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

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.

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Trichostatin A Promotes the Generation and Suppressive Functions of Regulatory T Cells

Clinical and Developmental Immunology ◽

10.1155/2013/679804 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 14

Author(s):

Cristian Doñas ◽

Macarena Fritz ◽

Valeria Manríquez ◽

Gabriela Tejón ◽

María Rosa Bono ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Trichostatin A ◽

Gene Promoter ◽

Treg Cells ◽

Specific Subset ◽

Global Increase ◽

And Function ◽

H3 Acetylation

Regulatory T cells are a specific subset of lymphocytes that suppress immune responses and play a crucial role in the maintenance of self-tolerance. They can be generated in the thymus as well as in the periphery through differentiation of naïve CD4+T cells. The forkhead box P3 transcription factor (Foxp3) is a crucial molecule regulating the generation and function of Tregs. Here we show that thefoxp3gene promoter becomes hyperacetylated inin vitrodifferentiated Tregs compared to naïve CD4+T cells. We also show that the histone deacetylase inhibitor TSA stimulated thein vitrodifferentiation of naïve CD4+T cells into Tregs and that this induction was accompanied by a global increase in histone H3 acetylation. Importantly, we also demonstrated that Tregs generated in the presence of TSA have phenotypical and functional differences from the Tregs generated in the absence of TSA. Thus, TSA-generated Tregs showed increased suppressive activities, which could potentially be explained by a mechanism involving the ectonucleotidases CD39 and CD73. Our data show that TSA could potentially be used to enhance the differentiation and suppressive function of CD4+Foxp3+Treg cells.

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Dendritic Cell-Induced T Cell Non-Responsiveness Is Mediated by FOXP3+ and TGF-beta+IL-10+ CD4+ T Cells.

Blood ◽

10.1182/blood.v108.11.3891.3891 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3891-3891

Author(s):

Zwi N. Berneman ◽

Nathalie Cools ◽

Viggo F.I. Van Tendeloo ◽

Marc Lenjou ◽

Griet Nijs ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Immunity ◽

Tgf Beta ◽

Cell Immunity

Abstract Dendritic cells (DC), the professional antigen presenting cells of the immune system, exert important functions both in induction of T cell immunity as well as of tolerance. Previously, it was accepted that the main function of immature DC (iDC) in their in vivo steady state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. However, a growing body of experimental evidence now indicates that traditional DC maturation can no longer be used to distinguish between tolerogenic and immunogenic properties of DC. In this study, we compared the in vitro stimulatory capacity of immature DC (iDC), cytokine cocktail-matured DC (CC-mDC) and poly I:C-matured DC (pIC-mDC) in the absence and presence of antigen. All investigated DC types could induce at least 2 subsets of regulatory T cells. We observed a significant increase in both the number of functionally suppressive transforming growth factor (TGF)-beta+ interleukin (IL)-10+ T cells as well as of CD4+CD25+FOXP3+ T cells within DC/T cell co-cultures as compared to T cell cultures without DC. The induction of these regulatory T cells correlates with in vitro T cell non-responsiveness after co-culture with iDC and CC-mDC, while stimulation with pIC-mDC resulted in reproducible cytomegalovirus pp65 or influenza M1 matrix peptide-specific T cell activation as compared to control cultures in the absence of DC. In addition, the T cell non-responsiveness after stimulation with iDC was shown to be mediated by TGF-beta and IL-10. Moreover, the suppressive capacity of CD4+ T cells activated by iDC and CC-mDC was shown to be transferable when these CD4+ T cells were added to an established T cell response. In contrast, addition of CD4+ T cells stimulated by pIC-mDC made responder T cells refractory to their suppressive activity. In conclusion, we hypothesize that DC have a complementary role in inducing both regulatory T cells and effector T cells, where the final result of antigen-specific T cell activation will depend on the activation state of the DC. This emphasizes the need for proper DC activation when T cell immunity is the desired effect, especially when used in clinical trials.

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

Blood ◽

10.1182/blood-2010-08-301275 ◽

2011 ◽

Vol 117 (11) ◽

pp. 3096-3103 ◽

Cited By ~ 56

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.

