• Journal of Periodontal and Implant Science
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• 제44권5호
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• pp.235-241
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• 2014

Purpose: Regulatory T cells (Tregs), expressing CD4 and CD25 as well as Foxp3, are known to play a pivotal role in immunoregulatory function in autoimmune diseases, cancers, and graft rejection. Dendritic cells (DCs) are considered the major antigen-presenting cells (APCs) for initiating these T-cell immune responses, of which $CD103^+$ DCs are derived from precursor human peripheral blood mononuclear cells (PBMCs). The aim of the present study was to evaluate the capacity of these PBMC-derived $CD103^+$ DCs to promote the differentiation of antigen-specific Tregs. Methods: Monocyte-derived DCs were induced from $CD14^+$ monocytes from the PBMCs of 10 healthy subjects. Once the $CD103^+$ DCs were purified, the cell population was enriched by adding retinoic acid (RA). Peptide numbers 14 and 19 of Porphyromonas gingivalis heat shock protein 60 (HSP60) were synthesized to pulse $CD103^+$ DCs as a tool for presenting the peptide antigens to stimulate $CD3^+$ T cells that were isolated from human PBMC. Exogenous interleukin 2 was added as a coculture supplement. The antigen-specific T-cell lines established were phenotypically identified for their expression of CD4, CD25, or Foxp3. Results: When PBMCs were used as APCs, they demonstrated only a marginal capacity to stimulate peptide-specific Tregs, whereas $CD103^+$ DCs showed a potent antigen presenting capability to promote the peptide-specific Tregs, especially for peptide 14. RA enhanced the conversion of $CD103^+$ DCs, which paralleled the antigen-specific Treg-stimulating effect, though the differences failed to reach statistical significance. Conclusions: We demonstrated that $CD103^+$ DCs can promote antigen-specific Tregs from naive T cells, when used as APCs for an epitope peptide from P. gingivalis HSP60. RA was an effective reagent that induces mature DCs with the typical phenotypic expression of CD103 that demonstrated the functional capability to promote antigen-specific Tregs.

• Molecules and Cells
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• 제38권11호
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• pp.998-1006
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• 2015

Retinols are metabolized into retinoic acids by alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (Raldh). However, their roles have yet to be clarified in hepatitis despite enriched retinols in hepatic stellate cells (HSCs). Therefore, we investigated the effects of retinols on Concanavalin A (Con A)-mediated hepatitis. Con A was injected into wild type (WT), Raldh1 knockout ($Raldh1^{-/-}$), $CCL2^{-/-}$ and $CCR2^{-/-}$ mice. For migration study of regulatory T cells (Tregs), we used in vivo and ex vivo adoptive transfer systems. Blockade of retinol metabolism in mice given 4-methylpyrazole, an inhibitor of ADH, and ablated Raldh1 gene manifested increased migration of Tregs, eventually protected against Con A-mediated hepatitis by decreasing interferon-${\gamma}$ in T cells. Moreover, interferon-${\gamma}$ treatment increased the expression of ADH3 and Raldh1, but it suppressed that of CCL2 and IL-6 in HSCs. However, the expression of CCL2 and IL-6 was inversely increased upon the pharmacologic or genetic ablation of ADH3 and Raldh1 in HSCs. Indeed, IL-6 treatment increased CCR2 expression of Tregs. In migration assay, ablated CCR2 in Tregs showed reduced migration to HSCs. In adoptive transfer of Tregs in vivo and ex vivo, Raldh1-deficient mice showed more increased migration of Tregs than WT mice. Furthermore, inhibited retinol metabolism increased survival rate (75%) compared with that of the controls (25%) in Con A-induced hepatitis. These results suggest that blockade of retinol metabolism protects against acute liver injury by increased Treg migration, and it may represent a novel therapeutic strategy to control T cell-mediated acute hepatitis.

• The Korean Journal of Physiology and Pharmacology
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• 제19권2호
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• pp.83-88
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• 2015

CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) have been shown to play a regulatory or suppressive role in the immune response and are possibly relevant to the pathogenesis of autoimmune diseases. In the present study, we attempted to investigate the frequency of CD4+CD25+ Tregs in peripheral blood (PB) of collagen-induced arthritis (CIA) rats during the development of arthritis, to determine whether their frequency is involved in the immunoregulation of this disease. The results showed that normal rats had similar frequencies of CD4+CD25+ Tregs in PB during the experiment time, expressed as a percentage of CD4+CD25+Foxp3+ T cells among the CD4+ T lymphocyte population. In contrast, the frequency of CD4+CD25+Foxp3+ T cells in CIA rats was found to change during the development of arthritis. In CIA rats, there is a significant negative correlation between the frequency of CD4+CD25+Foxp3+ T cells and paw swelling (r=-0.786, p< 0.01). The relationship between the frequency of CD4+CD25+Foxp3+ T and immune activation was not found in normal rats. During the time course, the frequency of CD4+CD25+Foxp3+ T was lower in CIA rats than in normal ones. The data suggest that the frequency of PB CD4+CD25+ Tregs may be a promising marker for arthritis activity.

