Cytotoxic Activity Of Bispecific Antibody-Redirected Human Regulatory T Cells: Fact Or Artifact

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5430-5430
Author(s):  
Stefanie Koristka ◽  
Marc Cartellieri ◽  
Anja Feldmann ◽  
Claudia Arndt ◽  
Irene Michalk ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Tumor Cell ◽  
Cell Lysis ◽  
In Vivo Studies ◽  
Target Cells ◽  
Cytotoxic Potential

Abstract Regulatory T cells (Tregs) play an inevitable role in immune homeostasis by maintaining self-tolerance as well as regulating the magnitude of immune responses against foreign antigens. Over the last few years, the enormous potential of adoptive Treg transfer for treatment of auto- and alloimmunity including Graft-versus-Host disease (GvHD) has been validated in a vast number of in vitro and in vivo studies. For their clinical application, all modes of action should be well understood. Regarding their cytotoxic potential, only few and conflicting data exist. On the one hand, it is assumed that Tregs are capable of inducing apoptosis of effector T cells (Teff) utilizing granzyme/perforin or FasL expression. Others claim that Tregs are not capable of suppressing Teff via programmed cell death pathways but rather induce apoptosis by cytokine deprivation. However, it is of importance to clarify whether Tregs possess a cytotoxic potential particularly when activating the cells antigen-specifically using bispecific antibodies (bsAb). In recent years, bsAb have emerged as promising tools for an antigen-specific immunotherapy of malignant diseases. Their tremendous potential for tumor therapy has been verified in a plethora of in vitro and in vivo studies as well as in first clinical trials. So far, our group was able to demonstrate that not only Teff but also Tregs can be redirected by CD3-engaging bsAb (Koristka et al., J Immunol. 2012; J Autoimmun. 2013). According to a recent presentation (Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research, 2012, abstract nr 4841), bsAb-redirected Tregs can act as killer cells and efficiently mediate cancer cell lysis. In order to shed light onto this controversial issue, we decided to analyze this question in more detail. According to our investigations tumor cell elimination of bsAb-engaged Tregs is largely dependent on the purity of isolated Treg fractions. Tregs isolated on the basis of CD25 expression exhibited a remarkable killing capacity which is most probably due to contaminating CD25+FOXP3- Teff, as highly pure (> 99 %), FACS-isolated CD4+CD25+CD127low Tregs did not display any considerable cytotoxic effect upon cross-linkage to tumor cells via bsAb. The same applies for CD45RA-sorted, expanded Tregs. In comparison to autologous, expanded Teff, tumor cell lysis was negligible. Moreover, the lack of cytotoxicity was independent of the chosen target antigen, as redirecting Tregs with two different bsAb did not result in tumor cell eradication. Besides, upon polyclonal stimulation with conventional aCD3/CD28-coated beads Tregs were not capable of eliminating target cells. Furthermore, as opposed to autologous Teff, Tregs showed only a marginal upregulation of the degranulation marker CD107a when being activated either antigen-specifically via bsAb or polyclonally via beads. Taken together, our findings clearly demonstrate that Tregs bear no considerable cytotoxic potential and hence do not contribute to cancer cell lysis, as recently claimed. On the other hand, the results show that Tregs can be activated by bsAb without the risk of cytotoxic effects against the recognized target cells. This provides the basis for the application of bsAb for a site-specific recruitment of Tregs aiming at attenuating Teff-mediated proinflammatory immune responses and tissue destruction in order to treat auto- and alloimmune diseases including GvHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD4+ CD25+ Regulatory T Cells Control T Helper Cell Type 1 Responses to Foreign Antigens Induced by Mature Dendritic Cells In Vivo

2003 ◽  
Vol 198 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Guillaume Oldenhove ◽  
Magali de Heusch ◽  
Georgette Urbain-Vansanten ◽  
Jacques Urbain ◽  
Charlie Maliszewski ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Peripheral Tolerance ◽  
T Helper Cell ◽  
Helper Cell ◽  
T Helper

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II–restricted interferon γ–producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

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

Blood ◽  
2006 ◽  
Vol 109 (9) ◽  
pp. 4071-4079 ◽  
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.

scholarly journals Targeting Antigen Presenting   Cells to Treat Autoimmune  Inflammation

2021 ◽  
Author(s):  
◽  
Aras Toker
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Antigen Presenting