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

Blood ◽

10.1182/blood.v116.21.3728.3728 ◽

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.

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Efficient Treatment of Murine Acute Graft-Versus-Host Disease with In Vitro Expanded CD4+CD25+ Regulatory T Cells

Blood ◽

10.1182/blood.v118.21.2987.2987 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2987-2987

Author(s):

Tina J Boeld ◽

Kristina Doser ◽

Corinna Lang-Schwarz ◽

Elisabeth Huber ◽

Reinhard Andreesen ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Acute Gvhd ◽

Treg Cells ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract Abstract 2987 Acute graft-versus-host disease (GVHD) is a frequent complication after allogeneic bone marrow transplantation (BMT). We previously showed that the adoptive transfer of donor-type CD4+CD25+ regulatory T cells (Treg) at the time of BMT prevents acute GVHD in murine models. However, the therapeutic potential of donor-derived Treg cells for the treatment of established acute GVHD has not yet been examined in detail. In analogy to potential clinical applications we now tested the capacity of in vitro expanded Treg cells to ameliorate acute GVHD after haploidentical BMT (BALB/c→CB6F1). CD4+CD25highCD62L+ Treg cells were purified by FACS and stimulated polyclonally using anti-CD3/CD28-coated beads. Cells expanded on average 130±19-fold (n=7) within 2 wks and maintained high levels of FoxP3 expression (96, 8±0, 8% FoxP3+ cells; n=7) as well as potent immunosuppressive activity in vitro. For the induction of acute GVHD CB6F1 recipients were lethally irradiated and transplanted with 2.5×106 BM cells in combination with 5×106 splenocytes. All animals developed severe GVHD by d11, as revealed by an increase of the GVHD severity score (2.3±0.4 in GVHD animals vs 0±0 in BM controls, p<0.001, n=1–11) and by histological analyses of the gut (score: 7.8±0.4 for the GVHD group vs 0.2±0.2 for BM controls, p =0.046, n=3). When animals with acute GVHD were treated with 5×106 expanded CD4+CD25highCD62L+ Treg cells on d11 after BMT, they initially developed progressive GVHD comparable to non-treated GVHD animals, as indicated by weight loss and an increase of the GVHD score. However from d44 post BMT onwards, Treg-treated GVHD animals regained body weight (d44: 75±3% vs 67±2% of initial weight; p <0.05; n=9–10) and their clinical GVHD score (d44: 6±0 vs 4.3±0.4; p <0.05; n=9–10) decreased. While all non-treated GVHD animals succumbed to disease by d67 after transplantation, 50% of Treg-treated GVHD animals survived for at least 100d (p =0, 002; n=16–21). As immune reconstitution and in particular reconstitution of the lymphocyte compartment is impaired in animals with GVHD, we analyzed the effect of Treg therapy on the reconstitution of the lymphoid and myeloid compartment. At d21 after BMT spleen and BM of non-treated as well as Treg-treated GVHD animals were completely lymphopenic as compared to control mice and both organs contained exceptionally high numbers of granulocytes. Unlike non-treated GVHD animals, however, Treg-treated recipients by d60 showed a recovery of the lymphocyte compartment in spleen (10±2.6×106 T cells and 23.5±12.5×106 B cells in Treg-treated vs 3.0±0.6×106 T cells and 1.5±0.4×106 B cells in non-treated GVHD animals vs 26.25±2.6×106 T cells and 63.9±9.1×106 B cells in BM controls) and BM (0.7±0.1×106 T cells and 8.6±4×106 B cells in Treg-treated vs 0.3±0.01×106 T cells and 0.7±0.4 ×106 B cells in non-treated GVHD animals vs 0.4±0.03×106 T cells and 11.2±0.6×106 B cells in BM controls), while the number of granulocytes decreased constantly. Successful treatment with Treg cells was finally accompanied by a reconstitution of the lymphatic system comparable to control mice. Furthermore, successfully treated mice showed only mild histological signs of gut GVHD at d100 that was significantly lower then those in non-treated GVHD animals with end-stage disease (score: 4.2±1 vs 9.9±1.5 in treated vs non-treated animals, p =0.006, n=4–6). Taken together, these results indicate that in vitro expanded natural Treg cells may not only be effective for the prevention, but also for the treatment of acute GVHD after allogeneic BMT. Disclosures: No relevant conflicts of interest to declare.

