scholarly journals Cas9-directed immune tolerance in humans—a model to evaluate regulatory T cells in gene therapy?

Gene Therapy ◽  
2021 ◽  
Author(s):  
Dimitrios Laurin Wagner ◽  
Lena Peter ◽  
Michael Schmueck-Henneresse
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Regulatory T Cells ◽  
Immune Tolerance ◽  
Neutralizing Antibodies ◽  
Treg Cells ◽  
Gene Therapies ◽  
Early Results ◽  
Cell Immunity

AbstractThe dichotomic nature of the adaptive immune response governs the outcome of clinical gene therapy. On the one hand, neutralizing antibodies and cytotoxic T cells can have a dramatic impact on the efficacy and safety of human gene therapies. On the other hand, regulatory T cells (Treg) can promote tolerance toward transgenes thereby enabling long-term benefits of in vivo gene therapy after a single administration. Pre-existing antibodies and T cell immunity has been a major obstacle for in vivo gene therapies with viral vectors. As CRISPR-Cas9 gene editing advances toward the clinics, the technology’s inherent immunogenicity must be addressed in order to guide clinical treatment decisions. This review summarizes the recent evidence on Cas9-specific immunity in humans—including early results from clinical trials—and discusses the risks for in vivo gene therapies. Finally, we focus on solutions and highlight the potential role of Cas9-specific Treg cells to promote immune tolerance. As a “beneficial alliance” beyond Cas9-immunity, antigen-specific Treg cells may serve as a living and targeted immunosuppressant to increase safety and efficacy of gene therapy.

Regulatory T Cells in Hepatic Immune Tolerance and Autoimmune Liver Diseases

2015 ◽  
Vol 33 (Suppl. 2) ◽  
pp. 70-74 ◽  
Author(s):  
Johannes Herkel
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Immune Tolerance ◽  
Liver Diseases ◽  
Liver Sinusoidal Endothelial Cells ◽  
Autoimmune Liver Diseases ◽  
Specific Control

Regulatory T cells (Tregs) have a profound ability to control immune responses. A majority of Tregs are derived from the thymus; yet a substantial Treg fraction is derived from the periphery. The liver seems to be an important source of peripherally derived Tregs. Indeed, the liver's well-known ability to induce immune tolerance is at least partly based on hepatic Treg generation. With recently developed tools to deliver antigens to tolerance-inducing liver cells, it is now possible to harness liver-derived Tregs for specific control of unwanted immune responses. Indeed, the selective delivery of autoantigens to liver sinusoidal endothelial cells could induce autoantigen-specific Tregs in vivo, providing effective treatment of autoimmune disease. Owing to the fundamental role Tregs play in controlling immune responses, an impairment of Tregs seems to be a plausible explanation for the development of autoimmune diseases, for example, in the liver. However, the actual role of Treg impairment in autoimmune liver diseases, such as autoimmune hepatitis (AIH), remains controversial. Major obstacles for clarifying the role of Tregs in autoimmune liver diseases are related to the difficulty to identify human Tregs unambiguously and to the difficulty to identify those Tregs and effector T cells that specifically recognize disease-driving autoantigens. However, even if AIH turned out to be a disease that is not driven by Treg impairment, Treg-based therapies for autoimmune liver diseases might still be effective, provided the Tregs for therapeutic use recognize the relevant antigens.

scholarly journals Toll-Like Receptor 4 Signaling Leads to Severe Fungal Infection Associated with Enhanced Proinflammatory Immunity and Impaired Expansion of Regulatory T Cells

2009 ◽  
Vol 78 (3) ◽  
pp. 1078-1088 ◽  
Author(s):  
Flávio V. Loures ◽  
Adriana Pina ◽  
Maíra Felonato ◽  
Eliseu F. Araújo ◽  
Katia R. M. Leite ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
T Cells ◽  
Regulatory T Cells ◽  
Treg Cells ◽  
Interleukin 12 ◽  
Necrosis Factor Alpha ◽  
Tlr4 Signaling ◽  
Inflammatory Reactions ◽  
Cell Immunity

