Mechanisms of Immunosuppression By FVIII-Specific TCR Engineered Human Regulatory T Cells

Abstract Expanded antigen-specific engineered regulatory T cells (Tregs) have been proposed for potential clinical application for the treatment of undesirable immune responses, such as inhibitor responses in hemophilia A patients and autoimmune diseases. By providing an antigen-specific T-cell receptor (TCR) to polyclonal natural Tregs, we suggested that antigen-specific engineered Tregs would migrate specifically to particular target tissues and induce antigen-specific immune tolerance in the local milieu. Previously, we developed FVIII C2-specific Tregs using a long-term stabilization protocol in vitro and demonstrated that these stabilized engineered Tregs successfully modulated FVIII-specific T-cell- and B-cell immune responses. Herein, we examined the mechanism of suppression by antigen-specific engineered Tregs compared to polyclonal normal natural Tregs. Initially, we tested whether these FVIII-specific engineered Tregs were able to suppress neighboring activated T-cell effectors locally. We found that FVIII C2-specific Tregs strongly suppressed myelin basic protein (MBP)-specific T effectors by presentation of both specific antigens in same APC population. However, we also observed that C2-specific Tregs could suppress MBP-specific T effectors presented on different APCs. These results imply contactless suppressive function of C2-specific engineered Tregs. Using a modified trans-well suppression assay, in which physical distance and clear separation between Tregs and a set of T effectors was created, we found that C2-specific activated Tregs showed significant contactless suppression only when T effectors were also present. In addition, and confirming previous studies with polyclonal Tregs, suppression by FVIII-specific engineered Tregs could be overcome by increasing the dose of IL-2 in co-culture media. This suggests that Tregs act, in part, by usurping IL-2 needed by T effectors to proliferate. Surprisingly, neutralization of CTLA-4 did not interfere with FVIII C2-specific suppression of engineered Tregs in contrast to the reversal seen with anti-CD3e-driven non-specific immunosuppression. Our data strongly suggest that suppressive function of FVIII-specific engineered Tregs is not restricted to cell-to-cell contact. Rather cross-talk of engineered Tregs and T effectors potentially generate a contactless suppressive mechanism to suppress other FVIII-specific multiple effector cells in the local milieu for effective immune tolerance. Understanding the mechanism of contactless suppression mechanism should provide critical clues to develop more effective engineered Tregs as a therapeutic tool in hemophilia A. (Supported by NIH grants HL061883 and HL126727) Disclosures Kim: Henry Jackson Foundation: Other: patent filed. Zhang:Henry Jackson Foundation: Other: patent filed. Scott:Henry Jackson Foundation: Other: patent filed.

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FVIII-Specific TCR Engineered Human Regulatory T Cells Suppress the Production of FVIII-Specific Responses Vitro and In Vivo

Blood ◽

10.1182/blood.v128.22.2586.2586 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2586-2586

Author(s):

Yong Chan Kim ◽

Aihong Zhang ◽

Jeong-Heon Yoon ◽

David W. Scott

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Hemophilia A ◽

Cell Receptor ◽

Cell Contact ◽

Therapeutic Trial

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CD25+CD4+ Regulatory T Cells from the Peripheral Blood of Asymptomatic HIV-infected Individuals Regulate CD4+ and CD8+ HIV-specific T Cell Immune Responses In Vitro and Are Associated with Favorable Clinical Markers of Disease Status

Journal of Experimental Medicine ◽

10.1084/jem.20032069 ◽

2004 ◽

Vol 200 (3) ◽

pp. 331-343 ◽

Cited By ~ 338

Author(s):

Audrey L. Kinter ◽

Margaret Hennessey ◽

Alicia Bell ◽

Sarah Kern ◽

Yin Lin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Cd4 T Cells ◽

Cellular Proliferation ◽

Cell Contact ◽

Hiv Disease ◽

T Cell Immune Responses

Human immunodeficiency virus (HIV) disease is associated with loss of CD4+ T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4+ T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25+CD4+ regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25+CD4+ T cells significantly suppressed cellular proliferation and cytokine production by CD4+ and CD8+ T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-β independent. Individuals with strong HIV-specific CD25+ T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4+: CD8+ T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25+CD4+ T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.

