• IMMUNE NETWORK
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• v.23 no.4
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• pp.35.1-35.16
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• 2023

Defining the molecular dynamics associated with T cell differentiation enhances our understanding of T cell biology and opens up new possibilities for clinical implications. In this study, we investigated the dynamics of CD5 expression in CD8⁺ T cell differentiation and explored its potential clinical uses. Using PBMCs from 29 healthy donors, we observed a stepwise decrease in CD5 expression as CD8⁺ T cells progressed through the differentiation stages. Interestingly, we found that CD5 expression was initially upregulated in response to T cell receptor stimulation, but diminished as the cells underwent proliferation, potentially explaining the differentiation-associated CD5 downregulation. Based on the proliferation-dependent downregulation of CD5, we hypothesized that relative CD5 expression could serve as a marker to distinguish the heterogeneous CD8⁺ T cell population based on their proliferation history. In support of this, we demonstrated that effector memory CD8⁺ T cells with higher CD5 expression exhibited phenotypic and functional characteristics resembling less differentiated cells compared to those with lower CD5 expression. Furthermore, in the retrospective analysis of PBMCs from 30 non-small cell lung cancer patients, we found that patients with higher CD5 expression in effector memory T cells displayed CD8⁺ T cells with a phenotype closer to the less differentiated cells, leading to favorable clinical outcomes in response to immune checkpoint inhibitor (ICI) therapy. These findings highlight the dynamics of CD5 expression as an indicator of CD8⁺ T cell differentiation status, and have implications for the development of predictive biomarker for ICI therapy.

• Clinical and Experimental Vaccine Research
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• v.12 no.4
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• pp.328-336
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• 2023

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.81-87
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• 2004

Background: In the thymus, developing thymocytes continually interact with thymic epithelial cell components. Self MHC restriction of mature T cells are imposed in the thymus through interaction of immature double positive thymocytes and thymic cortical epithelial cells. The site of negative selection, however, is a matter of debate. Through systemic injection of anti-TCR antibody or antigenic peptides, investigators suggested that most of the negative selection occurs in the thymic cortex. But the requirements for negative selection, i.e cellular counterparts and costimulatory molecules are more available in the medulla or cortico-medullary junction rather than in the thymic cortex. Methods: The direct and indirect pathways of thymocyte death after systemic anti-TCR antibody injection were separated through several experimental systems. B6 mice were either adrenalectomized or sham-adrenalectomized to evaluate the role of endogenous glucocorticoids from adrenal gland. Role of TNF were evaluated through using TNF receptor double knockout mice. Results: We found that without indirectly acting mediators such as $TNF-\alpha$ or corticosteroid, double positive thymocyte death were minimal by systemic injection of anti-TCR antibody in TNF receptor double knockout neonatal mice. Also by analyzing neonatal wild-type mice with adoptively transferred mature T cells, only peripheral activation of mature T cells could induce extensive double positive thymocyte death. Conclusion: Thus, systemically injected anti-TCR antibody mediated thymocyte death are mostly induced through indirect pathway.

• IMMUNE NETWORK
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• v.21 no.5
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• pp.34.1-34.16
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• 2021

Sjögren's syndrome (SS) is an autoimmune disease characterized by dryness of the mouth and eyes. The glandular dysfunction in SS involves not only T cell-mediated destruction of the glands but also autoantibodies against the type 3 muscarinic acetylcholine receptor or aquaporin 5 (AQP5) that interfere with the secretion process. Studies on the breakage of tolerance and induction of autoantibodies to these autoantigens could benefit SS patients. To break tolerance, we utilized a PmE-L peptide derived from the AQP5-homologous aquaporin of Prevotella melaninogenica (PmAqp) that contained both a B cell "E" epitope and a T cell epitope. Repeated subcutaneous immunization of C57BL/6 mice with the PmE-L peptide efficiently induced the production of Abs against the "E" epitope of mouse/human AQP5 (AQP5E), and we aimed to characterize the antigen specificity, the sequences of AQP5E-specific B cell receptors, and salivary gland phenotypes of these mice. Sera containing anti-AQP5E IgG not only stained mouse Aqp5 expressed in the submandibular glands but also detected PmApq and PmE-L by immunoblotting, suggesting molecular mimicry. Characterization of the AQP5E-specific autoantibodies selected from the screening of phage display Ab libraries and mapping of the B cell receptor repertoires revealed that the AQP5E-specific B cells acquired the ability to bind to the Ag through cumulative somatic hypermutation. Importantly, animals with anti-AQP5E Abs had decreased salivary flow rates without immune cell infiltration into the salivary glands. This model will be useful for investigating the role of anti-AQP5 autoantibodies in glandular dysfunction in SS and testing new therapeutics targeting autoantibody production.