• Molecules and Cells
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• 제43권5호
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• pp.438-447
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• 2020

The aim of this study was to explore the role of IL-6-miR-210 in the regulation of Tregs function and atrial fibrosis in atrial fibrillation (AF). The levels of interleukin (IL)-6 and IL-10 in AF patients were detected by using ELISA. Proportions of Treg cells were detected by fluorescence activated cell sorting analysis in AF patients. The expression of Foxp3, α-SMA, collagen I and collagen III were determined by western blot. The atrial mechanocytes were authenticated by vimentin immunostaining. The expression of miR-210 was performed by quantitative real-time polymerase chain reaction (qRT-PCR). TargetScan was used to predict potential targets of miR-210. The cardiomyocyte transverse sections in AF model group were observed by H&E staining. The myocardial filaments were observed by masson staining. The level of IL-6 was highly increased while the level of IL-10 (Tregs) was significantly decreased in AF patients as compared to normal control subjects, and IL-6 suppressed Tregs function and promoted the expression of α-SMA, collagen I and collagen III. Furthermore, miR-210 regulated Tregs function by targeting Foxp3, and IL-6 promoted expression of miR-210 via regulating hypoxia inducible factor-1α (HIF-1α). IL-6-miR-210 suppresses regulatory T cell function and promotes atrial fibrosis by targeting Foxp3.

• IMMUNE NETWORK
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• 제13권6호
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• pp.264-274
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• 2013

The unrestricted population of $CD4^+Foxp3^+$ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific $CD4^+Foxp3^+$ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect $CD4^+Foxp3^+$ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV $GP_{66-77}$-specific $CD4^+$ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV $GP_{66-77}$-specific $CD4^+$ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV $GP_{66-77}$-specific $CD4^+Foxp3^+$ Tregs using $Foxp3^{GFP}$ knock-in mouse, and found that LCMV $GP_{66-77}$-specific $CD4^+Foxp3^+$ Tregs and polyclonal $CD4^+Foxp3^+$ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific $CD4^+Foxp3^+$ Treg in various models.

• IMMUNE NETWORK
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• 제23권5호
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• pp.37.1-37.11
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• 2023

Forkhead box P3-positive (Foxp3⁺)-inducible Tregs (iTregs) are readily generated by TGF-β1 at low TCR signaling intensity. TGF-β1-mediated Foxp3 expression is further enhanced by retinoic acid (RA) and lactoferrin (LF). However, the intensity of TCR signaling required for induction of Foxp3 expression by TGF-β1 in combination with RA and LF is unknown. Here, we found that either RA or LF alone decreased TGF-β1-mediated Foxp3 expression at low TCR signaling intensity. In contrast, at high TCR signaling intensity, the addition of either RA or LF strongly increased TGF-β1-mediated Foxp3 expression. Moreover, decreased CD28 stimulation was more favorable for TGF-β1/LF-mediated Foxp3 expression. Lastly, we found that at high signaling intensities of both TCR and CD28, combined treatment with TGF-β1, RA, and LF induced robust expression of Foxp3, in parallel with powerful suppressive activity against responder T cell proliferation. Our findings that TGFβ/RA/LF strongly generate high affinity Ag-specific iTreg population would be useful for the control of unwanted hypersensitive immune reactions such as various autoimmune diseases.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.136-145
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• 2024