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Regulatory T-cell: Regulator of Host Defense in Infection

Journal of Molecular Biology Research ◽

10.5539/jmbr.v7n1p9 ◽

2017 ◽

Vol 7 (1) ◽

pp. 9 ◽

Cited By ~ 2

Author(s):

Mousa Mohammadnia-Afrouzi ◽

Mehdi Shahbazi ◽

Sedigheh Baleghi Damavandi ◽

Ghasem Faghanzadeh Ganji ◽

Soheil Ebrahimpour

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Regulatory T Cell ◽

Critical Role ◽

The Body ◽

Cell Contact ◽

Infectious Agents ◽

Activated T Cells

Based on diverse activities and production of several cytokines, T lymphocytes and T helper cells are divided into Th1, Th2, Th17 and regulatory T-cell (T regs) subsets based on diverse activities and production of several cytokines. Infectious agents can escape from host by modulation of immune responses as effector T-cells and Tregs. Thus, regulatory T-cells play a critical role in suppression of immune responses to infectious agents such as viruses, bacteria, parasites and fungi and as well as preserving immune homeostasis. However, regulatory T-cell responses can advantageous for the body by minimizing the tissue-damaging effects. The following subsets of regulatory T-cells have been recognized: natural regulatory Tcells, Th3, Tr1, CD8+ Treg, natural killer like Treg (NKTreg) cells. Among various markers of Treg cells, Forkhead family transcription factor (FOXP3) as an intracellular protein is used for discrimination between activated T reg cells and activated T-cells. FOXP3 has a central role in production, thymocyte differentiation and function of regulatory Tcells. Several mechanisms have been indicated in regulation of T reg cells. As, the suppression of T-cells via regulatory T-cells is either mediated by Cell-cell contact and Immunosuppressive cytokines (TGF-Beta, IL-10) mediated.

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New differentiation pathway for double-negative regulatory T cells that regulates the magnitude of immune responses

Blood ◽

10.1182/blood-2006-10-050625 ◽

2006 ◽

Vol 109 (9) ◽

pp. 4071-4079 ◽

Cited By ~ 64

Author(s):

Dong Zhang ◽

Wei Yang ◽

Nicolas Degauque ◽

Yan Tian ◽

Allison Mikita ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cell Surface ◽

Immune Responses ◽

Lymphoid Tissues ◽

Double Negative ◽

Healthy Humans

Abstract Recent studies have demonstrated that in peripheral lymphoid tissues of normal mice and healthy humans, 1% to 5% of αβ T-cell receptor–positive (TCR+) T cells are CD4−CD8− (double-negative [DN]) T cells, capable of down-regulating immune responses. However, the origin and developmental pathway of DN T cells is still not clear. In this study, by monitoring CD4 expression during T-cell proliferation and differentiation, we identified a new differentiation pathway for the conversion of CD4+ T cells to DN regulatory T cells. We showed that the converted DN T cells retained a stable phenotype after restimulation and that furthermore, the disappearance of cell-surface CD4 molecules on converted DN T cells was a result of CD4 gene silencing. The converted DN T cells were resistant to activation-induced cell death (AICD) and expressed a unique set of cell-surface markers and gene profiles. These cells were highly potent in suppressing alloimmune responses both in vitro and in vivo in an antigen-specific manner. Perforin was highly expressed by the converted DN regulatory T cells and played a role in DN T-cell–mediated suppression. Our findings thus identify a new differentiation pathway for DN regulatory T cells and uncover a new intrinsic homeostatic mechanism that regulates the magnitude of immune responses. This pathway provides a novel, cell-based, therapeutic approach for preventing allograft rejection.

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Endometrial mesenchymal stem/stromal cell modulation of T cell proliferation

Reproduction ◽

10.1530/rep-18-0266 ◽

2018 ◽

Cited By ~ 3

Author(s):

Xiaoqing Yang ◽

Meivita Devianti ◽

Yuan H Yang ◽

Yih Rue Ong ◽

Ker Sin Tan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Stromal Cells ◽

Lymphocyte Proliferation ◽

Interleukin 10 ◽

Immunomodulatory Properties ◽

Tgf Β1

Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2, and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-β1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-β receptors, despite a reduction in regulatory T cells. Thus the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-β1, and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSC were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

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Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients

Blood ◽

10.1182/blood-2006-03-011353 ◽

2006 ◽

Vol 108 (8) ◽

pp. 2655-2661 ◽

Cited By ~ 221

Author(s):

Devi K. Banerjee ◽

Madhav V. Dhodapkar ◽

Elyana Matayeva ◽

Ralph M. Steinman ◽

Kavita M. Dhodapkar

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Regulatory T Cells ◽

Interleukin 2 ◽

Cell Contact ◽

Myeloid Dendritic Cells ◽

Healthy Donors

AbstractCD4+CD25+FOXP3+ regulatory T cells (Treg's) play an important role in the maintenance of immune tolerance. The mechanisms controlling the induction and maintenance of Treg's in humans need to be defined. We find that human myeloid dendritic cells (DCs) are superior to other antigen presenting cells for the maintenance of FOXP3+ Treg's in culture. Coculture of DCs with autologous T cells leads to an increase in both the number of Treg's, as well as the expression of FOXP3 protein per cell both in healthy donors and myeloma patients. DC-mediated expansion of FOXP3high Treg's is enhanced by endogenous but not exogenous interleukin-2 (IL-2), and DC-T-cell contact, including the CD80/CD86 membrane costimulatory molecules. DCs also stimulate the formation of Treg's from CD25- T cells. The efficacy of induction of Treg's by DCs depends on the nature of the DC maturation stimulus, with inflammatory cytokine-treated DCs (Cyt-DCs) being the most effective Treg inducers. DC-induced Treg's from both healthy donors and patients with myeloma are functional and effectively suppress T-cell responses. A single injection of cytokine-matured DCs led to rapid enhancement of FOXP3+ Treg's in vivo in 3 of 3 myeloma patients. These data reveal a role for DCs in increasing the number of functional FOXP3high Treg's in humans.

