Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3815-3821 ◽  
Author(s):  
Eric S. Yvon ◽  
Stephane Vigouroux ◽  
Raphael F. Rousseau ◽  
Ettore Biagi ◽  
Persis Amrolia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Notch Pathway ◽  
Cell Phenotype ◽  
Hematopoietic Stem ◽  
Notch Ligand

Abstract Graft-versus-host disease (GVHD) represents one of the major complications of allogeneic hematopoietic stem cell transplantation. Techniques to prevent GVHD have included ex vivo T-cell depletion of the graft or prolonged in vivo immunosuppression. Both reduce the frequency and severity of GVHD but also reduce T-cell-mediated graft-versus-malignancy effect, and increase the risk of infection. A major goal in transplantation is to prevent alloreactivity while preserving activity against tumors and infectious agents. We have used activation of the Notch pathway to try to generate T cells able to specifically regulate alloantigen responses. We used allogeneic Epstein-Barr virus lymphoblastoid B cells (EBV-LCLs) as stimulator cells. Such LCLs are excellent (allo) antigen-presenting cells and can be obtained in large numbers even from donors who have received extensive chemo/radiotherapy. We overexpressed a Notch ligand, Jagged-1, in these cells by adenoviral vector transduction. Stimulation of CD45RA+ naive T cells by Jagged-1 EBV-LCL reduces production of interferon-γ, interleukin-2, and interleukin-5, but up-regulates transforming growth factor-β1 synthesis, consistent with induction of a regulatory T-cell phenotype. Transfer of these T cells to fresh lymphocyte cultures inhibits proliferative and cytotoxic immune responses to the priming alloantigens while sparing responses to third-party stimulator cells. Notch activation in the presence of alloantigen-presenting cells may therefore be a means of inducing specific regulatory T cells while preserving other T-cell functionality. (Blood. 2003;102:3815-3821)

Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3222-3222
Author(s):  
Jenny Zilberberg ◽  
Kira Goldgirsh ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Clinical Situation ◽  
Post Transplantation ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Pcr Products

Abstract CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.

Functional Comparison of Freshly Isolated and Ex-Vivo Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Suppressing Murine Acute GvHD

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 812-812 ◽  
Author(s):  
Emanuela I Sega ◽  
Dennis Leveson-Gower ◽  
Vu H. Nguyen ◽  
Robert Negrin
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Acute Gvhd ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Allogeneic Bone ◽  
Cell Reactivity ◽  
Hematopoietic Stem

Abstract Graft versus host disease (GVHD) is a major complication of hematopoietic stem cell transplantation resulting from donor T cell reactivity against host tissue antigens. CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to be important in maintaining self tolerance and preventing autoimmunity. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown that donor Treg are able to suppress GVHD induced by donor allogeneic T cells and dramatically improve survival. Treg are rare and suppression of GVHD requires adequate numbers of Treg in relation to the number of conventional T cells (Tcon). To overcome this problem, expansion of Treg has been performed, however there has not been a head to head comparison of the function of expanded vs fresh Treg. Highly purified CD4+CD25+Foxp3+ T cells (>98% purity) were expanded using anti-CD3/anti-CD28 dynabeads and 1000 U/ml IL-2. Under these conditions, after five days Treg expanded up to 13 fold while maintaining high Foxp3 expression levels (85–90%). Longer expansion periods result in more T cell expansion but an overgrowth of Foxp3 negative T cells. In a mixed lymphocyte reaction assay, the ex-vivo expanded Treg efficiently suppressed the proliferation of alloreactive T cells. The expanded Treg were evaluated in an in vivo acute GVHD mouse model in direct comparison with freshly isolated Treg using a novel bioluminescent imaging assay that allowed for assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight gain, survival and GVHD score. Initial experiments show that, similar to freshly isolated Treg, the ex-vivo expanded Treg suppress GVHD symptoms and improve survival, although a greater number of expanded Treg were required comparable to freshly isolated Treg. The mean GVHD score for the Tcon alone group was 5.8±1.02. Fresh Treg added at 1:1 ratio decreased the GVHD score to 0.75±0.25 (p=0.0036). Ex-vivo expanded Treg demonstrated a dose-dependent decrease in GVHD score, although four times more expanded Treg were needed to obtain a similar reduction in GVHD score (0.50±0.5, p=0.0036). This observed difference in potency was not due to the ex-vivo expanded Treg being short-lived when infused in mice. Bioluminescence imaging of luciferase positive (luc+) cultured Treg showed the same in vivo persistence as freshly isolated Treg. The ability to expand ex-vivo generated Treg is greater than the difference in potency, making ex-vivo expanded Treg potentially a viable option for treatment of GVHD, however, increased ratios of Treg:Tcon are likely to be required.