<p>Glatiramer acetate (GA) is approved for the treatment of relapsing-remitting multiple sclerosis (MS), and can suppress experimental autoimmune encephalomyelitis (EAE), a murine model of human MS. GA treatment is associated with the induction of anti-inflammatory TH2 responses and with the antigen specific expansion of regulatory T cells that counteract or inhibit pathogenic events in MS and EAE. These T cell mediated mechanisms of protection are considered to be a result of modulation of antigen presenting cells (APCs) by GA, rather than direct effects on T cells. However, it is unknown if GA preferentially targets a specific APC subset or can act through multiple APCs in vivo. In addition, GA-modulated innate cells may also exhibit direct antigen non-specific suppression of autoreactive cells. One objective of this study was to identify the in vivo target cell population of GA and to assess the potential of the target cells to antigen non-specifically suppress immune responses. Fluorophor-labelled GA bound to monocytes after intravenous injections, suggesting that monocytes may be the primary target of GA in vivo. In addition, intravenous GA treatment enhanced the intrinsic ability of monocytes to suppress T cell proliferation, both in vitro and in vivo. The findings of this study therefore suggest that GA-induced monocytes may contribute to GA therapy through direct mechanisms of antigen non-specific T cell immunosuppression. A further objective of this work was to investigate the potential of an in vivo drug targeting approach. This approach was hypothesised to increase the uptake of GA by the target cells and substantially improve GA treatment through antigen specific mechanisms such as induction of TH2 or regulatory T cells. Targeting antigens to professional APCs with an anti-MHC class II antibody resulted in significantly enhanced T cell proliferation in vitro. However, no EAE suppression occurred when GA was targeted to MHC class II in vivo. In addition, targeting GA specifically to monocytes also failed to suppress EAE. These findings suggest that GA treatment may selectively modulate monocytes to enhance their ability to inhibit autoreactive T cells, which could be part of the mechanism by which GA ameliorates MS. Targeting GA to a specific cell type may not be a powerful approach to improve treatment, because increased proliferation of GA specific T cells is not sufficient for disease suppression, and conjugation to antibodies may functionally reduce GA to a mere antigen devoid of immunomodulatory capacity.</p>

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

Blood ◽  
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

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 in vitro. Furthermore, these engineered Tregs could suppress T-effectors specific for additional epitopes in local milieu in both a cell contact and contactless manner. From these data, we hypothesized that IL-2 and related signaling pathways are major regulatory mechanisms of the suppression. To further investigate how IL-2R signaling is engaged to control T effectors and Tregs, we followed the phospho-STAT5 status of these cells kinetically. Our results showed clearly that IL-2 from activated T effectors is a key requirement for Treg activation, inducing subsequent blockage of STAT5 signal in T effectors by activated Tregs. As further evidence of the efficacy of these specific Tregs, we then determined whether FVIII C2-specific Tregs could suppress the induction of FVIII inhibitor antibody in vivo. Thus, we transferred FVIII C2-specific human Tregs into HLA DR1 hemophilic mice and challenged them with FVIII in vivo. Our results showed that induction of FVIII-specific antibodies was inhibited for over 8 weeks. Taken together, our results suggest a potential therapeutic trial of FVIII-specific engineered Tregs in hemophilia A. Disclosures Kim: Henry Jackson Foundation: Patents & Royalties: Provisional submitted.

Mechanisms of self–nonself discrimination and possible clinical relevance

Immunotherapy ◽  
2009 ◽  
Vol 1 (4) ◽  
pp. 631-644
Author(s):  
Carolin Daniel ◽  
Jens Nolting ◽  
Harald von Boehmer
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Clinical Relevance ◽  
Molecular Mechanisms ◽  
Immunological Tolerance ◽  
Molecular Pathways ◽  
Clinical Protocols

This review discusses different mechanisms that result in immunological tolerance, such as intrathymic deletion of immature T cells, intrathymic and extrathymic generation of regulatory T cells, effector mechanisms of regulatory T cells as well as molecular pathways involved in extrathymic generation of regulatory T cells in vivo and in vitro. These molecular mechanisms should enable investigators to develop clinical protocols aiming at the specific prevention of unwanted immune responses, thereby replacing indiscriminate immunosuppression that often has fatal consequences.

scholarly journals Targeting Antigen Presenting   Cells to Treat Autoimmune  Inflammation

2021 ◽  
Author(s):  
◽  
Aras Toker
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Antigen Presenting

<p>Glatiramer acetate (GA) is approved for the treatment of relapsing-remitting multiple sclerosis (MS), and can suppress experimental autoimmune encephalomyelitis (EAE), a murine model of human MS. GA treatment is associated with the induction of anti-inflammatory TH2 responses and with the antigen specific expansion of regulatory T cells that counteract or inhibit pathogenic events in MS and EAE. These T cell mediated mechanisms of protection are considered to be a result of modulation of antigen presenting cells (APCs) by GA, rather than direct effects on T cells. However, it is unknown if GA preferentially targets a specific APC subset or can act through multiple APCs in vivo. In addition, GA-modulated innate cells may also exhibit direct antigen non-specific suppression of autoreactive cells. One objective of this study was to identify the in vivo target cell population of GA and to assess the potential of the target cells to antigen non-specifically suppress immune responses. Fluorophor-labelled GA bound to monocytes after intravenous injections, suggesting that monocytes may be the primary target of GA in vivo. In addition, intravenous GA treatment enhanced the intrinsic ability of monocytes to suppress T cell proliferation, both in vitro and in vivo. The findings of this study therefore suggest that GA-induced monocytes may contribute to GA therapy through direct mechanisms of antigen non-specific T cell immunosuppression. A further objective of this work was to investigate the potential of an in vivo drug targeting approach. This approach was hypothesised to increase the uptake of GA by the target cells and substantially improve GA treatment through antigen specific mechanisms such as induction of TH2 or regulatory T cells. Targeting antigens to professional APCs with an anti-MHC class II antibody resulted in significantly enhanced T cell proliferation in vitro. However, no EAE suppression occurred when GA was targeted to MHC class II in vivo. In addition, targeting GA specifically to monocytes also failed to suppress EAE. These findings suggest that GA treatment may selectively modulate monocytes to enhance their ability to inhibit autoreactive T cells, which could be part of the mechanism by which GA ameliorates MS. Targeting GA to a specific cell type may not be a powerful approach to improve treatment, because increased proliferation of GA specific T cells is not sufficient for disease suppression, and conjugation to antibodies may functionally reduce GA to a mere antigen devoid of immunomodulatory capacity.</p>