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Regulatory T cells in rheumatoid arthritis showed increased plasticity toward Th17 but retained suppressive function in peripheral blood

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2013-204228 ◽

2014 ◽

Vol 74 (6) ◽

pp. 1293-1301 ◽

Cited By ~ 53

Author(s):

Tian Wang ◽

Xiaolin Sun ◽

Jing Zhao ◽

Jing Zhang ◽

Huaqun Zhu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Peripheral Blood ◽

Negative Regulator ◽

Suppressive Function ◽

Treg Population ◽

Treg Subset

ObjectiveRegulatory T cells (Tregs) with the plasticity of producing proinflammatory cytokine IL-17 have been demonstrated under normal and pathogenic conditions. However, it remains unclear whether IL-17-producing Tregs lose their suppressive functions because of their plasticity toward Th17 in autoimmunity. The aim of this study was to investigate IL-17-producing Tregs from patients with rheumatoid arthritis (RA), and characterise their regulatory capacity and clinical significance.MethodsFoxp3 and IL-17 coexpression were evaluated in CD4 T lymphocytes from RA patients. An in vitro T cell polarisation assay was performed to investigate the role of proinflammatory cytokines in IL-17-producing Treg polarisation. The suppressive function of IL-17-producing Tregs in RA was assessed by an in vitro suppression assay. The relationship between this Treg subset and clinical features in RA patients was analysed using Spearman's rank correlation test.ResultsA higher frequency of IL-17-producing Tregs was present in the peripheral blood of RA patients compared with healthy subjects. These cells from peripheral blood showed phenotypic characteristics of Th17 and Treg cells, and suppressed T cell proliferation in vitro. Tregs in RA synovial fluid lost suppressive function. The Th17 plasticity of Tregs could be induced by IL-6 and IL-23. An increased ratio of this Treg subset was associated with decreased levels of inflammatory markers, including the erythrocyte sedimentation rate and C-reactive protein level, in patients with RA.ConclusionsIncreased levels of IL-17-producing Tregs were identified in RA patients. This Treg subset with Th17 plasticity in peripheral blood retained suppressive functions and was associated with milder inflammatory conditions, suggesting that this Treg population works as a negative regulator in RA, but in RA synovial site it may be pathogenic.

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Faculty Opinions recommendation of CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020455.232596 ◽

2004 ◽

Author(s):

Sarah Rowland-Jones

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Peripheral Blood ◽

Disease Status ◽

Clinical Markers ◽

T Cell Immune Responses ◽

Asymptomatic Hiv

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Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD4+CD25+ regulatory T cells

Blood ◽

10.1182/blood-2009-02-206730 ◽

2009 ◽

Vol 114 (6) ◽

pp. 1263-1269 ◽

Cited By ~ 55

Author(s):

Christian Becker ◽

Christian Taube ◽

Tobias Bopp ◽

Christoph Becker ◽

Kai Michel ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Graft Versus Host Disease ◽

Adenosine Monophosphate ◽

Host Disease ◽

Graft Versus Host

AbstractNaturally occurring CD4+CD25+ regulatory T cells (Tregs) represent a unique T-cell lineage that is endowed with the ability to actively suppress immune responses. Therefore, approaches to modulate Treg function in vivo could provide ways to enhance or reduce immune responses and lead to novel therapies. Here we show that the CD4 binding human immunodeficiency virus-1 envelope glycoprotein gp120 is a useful and potent tool for functional activation of human Tregs in vitro and in vivo. Gp120 activates human Tregs by binding and signaling through CD4. Upon stimulation with gp120, human Tregs accumulate cyclic adenosine monophosphate (cAMP) in their cytosol. Inhibition of endogeneous cAMP synthesis prevents gp120-mediated Treg activation. Employing a xenogeneic graft versus host disease model that has been shown to be applicable for the functional analysis of human Tregs in vivo, we further show that a single dose of gp120 is sufficient to prevent lethal graft versus host disease and that the tolerizing effect of gp120 is strictly dependent on the presence of human Tregs and their up-regulation of cAMP upon gp120-mediated activation. Our findings demonstrate that stimulation via the CD4 receptor represents a T-cell receptor–independent Treg activating pathway with potential to induce immunologic tolerance in vivo.

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

Blood ◽

10.1182/blood.v104.11.110.110 ◽

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.

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