ABSTRACT Toll-like receptors (TLRs) present in innate immune cells recognize pathogen molecular patterns and influence immunity to control the host-parasite interaction. The objective of this study was to characterize the involvement of TLR4 in the innate and adaptive immunity to Para co c cidioides brasiliensis, the most important primary fungal pathogen of Latin America. We compared the responses of C3H/HeJ mice, which are naturally defective in TLR4 signaling, with those of C3H/HePas mice, which express functional receptors, after in vitro and in vivo infection with P. brasiliensis. Unexpectedly, we verified that TLR4-defective macrophages infected in vitro with P. brasiliensis presented decreased fungal loads associated with impaired synthesis of nitric oxide, interleukin-12 (IL-12), and macrophage chemotactic protein 1 (MCP-1). After intratracheal infection with 1 million yeasts, TLR4-defective mice developed reduced fungal burdens and decreased levels of pulmonary nitric oxide, proinflammatory cytokines, and antibodies. TLR4-competent mice produced elevated levels of IL-12 and tumor necrosis factor alpha (TNF-α), besides cytokines of the Th17 pattern, indicating a proinflammatory role for TLR4 signaling. The more severe infection of TLR4-normal mice resulted in increased influx of activated macrophages and T cells to the lungs and progressive control of fungal burdens but impaired expansion of regulatory T cells (Treg cells). In contrast, TLR4-defective mice were not able to clear their diminished fungal burdens totally, a defect associated with deficient activation of T-cell immunity and enhanced development of Treg cells. These divergent patterns of immunity, however, resulted in equivalent mortality rates, indicating that control of elevated fungal growth mediated by vigorous inflammatory reactions is as deleterious to the hosts as low fungal loads inefficiently controlled by limited inflammatory reactions.

scholarly journals Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3096-3103 ◽  
Author(s):  
Kenrick Semple ◽  
Antony Nguyen ◽  
Yu Yu ◽  
Honglin Wang ◽  
Claudio Anasetti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Protein Tyrosine Kinase ◽  
Cell Receptor ◽  
Specific Protein ◽  
Cell Immunity ◽  
Cd28 Costimulation

Abstract CD28 costimulation is required for the generation of naturally derived regulatory T cells (nTregs) in the thymus through lymphocyte-specific protein tyrosine kinase (Lck) signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naive CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naive CD4 T cells (CD25−Foxp3−) by T-cell receptor stimulation with additional transforming growth factorβ (TGFβ) in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. Using a series of transgenic mice on a CD28-deficient background that bears wild-type or mutated CD28 in its cytosolic tail that is incapable of binding to Lck, phosphoinositide 3-kinase (PI3K), or IL-2–inducible T-cell kinase (Itk), we found that CD28-mediated Lck signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide a rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28 signaling.

scholarly journals In vivo induction of regulatory T cells for immune tolerance in hemophilia

2016 ◽  
Vol 301 ◽  
pp. 18-29 ◽  
Author(s):  
Xiaomei Wang ◽  
Cox Terhorst ◽  
Roland W. Herzog
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Immune Tolerance ◽  
In Vivo Induction

scholarly journals Immune Tolerance Developed in Platelet-Targeted FVIII Gene Therapy in Hemophilia Mice Is CD4+ T Cell-Mediated

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1071-1071
Author(s):  
Yingyu Chen ◽  
Xiaofeng Luo ◽  
Juan Chen ◽  
Jocelyn Schroeder ◽  
Christina K Baumgartner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Tolerance ◽  
Cd4 T Cells ◽  
Memory B Cells ◽  
Cd4 T Cell ◽  
Control Group