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

Gene Therapy ◽

10.1038/s41434-021-00232-2 ◽

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.

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The relationship between human thymus-derived regulatory T cells, TGF-β-induced regulatory T cells and conventional CD4+ T cells as revealed by proteomics

10.1101/2021.10.30.466569 ◽

2021 ◽

Author(s):

Mark Mensink ◽

Ellen Schrama ◽

Maartje van den Biggelaar ◽

Derk Amsen ◽

Jannie Borst ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Protein Expression ◽

Treg Cell ◽

Ex Vivo ◽

Foxp3 Expression ◽

Treg Cells ◽

Cell Populations ◽

The Relationship

The CD4+ regulatory T (Treg) cell lineage, as defined by FOXP3 expression, comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. In human, naive tTreg cells can be purified from blood, but occur in low abundance, while effector pTreg and tTreg cell populations cannot be purified for lack of discriminating cell surface markers. Therefore, studies often employ TGF-β-induced (i)Treg cells that are generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells, as optimally purified from human blood. Global proteomic analysis revealed that iTreg, tTreg and Tconv cell populations each have a unique protein expression pattern. We next used as a benchmark a previously defined proteomic signature that discerns ex vivo naive and effector phenotype Treg cells from Tconv cells and reflects unique Treg cell properties. This Treg cell core signature was largely absent from iTreg cells, while clearly present in simultaneously analyzed tTreg cells. In addition, we used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naive Treg cells. This effector Treg cell signature was partially present in iTreg cells. Thus, iTreg cells are distinct from tTreg cells and largely lack the common Treg cell proteomic signature. However, they do have certain protein expression features in common with ex vivo effector Treg cells. These data demonstrate the utility of the core and effector Treg cell signatures as tools to define Treg cell populations and encourage the use of ex vivo Treg cells for functional analyses.

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

Frontiers in Immunology ◽

10.3389/fimmu.2021.766757 ◽

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.

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Role of regulatory T-cells in autoimmunity

Clinical Science ◽

10.1042/cs20080200 ◽

2009 ◽

Vol 116 (8) ◽

pp. 639-649 ◽

Cited By ~ 20

Author(s):

Richard J. Mellanby ◽

David C. Thomas ◽

Jonathan Lamb

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Treg Cell ◽

Cell Function ◽

Cell Subset ◽

Treg Cells ◽

Self Tolerance

There has been considerable historical interest in the concept of a specialist T-cell subset which suppresses over-zealous or inappropriate T-cell responses. However, it was not until the discovery that CD4+CD25+ T-cells had suppressive capabilities both in vitro and in vivo that this concept regained credibility and developed into one of the most active research areas in immunology today. The notion that in healthy individuals there is a subset of Treg-cells (regulatory T-cells) involved in ‘policing’ the immune system has led to the intensive exploration of the role of this subset in disease resulting in a number of studies concluding that a quantitative or qualitative decline in Treg-cells is an important part of the breakdown in self-tolerance leading to the development of autoimmune diseases. Although Treg-cells have subsequently been widely postulated to represent a potential immunotherapy option for patients with autoimmune disease, several studies of autoimmune disorders have demonstrated high numbers of Treg-cells in inflamed tissue. The present review highlights the need to consider a range of other factors which may be impairing Treg-cell function when considering the mechanisms involved in the breakdown of self-tolerance rather than focussing on intrinsic Treg-cell factors.

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Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells

APOPTOSIS ◽

10.1007/s10495-020-01629-x ◽

2020 ◽

Vol 25 (9-10) ◽

pp. 715-729 ◽

Cited By ~ 1

Author(s):

Lilla Prenek ◽

Tímea Litvai ◽

Noémi Balázs ◽

Réka Kugyelka ◽

Ferenc Boldizsár ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Treg Cells ◽

High Dose ◽

Apoptosis Resistance ◽

Apoptotic Pathway ◽

Glucocorticoid Hormone ◽

Induced Apoptosis

Abstract Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

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