95% versus untreated 0.99±0.11%, n=10, p<0.0001) and CD4+CD25high FoxP3 T cells (rATG 2.24±0.11% versus untreated 0.90±0.10%, n=10, p<0.0001). rATG exposure converted CD4+CD25− T cells into CD4+CD25+ T cells, which proliferated better in comparison to CD4+CD25− T cells. In immunoblots of protein extracted from PBMC treated with rATG, there was increased expression of FoxP3 and nuclear factor of activated T cells (NFAT1) in CD4+CD25− and CD4+CD25+ T cells; rATG-induced NFAT1 expression correlated with FoxP3 expression. Expanded Treg suppressed autologous T-cell proliferation after T-cell receptor (TCR) stimulation by 64% when cultured with autologous PBMC at a 1:1 ratio, consistent with functional activity. Culture supernatants of PBMC treated with rATG showed increased levels of IL-10, compared with supernatants of PBMC treated with hATG or CsA, but no differences in INF-γ, IL-2, and IL-4. Unexpectedly, hATG did not expand but rather decreased Treg [For CD4+CD25high T cells (n=10): hATG 0.67±0.10% versus untreated 0.99±0.11%, p=0.0386; For CD4+CD25high FoxP3 T cells (n=10): hATG 0.62±0.08% versus untreated 0.90±0.10%, p=0.0435]. Furthermore, rATG and hATG showed differences in binding to lymphocytes, they contained different amounts of CD3 and TCRαβ antibodies, and they induced different activation states (expression of glucocorticoid-induced tumor necrosis factor receptor, cytotoxic T lymphocyte-associated antigen-4, and CD62L) for CD4+ T cells. In vitro, Treg expansion mediated by rATG occurred at submitogenic concentrations (< 50 μg/ml) rather than at lymphocyte depletion levels (50–100 μg/ml). Our findings suggest that rATG expanded Treg by converting CD4+CD25− T cells into CD4+CD25+ T cells, probably through a mechanism of transcription regulation, and enhanced NFAT1 expression, in turn conferring on CD4+CD25− T cells FoxP3 expression and regulatory activity. The therapeutic effects of rATG in the treatment of autoimmune diseases and GVHD may occur due to not only lymphocyte depletion but also enhanced Treg cell number and function. Our observation may provide a useful method for expansion of Treg in cellular treatment in transplantation and autoimmune diseases.

Rap1-GTP Promotes the Generation of Regulatory T Cells in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 110-110
Author(s):  
Lequn Li ◽  
Rebecca Greenwald ◽  
Esther M. Lafuente ◽  
Dimitrios Tzachanis ◽  
Alla Berezovskaya ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cd4 Cells

Abstract Elucidating the mechanisms that regulate T cell activation and tolerance in vivo will provide insights into the maintenance of physiologic homeostasis and will facilitate development novel strategies for induction of transplantation tolerance. Transient activation of the small GTPase Rap1 is one of the physiologic consequences of TCR ligation and is mandatory for β1 and β2 integrin-mediated adhesion. In contrast, sustained increase of active Rap1 inhibits T cell activation and IL-2 transcription in vitro. In order to understand the role of Rap1 in the immune responses of the intact host we generated transgenic (Tg) mice, which express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg mice had no defects in thymocyte development or maturation. Rap1E63-Tg thymocytes were capable of activating Ras and Erk1/2 and, compared to wild type (WT) thymocytes, displayed enhanced LFA-1:ICAM-1-mediated adhesion and increased proliferation in response to anti-CD3. Surprisingly, although lymph node and splenic CD4+ cells from the Rap1E63-Tg mice also displayed increased LFA-1:ICAM-1-mediated adhesion, they had significantly impaired activation of Erk1/2 and dramatically reduced proliferation and IL-2 production in response to anti-CD3 and WT antigen presenting cells (APC). The defective responses of CD4+ T cells suggest that Rap1E63-Tg mice may have impaired helper function in vivo. To address this issue we immunized Rap1E63-Tg and WT mice with TNP-OVA, a T-cell dependent antigen. Total IgG, IgG1 and IgG2a were dramatically reduced, indicating that Rap1E63-Tg mice had a defect in immunoglobulin class switching, consistent with defective helper T cell-dependent B cell activation. Because these results suggest that Rap1E63-Tg CD4+ cells may have an anergic phenotype, we tested rechallenge responses. We immunized Rap1E63-Tg and WT mice with TNP-OVA in vivo and subsequently we rechallenged T cells in vitro with WT APC pulsed with OVA. Compared with WT, Rap1E63-Tg T cells had dramatically reduced proliferation, IFN- γ and IL-2 production on rechallenge, findings consistent with T cell anergy. Using suppression subtraction hybridization we determined that Rap1E63 induced mRNA expression of CD103, a marker that defines a potent subset of regulatory T cells (Treg). Strikingly, Rap1E63-Tg mice had a 5-fold increase of CD103+CD25+CD4+ Treg compared to WT mice. Rap1E63-Tg CD103+CD25+CD4+ Treg expressed the highest level of Foxp3 among all T cell subsets and had the most potent inhibitory effect on proliferation and IL-2 production when added into cultures of WT CD4+CD25− cells. Importantly, removal of the CD103+ cells significantly restored Erk1/2 activation, proliferation and IL-2 production of Rap1E63-Tg CD4+ T cells. Generation of CD103+ Treg occurs after thymic development and requires encounter of peripheral autoantigen. Consistent with this, differences in CD103+ Treg were detected only between lymph node and splenic cells and not between thymocytes from Rap1E63-Tg and WT mice. Since generation of CD103+ Treg depends on the strength of TCR signal, these results suggest that by enhancing adhesion, active Rap1 regulates the generation of Treg. Moreover, these results provide evidence that active Rap1 is a potent negative regulator of immune responses in vivo and have significant implications for the development of immune-based therapies geared towards tolerance induction.