Abstract Immune response to factor VIII (FVIII) is not only a severe complication in protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Our previous studies have demonstrated that platelet-targeted FVIII (2bF8) gene therapy together with in vivo drug-selection of transduced cells can not only rescue the bleeding diathesis but also induce anti-FVIII specific immune tolerance in FVIIInull mice. In the current study, we investigated 1) whether our non-selectable lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and 2) which cell compartment is tolerized after platelet gene therapy. Platelet-specific FVIII expression was introduced by 2bF8LV-transduction of hematopoietic stem cells followed by syngeneic transplantation into FVIIInull mice preconditioned with 660 cGy total body irradiation (TBI) or Busulfan (Bu) plus ATG (anti-thymocyte globulin). After bone marrow transplantation and reconstitution, animals were analyzed by PCR, qPCR, FVIII:C assay, and tail clipping test to confirm the success of 2bF8 gene therapy. Sixteen weeks after transplantation, animals were challenged with recombinant human FVIII (rhF8) via retro-orbital venous administration at a dose of 50 U/kg weekly for 4 weeks. The titers of anti-FVIII inhibitory antibodies (inhibitors) were determined by Bethesda assay. The CFSE-labeled CD4 T cell proliferation assay and ELISPOT-based memory B cell maturation assay were used to determine which cell compartment is tolerized to FVIII after 2bF8 gene therapy. The level of platelet-FVIII expression was 1.44 ± 0.39 mU/108 platelets (n = 6) in the 660 cGy group, which is not significantly different from the level obtained from the Bu+ATG group [3.04 ± 1.19 mU/108 platelets (n = 6)]. Even after rhF8 challenge, no antibodies were detected in 2bF8LV-transduced recipients in either group. In contrast, all animals in the control group that did not undergo gene therapy developed various levels of inhibitors (204±97 BU/ml, n=7). The frequency of regulatory T cells in both 660 cGy TBI (11.01±0.52%) and Bu+ATG (11.02±0.68%) groups were significantly higher than the control group (8.05±0.57%). T cell proliferation assay demonstrated that CD4+ T cells from 2bF8 LV-transduced recipients that had been challenged with rhF8 did not proliferate when restimulated with rhF8 in vitro. The daughter CD4+ T cells in the group with 10 U/ml of rhF8 were 5.84 ± 2.49% (n = 6), which was not significantly different from the control group without no rhF8 stimulation (0 U/ml) (5.33 ± 1.72%). CD4+ T cells from primed FVIIInull mice did proliferate after rhF8 restimulation. The proliferated daughter cell was 13.12 ± 6.76% (n = 7) in the group with rhF8 (10 U/ml) re-stimulation, which is significantly higher than the group without rhF8 co-culture (4.99 ± 1.16%). Since FVIII-specific memory B cell maturation is CD4+ T cell dependent, we isolated CD4+ T and memory B cells from 2bF8LV-transduced or FVIIInull mice after rhF8 immunization and co-cultured with rhF8 followed by ELISPOT assay to examine the antibody secreting cells. No spots were detected when memory B cells from rhF8-primed FVIIInull mice were co-cultured with CD4+ T cells from 2bF8LV-transduced recipients. In contrast, when memory B cells from either rhF8 immunized 2bF8LV-transduced or untreated FVIIInull mice were cultured with CD4+ T cells from rhF8-primed FVIIInull mice, there were 142 and 205 anti-FVIII antibody secreting cells, respectively, detected per 106 cells seeded. These results indicate that CD4+ T cells from 2bF8LV-transduced mice are tolerized to rhF8 stimulation. In conclusion, 2bF8 lentiviral gene transfer without in vivo selection of genetically manipulated cells can introduce FVIII-specific immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell-mediated. Disclosures Baumgartner: Novo Nordisk: Research Funding. Shi:BloodCenter of Wisconsin: Patents & Royalties: METHOD OF INDUCING IMMUNE TOLERANCE THROUGH TARGETTED GENE EXPRESSION..

Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3728-3728
Author(s):  
Kenrick Semple ◽  
Antony Nguyen ◽  
Yu Yu ◽  
Claudio Anasetti ◽  
Xue-Zhong Yu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Graft Versus Host ◽  
Cell Immunity ◽  
Cd28 Costimulation ◽  
Insight Into

Abstract Abstract 3728 CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Involvement of Regulatory T Cells in the Immunosuppression Characteristic of Patients with Paracoccidioidomycosis

2010 ◽  
Vol 78 (10) ◽  
pp. 4392-4401 ◽  
Author(s):  
Maria Carolina Ferreira ◽  
Rômulo Tadeu Dias de Oliveira ◽  
Rosiane Maria da Silva ◽  
Maria Heloisa Souza Lima Blotta ◽  
Ronei Luciano Mamoni
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Neutralizing Antibodies ◽  
Transforming Growth Factor ◽  
Paracoccidioides Brasiliensis ◽  
Treg Cells ◽  
Delayed Type Hypersensitivity ◽  
Cell Contact ◽  
Regulatory Activity ◽  
Cell Cell