Induction of allopeptide-specific human CD4+CD25+ regulatory T cells ex vivo

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2180-2186 ◽  
Author(s):  
Shuiping Jiang ◽  
Niels Camara ◽  
Giovanna Lombardi ◽  
Robert I. Lechler
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Line ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Cell Lineage ◽  
Human Leukocyte ◽  
Regulatory Cells ◽  
Antigen Specificity

Abstract Although CD4+CD25+ regulatory T cells are pivotal in the prevention of autoimmunity and appear to mediate transplantation tolerance, little is known concerning their antigen specificity. Here we describe the induction of a human CD4+CD25+ regulatory T-cell line specific for a defined peptide alloantigen (human leukocyte antigen A2 [HLA-A2] 138-170) by priming purified CD4+CD25+ cells ex vivo. The regulatory cells were anergic and retained their ability to suppress antigen-driven responses of CD4+CD25– cells. They inhibited not only interleukin 2 (IL-2) secretion by CD4+CD25– T cells specific for the same peptide but also direct alloresponse of naive CD4+CD25– T cells stimulated by semiallogeneic dendritic cells (DCs) in the presence of the peptide (“linked suppression”). They also suppressed the response of CD4+ T cells specific for viral and bacterial antigens. The suppressive T-cell line showed sustained high CD25 expression. These findings suggest that peripheral CD4+CD25+ regulatory cells are a precommitted cell lineage from which cells with specificity for non–self-peptides can be selected. This may pave the way for inducing and expanding peptide antigen-specific regulatory T cells ex vivo for cell therapy in transplantation, allergy, and autoimmune disease.

Human CD4+CD25+ cells: a naturally occurring population of regulatory T cells

Blood ◽  
2001 ◽  
Vol 98 (9) ◽  
pp. 2736-2744 ◽  
Author(s):  
Wan Fai Ng ◽  
Phillip J. Duggan ◽  
Frederique Ponchel ◽  
Giuseppe Matarese ◽  
Giovanna Lombardi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Interleukin 2 ◽  
Notch Pathway ◽  
Regulatory Function ◽  
Cell Contact ◽  
Transcriptional Level ◽  
Fetal Life

Abstract Despite thymic deletion of cells with specificity for self-antigens, autoreactive T cells are readily detectable in the normal T-cell repertoire. In recent years, a population of CD4+ T cells that constitutively express the interleukin-2 receptor-α chain, CD25, has been shown to play a pivotal role in the maintenance of self-tolerance in rodent models. This study investigated whether such a regulatory population exists in humans. A population of CD4+CD25+ T cells, taken from the peripheral blood of healthy individuals and phenotypically distinct from recently activated CD4+ T cells, was characterized. These cells were hyporesponsive to conventional T-cell stimuli and capable of suppressing the responses of CD4+CD25− T cells in vitro. Addition of exogenous interleukin-2 abrogated the hyporesponsiveness and suppressive effects of CD4+CD25+ cells. Suppression required cell-to-cell contact but did not appear to be via the inhibition of antigen-presenting cells. In addition, there were marked changes in the expression of Notch pathway molecules and their downstream signaling products at the transcriptional level, specifically in CD4+CD25+ cells, suggesting that this family of molecules plays a role in the regulatory function of CD4+CD25+ cells. Cells with similar phenotype and function were detected in umbilical venous blood from healthy newborn infants. These results suggest that CD4+CD25+ cells represent a population of regulatory T cells that arise during fetal life. Comparison with rodent CD4+CD25+ cells suggests that this population may play a key role in the prevention of autoimmune diseases in humans.