ANTIGEN-PRIMED CD4+CD25+ REGULATORY T CELLS PREVENT SKIN GRAFT REJECTION: IN VITRO AND IN VIVO STUDIES

2010 ◽  
Vol 90 ◽  
pp. 523
Author(s):  
S. GU ◽  
J. ZHENG ◽  
S. YI ◽  
W. HE ◽  
X. CHEN ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Skin Graft ◽  
Graft Rejection ◽  
In Vivo Studies

723 Lymph node-targeted AMP-vaccine enables tumor-directed mKRAS-specific immune responses with potent polyfunctional and cytolytic activity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A766-A766
Author(s):  
Martin Steinbuck ◽  
Peter DeMuth ◽  
Lochana Seenappa
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Immune Cells ◽  
Tumor Antigens ◽  
Large Fraction ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Subunit Vaccines

BackgroundSubunit vaccines targeting tumor antigens have shown limited capacity for expanding cytotoxic T-cells against tumors in the clinic. Especially in the case of KRAS-driven tumors, responses elicited by conventional vaccines have been exceedingly weak. For molecular immunogens including peptides and oligonucleotides, inefficient delivery to immune cells residing in the lymphatics is a significant challenge limiting their ability to induce cancer-directed immune responses of sufficient strength and functionality to impact tumors. Improving the targeting of immunogens to lymph nodes (LN), where resident immune cells potently orchestrate immunity, can substantially amplify their ability to induce effective tumor-directed immunity. Here, we demonstrate such an approach for significantly enhancing mKRAS-directed T-cell responses by precisely targeting antigens and adjuvants directly to the draining LN through a simple one-step conjugation to albumin-binding lipids. These amphiphilic conjugates (‘Amphiphiles’, or AMP) then ‘hitch-hike’ on albumin into the LNs where they elicit strong immune responses. LN accumulation of structurally optimized amphiphiles in mice is greatly improved over soluble equivalents.MethodsC57BL/6J mice received two or more doses of benchmark or amphiphile-modified vaccines, comprised of mKRAS peptide and CpG adjuvant, subcutaneously injected into the tail base in two-week intervals. Immunological readouts were performed 7 days post dosing. For ELISpot analysis of IFNγ and Granzyme B production and flowcytometric bead array analysis of Th1/2 cytokines, splenocytes were harvested and re-stimulated with antigen overnight. In vivo, cytolytic capabilities of antigen-specific T-cells were evaluated by pulsing CFSE-stained splenocytes from naïve mice with mKRAS antigen and injecting these cells intravenously into immunized mice. Recovery of CFSE-labeled target cells from immunized mice was performed 24h later and analyzed flowcytometrically.ResultsWe show robust immune responses that yield strong activation against all common mutations in the mKRAS protein compared to low or undetectable responses generated by soluble or benchmark treatments. Further, this response is composed of CD4+ as well as CD8+ T-cells resulting in the production of high levels of TH1-associated cytokines upon re-stimulation with mKRAS-specific peptides in vitro. In vivo, robust cytolytic function towards mKRAS-presenting targets can be measured in T-cells.ConclusionsBy targeting immunogens directly and precisely to the LNs, the Amphiphile platform can significantly amplify the potency of subunit vaccines. In the case of mKRAS, substantially improved cytolytic immune responses represent a promising therapeutic strategy for targeting mKRAS-driven tumor growth and survival in a large fraction of human tumors. Furthermore, this platform technology is simple, rapid and scalable for broad clinical application.

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

Blood ◽  
2009 ◽  
Vol 114 (6) ◽  
pp. 1263-1269 ◽  
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.

scholarly journals Replication of Murine Cytomegalovirus in Differentiated Macrophages as a Determinant of Viral Pathogenesis

1999 ◽  
Vol 73 (7) ◽  
pp. 5970-5980 ◽  
Author(s):  
Laura K. Hanson ◽  
Jacquelyn S. Slater ◽  
Zaruhi Karabekian ◽  
Herbert W. Virgin ◽  
Christine A. Biron ◽  
...  
Keyword(s):  
Immune Responses ◽  
In Vivo Studies ◽  
Murine Cytomegalovirus ◽  
Target Cells ◽  
Blood Monocytes ◽  
Cell Type ◽  
Mcmv Infection ◽  
Necrosis Factor Alpha

ABSTRACT Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.

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