ABSTRACT Patients with paracoccidioidomycosis (PCM) exhibit a suppression of the cellular immune response characterized by negative delayed-type hypersensitivity (DTH) to Paracoccidioides brasiliensis antigens, the apoptosis of lymphocytes, and high levels of expression of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). The aim of this study was to investigate whether and how regulatory T cells (Treg cells) are involved in this immunosuppression by analyzing the number, phenotype, and activity of these cells in patients with active disease (AD group) and patients who had received treatment (TD group). Our results showed that the AD patients had more Treg cells than the TD patients or controls (C group) and also had elevated levels of expression of regulatory markers (glucocorticoid-induced tumor necrosis factor [TNF] receptor-related protein [GITR], CTLA-4, CD95L, LAP-1, and CD38). An analysis of regulatory activity showed that Treg cells from the AD group had greater activity than did cells from the other groups and that cell-cell contact is mandatory for this activity in the C group but was only partially involved in the regulatory activity of cells from AD patients. The addition of anti-IL-10 and anti-TGF-β neutralizing antibodies to the cultures showed that the production of cytokines may be another mechanism used by Treg cells. In conclusion, the elevated numbers of these cells with an increased regulatory phenotype and strong suppressive activity suggest a potential role for them in the immunosuppression characteristic of paracoccidioidomycosis. In addition, our results indicate that while Treg cells act by cell-cell contact, cytokine production also plays an important role.

scholarly journals MicroRNA-21 Regulates Diametrically Opposed Biological Functions of Regulatory T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Jijun Sun ◽  
Ruiling Liu ◽  
Xiaozhen He ◽  
Jiang Bian ◽  
Wenbo Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
Autoimmune Disease ◽  
Regulatory T Cells ◽  
Cell Activation ◽  
Treg Cells ◽  
Mechanistic Study ◽  
Dendritic Cell Activation ◽  
Adenoviral Delivery

Regulatory T cells (Tregs) are considered important for controlling the onset and development of autoimmune disease. Although studies have shown that miR-21 is expressed at higher levels in Treg cells, it remains largely elusive whether miR-21 regulates the immune-suppressive function of Tregs. In the current study, we generated mice lacking miR-21 specifically in their Tregs and investigated the role of miR-21 in regulating Treg function both in vitro and in vivo. Our study revealed that Tregs lacking miR-21 exhibit normal phenotype and unaltered function in suppressing T cell proliferation and dendritic cell activation in vitro. However, compared with miR-21-sufficient Tregs, they produce significant more IL-17 and IL-10 when under pathogenic Th17-priming condition. Adenoviral delivery of miR-21 into Treg cells is able to reduce the expression of both IL-17 and IL-10. Mechanistic study revealed that miR-21 down-regulates IL-10 expression through direct targeting of IL-10, and suppresses reprogramming of Tregs into IL-17-secreting cells through down-regulating Stat3 activity. However, we detected no significant or marginal difference in the development of various autoimmune diseases between wild type mice and mice with Treg-specific deletion of miR-21. In conclusion, our study demonstrated that miR-21 in Tregs regulates diametrically opposed biological Treg functions and is largely dispensable for the development of autoimmune disease.

In vivo peripheral expansion of naive CD4+CD25highFoxP3+ regulatory T cells in patients with multiple myeloma

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3940-3949 ◽  
Author(s):  
Marc Beyer ◽  
Matthias Kochanek ◽  
Thomas Giese ◽  
Elmar Endl ◽  
Martin R. Weihrauch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cancer Patients ◽  
Treg Cells ◽  
Effector Memory ◽  
Autoreactive T Cell ◽  
Peripheral Expansion

In solid tumors, leukemias, and lymphomas, increased frequencies of functional CD4+CD25high regulatory T cells (Treg cells) have been previously demonstrated. In healthy individuals, Treg cells consist not only of memory but also of naive T cells, which can undergo peripheral expansion and are characterized by a relative enrichment for autoreactive T-cell receptors. Here, we demonstrate in patients with premalignant monoclonal gammopathy of undetermined significance and patients with multiple myeloma that functional FoxP3+ Treg cells of naive, central, and effector memory phenotype as determined by CCR7 and CD45RA expression are significantly expanded. Low frequencies of T-cell receptor excision circles in naive Treg cells in both healthy controls and multiple myeloma patients point to peripheral expansion as the prominent mechanism of increased frequencies of naive Treg cells in these cancer patients. These findings strongly suggest that the increase of functional Treg cells in cancer patients is a response to the process of malignant transformation.

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