People with obesity maintain low levels of inflammation; therefore, their exposure to foreign antigens can trigger an excessive immune response. In people with obesity or allergic contact dermatitis (ACD), symptoms are exacerbated by a reduction in the number of regulatory T cells (Tregs) and IL-10/TGF-β-modified macrophages (M2 macrophages) at the inflammatory site. Benefits of intermittent fasting (IF) have been demonstrated for many diseases; however, the immune responses regulated by macrophages and CD4⁺T cells in obese ACD animal models are poorly understood. Therefore, we investigated whether IF suppresses inflammatory responses and upregulates the generation of Tregs and M2 macrophages in experimental ACD animal models of obese mice. The IF regimen relieved various ACD symptoms in inflamed and adipose tissues. We showed that the IF regimen upregulates Treg generation in a TGF-β-dependent manner and induces CD4⁺T cell hypo-responsiveness. IF-M2 macrophages, which strongly express TGF-β and inhibit CD4⁺T cell proliferation, directly regulated Treg differentiation from CD4⁺T cells. These results indicate that the IF regimen enhances the TGF-β-producing ability of M2 macrophages and that the development of Tregs keeps mice healthy against ACD exacerbated by obesity. Therefore, the IF regimen may ameliorate inflammatory immune disorders caused by obesity.

• IMMUNE NETWORK
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• 제20권4호
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• pp.34.1-34.8
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• 2020

Cryopreservation and thawing of PBMCs are inevitable processes in expanding the scale of experiments in human immunology. Here, we carried out a fundamental study to investigate the detailed effects of PBMC cryopreservation and thawing on transcriptomes. We sorted Tregs from fresh and cryopreserved/thawed PBMCs from an identical donor and performed single-cell RNA-sequencing (scRNA-seq). We found that the cryopreservation and thawing process minimally affects the key molecular features of Tregs, including FOXP3. However, the cryopreserved and thawed sample had a specific cluster with up-regulation of genes for heat shock proteins. Caution may be warranted in interpreting the character of any cluster of cells with heat shock-related properties when cryopreserved and thawed samples are used for scRNA-seq.

• IMMUNE NETWORK
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• 제11권5호
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• pp.299-306
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• 2011

Background: $CD4^+Fop3^+$ regulatory T cells (Tregs) are needed to maintain peripheral tolerance, but their role in the development of autoimmune arthritis is still debated. The present study was undertaken to investigate the mechanism by which Tregs influence autoimmune arthritis, using a mouse model entitled K/BxN. Methods: We generated Treg-deficient K/BxNsf mice by congenically crossing K/BxN mice with Foxp3 mutant scurfy mice. The arthritic symptoms of the mice were clinically and histopathologically examined. The proportions and activation of $CD4^+$ T cells and/or dendritic cells were assessed in the spleens, draining lymph nodes and synovial tissue of these mice. Results: K/BxNsf mice exhibited earlier onset and more aggressive progression of arthritis than their K/BxN littermates. In particular, bone destruction associated with the influx of numerous RANKL+ cells into synovia was very prominent. They also contained more memory phenotype $CD4^+$ T cells, more Th1 and Th2 cells, and fewer Th17 cells than their control counterparts. Plasmacytoid dendritic cells expressing high levels of CD86 and CD40 were elevated in the K/BxNsf synovia. Conclusion: We conclude that Tregs oppose the progression of arthritis by inhibiting the development of $RANKL^+$ cells, homeostatically proliferating $CD4^+$ T cells, Th1, Th2 and mature plasmacytoid dendritic cells, and by inhibiting their influx into joints.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7681-7684
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• 2013

Background: Human leukocyte antigen (HLA)-G-positive gastric cancers are associated with poor survival, but links with tumor escape mechanisms remain to be determined. Materials and Methods: We used immunohistochemistry to investigate HLA-G expression, tumor infiltrating CD8+ T lymphocytes, and Treg cells in 52 gastric cancer patients. Results: There were 29 cancer-related deaths during the follow-up period. Kaplan-Meier analysis indicated that patients with HLA-G-positive (n=16) primary tumors had a significantly poorer prognosis than patients with HLA-G-negative tumors (n=36, p=0.008). The median survival time was 14 months and 47 months, respectively. Patients with high numbers of Tregs and low numbers of CD8+T lymphocytes in the primary tumor had a poorer prognosis than those with low numbers of Tregs and high numbers of CD8+T lymphocytes (p=0.034, p=0.043). Multivariate Cox proportional hazard regression analysis showed that HLA-G expression (hazard ratio: 2.662; 95% confidence interval: 1.242-5.723; p=0.012) and stage (hazard ratio: 2.012;95% confidence interval: 1.112-3.715; p=0.041) were independent unfavorable factors for patient survival. Conclusions: We found a significant positive correlation between HLA-G expression and the number of tumor infiltrating Tregs (p=0.01) and a negative correlation with the number of CD8+T lymphocytes (p=0.041). HLA-G may protect gastric cancer cells from cytolysis by inducing Foxp3+Treg lymphocytes and suppressing CD8+T lymphocytes.