FcRγ-Dependent Facilitating Cells Are Direct Inducers of Regulatory T Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 65-65
Author(s):  
Kendra N. Taylor ◽  
Vivek R. Shinde Patil ◽  
Meredith Chittenden ◽  
Yolonda L. Colson
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Regulatory T Cell ◽  
In Vivo Studies ◽  
Pcr Analysis

Abstract Facilitating cells (FC) are CD8+/αβγδTCR− bone marrow-derived cells that promote allogeneic stem cell (SC) engraftment without graft vs. host disease (GVHD) and induce donor-specific transplantation tolerance. However, the mechanism of FC-mediated allogeneic SC engraftment is not known. We have previously demonstrated that allogeneic SC engraftment promoted by FC result in no clinical evidence of GVHD and is associated with increased number of CD4+25+ regulatory T cells post transplant compared to recipients of bone marrow T cells that develop severe GVHD. This is consistent with the hypothesis that FC facilitate allogeneic SC engraftment and induce tolerance through the induction of a regulatory T cell network. Here we report that CpG activation of toll-like receptor 9 (TLR9) on FC induce CD4+25− naïve T cell differentiation into CD4+25+ regulatory T cells. CpG stimulated and unstimulated CD8+αβγδTCR− FC isolated from bone marrow of C57/BL6 mice by flow cytometric cell sorting FC were co-cultured with splenic CD4+25− T cells for 5 days. CpG stimulated FC co-culture gave rise to CD4+25+ T cells as determined by mRNA expression of the CD4+25+ regulatory T cell marker, FoxP3. In contrast, unstimulated FC in co-culture did not induce regulatory T cells. Because FcRγ is the dominant ITAM receptor (immunoreceptor tyrosine-based activating motif), on FC and given that in vivo studies have demonstrated a requirement for FcRγ expression on FC, we hypothesized that FcRγ gene expression is increased within activated FC. FcRγ gene expression in CpG stimulated and unstimulated FC were compared by real-time PCR analysis. CpG-mediated TLR signaling within FC result in increased gene expression of FcRγ. Taken together, these studies demonstrate that CpG stimulated FC induce the generation of CD4+25+ regulatory T cells in vitro and CpG activation results in increased gene expression of FcRγ, suggesting a requirement for FcRγ signaling in FC-mediated induction of regulatory T cells. These findings provide the first mechanistic evidence that FC are direct inducers of regulatory T cells. Further characterization of cooperative TLR and FcRγ signaling pathways in FC will be critical to defining the mechanism of FC-mediated SC engraftment and the identification of potential therapeutic targets for the clinical induction of tolerance in the future.