scholarly journals A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 251-259 ◽  
Author(s):  
Paul E. Zarek ◽  
Ching-Tai Huang ◽  
Eric R. Lutz ◽  
Jeanne Kowalski ◽  
Maureen R. Horton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Transforming Growth Factor ◽  
Interleukin 2 ◽  
Peripheral Tolerance ◽  
Negative Regulator ◽  
T Cell Anergy ◽  
A2a Receptor ◽  
Cell Anergy

Tissue-derived adenosine, acting via the adenosine A2A receptor (A2AR), is emerging as an important negative regulator of T-cell function. In this report, we demonstrate that A2AR stimulation not only inhibits the generation of adaptive effector T cells but also promotes the induction of adaptive regulatory T cells. In vitro, antigen recognition in the setting of A2AR engagement induces T-cell anergy, even in the presence of costimulation. T cells initially stimulated in the presence of an A2AR agonist fail to proliferate and produce interleukin-2 and interferon (IFN)-γ when rechallenged in the absence of A2AR stimulation. Likewise, in an in vivo model of autoimmunity, tissue-derived adenosine promotes anergy and abrogates tissue destruction. Indeed, A2AR stimulation inhibits interleukin-6 expression while enhancing the production of transforming growth factor-β. Accordingly, treating mice with A2AR agonists not only inhibits Th1 and Th17 effector cell generation but also promotes the generation of Foxp3+ and LAG-3+ regulatory T cells. In this regard, A2AR agonists fail to prevent autoimmunity by LAG-3−/− clonotypic T cells, implicating an important role for LAG-3 in adenosine-mediated peripheral tolerance. Overall, our findings demonstrate that extracellular adenosine stimulates the A2AR to promote long-term T-cell anergy and the generation of adaptive regulatory T cells.

scholarly journals Identification of an immediate Foxp3− precursor to Foxp3+ regulatory T cells in peripheral lymphoid organs of nonmanipulated mice

2010 ◽  
Vol 207 (7) ◽  
pp. 1393-1407 ◽  
Author(s):  
Sonja Schallenberg ◽  
Pei-Yun Tsai ◽  
Julia Riewaldt ◽  
Karsten Kretschmer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Transforming Growth Factor ◽  
Interleukin 2 ◽  
Transforming Growth Factor Β ◽  
Cell Receptor ◽  
Lymphoid Organs ◽  
Minor Role ◽  
A Minor

CD4+CD25+ regulatory T cells (T reg cells) expressing the transcription factor Foxp3 can be induced from peripheral T cell receptor (TCR) transgenic CD4+CD25−Foxp3− T cells stimulated with noninflammatory dendritic cells presenting low amounts of agonist cognate antigen. However, limited evidence exists for extra-thymic T reg cell generation from non-TCR transgenic T cells in unmanipulated mice. We compared events early during agonist-driven generation of Foxp3+ TCR transgenic T cells to polyclonal CD4+ T cell populations in unmanipulated mice. We identified an interleukin-2– and phosphatidylinositol-3-kinase–dependent precommitted Foxp3− precursor to Foxp3+ T reg cells in peripheral lymphoid organs. Transforming growth factor β signaling played a minor role in the generation and subsequent differentiation of these T reg precursor cells.

scholarly journals Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

2020 ◽  
Vol 4 (10) ◽  
pp. 2143-2157 ◽  
Author(s):  
Alak Manna ◽  
Timothy Kellett ◽  
Sonikpreet Aulakh ◽  
Laura J. Lewis-Tuffin ◽  
Navnita Dutta ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Lymphocytic Leukemia ◽  
Dependent Manner

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

scholarly journals Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT

2020 ◽  
Vol 11 ◽  
Author(s):  
Mahinbanu Mammadli ◽  
Weishan Huang ◽  
Rebecca Harris ◽  
Aisha Sultana ◽  
Ying Cheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Interleukin 2 ◽  
Treatment Strategies ◽  
Receptor Expression ◽  
Hematopoietic Stem ◽  
Target Organs ◽  
Donor T Cells ◽  
Inducible T Cell Kinase

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8+ and CD4+ donor T cells from Itk-/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk-/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

scholarly journals Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

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