Lenalidomide Decreases PD-1 Expression, Depletes Regulatory T-Cells and Improves Cellular Response to a Multiple Myeloma/Dendritic Cell Fusion Vaccine In Vitro

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 492-492 ◽  
Author(s):  
Katarina Luptakova ◽  
Brett Glotzbecker ◽  
Heidi Mills ◽  
Dina Stroopinsky ◽  
Baldev Vasir ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Fold Increase ◽  
Cellular Immunotherapy ◽  
Cd4 Cells

Abstract Abstract 492 Introduction: We have developed a cancer vaccine for multiple myeloma in which patient derived tumor cells are fused with dendritic cells (DCs) such that a broad array of tumor antigens are presented in the context of DC mediated costimulation. In clinical studies, we have demonstrated that vaccination results in the induction of anti-tumor immunity and disease response in a subset of patients. A fundamental challenge limiting the efficacy of cellular immunotherapy is the immunosuppressive milieu that characterizes patients with myeloma. We have previously reported that the PD-1/PDL-1 pathway plays an important role in suppressing T cell immunity in patients with myeloma, PD-1 expression is upregulated on T cells isolated from patients with multiple myeloma, and PD-1 blockade is associated with enhancement of T-cell response to the vaccine. Lenalidomide is a potent anti-myeloma agent whose activity may be linked, in part, to its immunomodulatory properties. We hypothesized that lenalidomide would augment the capacity to elicit anti-myeloma immunity. In our current study, we examined the effect of lenalidomide on T-cell activation and polarization, PD-1 signaling, and vaccine-induced responses in vitro. Methods and results: Peripheral blood mononuclear cells were cultured in media containing IL-2 with and without 1μM lenalidomide. The expression of cell surface molecules and intracellular cytokines was assessed using flow cytometry. Exposure of unstimulated T cells to lenalidomide resulted in a decrease in the percentage of CD4+ T cells expressing PD-1 (from 8.0% to 5.6%, p=0.04) and a 2 fold increase in T-cell proliferation as measured by incorporation of tritiated thymidine. We then examined the effect of lenalidomide on T cell activation by ligation of the costimulatory complex using antibodies directed against CD3 and anti CD28. Most notably, the upregulation of PD-1 by CD3/CD28 ligation was markedly decreased in the presence of lenalidomide as measured in CD4+ cells (from 26% to 15%, p<0.0001) and in CD8+ cells (from 16% to 10% p<0.01). Ligation of CD3/CD28 in the presence of lenalidomide resulted in greater degree of Th1 polarization as manifested by a 2 fold increase in the percentage of CD8+ T cells expressing IFNγ (p=0.02) and a decrease in the percentage of regulatory T-cells (CD4+CD25+FoxP3+) from 6.88% to 3.13% (p=0.02). In addition, the percentage of NK cells (CD3-CD56+) expressing IFNγ following CD3/CD28 ligation was 5-fold greater (p=0.03) in the presence of lenalidomide. Lastly, we studied the effect of lenalidomide on T-cells stimulated in vitro by the DC/myeloma fusion vaccine. DC/myeloma fusions were generated as previously described. Fusion mediated stimulation of autologous T cells in the presence of lenalidomide resulted in an increase in the percentage CD4+ and CD8+ T cells expressing IFNγ, (5.35% to 8.79%, p=0.06; and 6.37% to 9.85%, p=0.03, respectively). The proportion of regulatory T-cells decreased from 9.57% to 4.43% in the presence of lenalidomide (p<0.01). As with non-specific stimulation, PD-1 expression on CD4+ cells in the presence of lenalidomide decreased from 24% to 19%. In concert with these findings, exposure to lenalidomide resulted in increased cytotoxic T lymphocyte mediated lysis of autologous tumor targets (from 25% to 36%). Conclusions: In vitro exposure to lenalidomide results in enhanced T-cell activation in response to direct ligation of the co-stimulatory complex and stimulation by the DC/myeloma fusion vaccine. Exposure to lenalidomide suppresses T cell expression of PD-1 and expansion of regulatory T cells, 2 critical pathways responsible for tumor mediated immune suppression. To our knowledge, this is the first demonstration of an interaction between lenalidomide and the PD-1/PDL-1 pathway. These findings support the development of cellular immunotherapy in conjunction with lenalidomide, including its use with the DC/myeloma fusion vaccine. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document