• Animal Bioscience
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• v.34 no.10
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• BMB Reports
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• v.35 no.1
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• pp.7-18
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• 2002

The major function of the thymus is to eliminate developing thymocytes that are potentially useless or autoreactive, and select only those that bear functional T cell antigen receptors (TCRs) through fastidious screening. It is believed that glucocorticoids (GCs) are at least in part responsible for cell death during death by neglect. In this review, we will mainly cover the topic of the GC-induced apoptosis of developing thymocytes. We will also discuss how thymocytes that are fated to die by GCs can be rescued from GC-induced apoptosis in. response to a variety of signals with antagonizing properties for GC receptor (GR) signaling. Currently, a lot of evidence supports the notion that the decision is made as a result of the integration of the multiple signal transduction networks that are triggered by GR, TCR, and Notch. A few candidate molecules at the converging point of these multiple signaling pathyways will be discussed. We will particularly describe the role of the SRG3 protein as a potent modulator of GC-induced apoptosis in the crosstalk.

• Parasites, Hosts and Diseases
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• v.50 no.4
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• pp.281-286
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• 2012

Helminthic infections afflict over 1.5 billion people worldwide, while Mycobacterium tuberculosis infects one third of the world's population, resulting in 2 million deaths per year. Although tuberculosis and helminthic infections coexist in many parts of the world, and it has been demonstrated that the T-helper 2 and T-regulatory cell responses elicited by helminths can affect the ability of the host to control mycobacterial infection, it is still unclear whether helminth infections in fact affect tuberculosis disease. In this review article, current progress in the knowledge about the immunomodulation induced by helminths to diminish the protective immune responses to bacille Calmette-Guerin vaccination is reviewed, and the knowledge about the types of immune responses modulated by helminths and the consequences for tuberculosis are summarized. In addition, recent data supporting the significant reduction of both M. tuberculosis antigen-specific Toll-like receptor (TLR) 2 and TLR9 expression, and pro-inflammatory cytokine responses to TLR2 and TLR9 ligands in individuals with M. tuberculosis and helminth co-infection were discussed. This examination will allow to improve understanding of the immune responses to mycobacterial infection and also be of great relevance in combating human tuberculosis.

• BMB Reports
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• v.47 no.9
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• pp.512-517
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• 2014

In this study, we showed that Mycobacterium abscessus MAB2560 induces the maturation of dendritic cells (DCs), which are representative antigen-presenting cells (APCs). M. abscessus MAB2560 stimulate the production of pro-inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-${\alpha}$, IL-$1{\beta}$, and IL-12p70] and reduce the endocytic capacity and maturation of DCs. Using $TLR4^{-/-}$ DCs, we found that MAB2560 mediated DC maturation via Toll-like receptor 4 (TLR4). MAB2560 also activated the MAPK signaling pathway, which was essential for DC maturation. Furthermore, MAB2560-treated DCs induced the transformation of $na\ddot{i}ve$ T cells to polarized $CD4^+$ and $CD8^+$ T cells, which would be crucial for Th1 polarization of the immune response. Taken together, our results indicate that MAB2560 could potentially regulate the host immune response to M. abscessus and may have critical implications for the manipulation of DC functions for developing DC-based immunotherapy.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.61-69
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• 2001

Background: To determine the molecular structure of type II collagen-specific T-cell receptors associated with rheumatoid arthritis (RA). Methods: We generated CII-specific T-cell lines of 8 RA patients by prolonged in vitro culture with bovine CII (bCII) and the immunogenic peptide (256-270) of human CII. The proliferation response towards CII stimulation was measured from the uptake of 3H-thymidine. Changes in the secretion of Th 1 and Th2 cytokines in the culture supernatent were measured by ELISA. The TCR clonotypes of these T-cells were examined by RT-PCR/SSCP analyses of all 22 $V_{\beta}$ chains. Results: T-cells from patients' tissue exhibited strong proliferation index upon CII stimulation, which was maintained up to 6 months in the culture. The secretion of INF-$\gamma$from these T-cells increased along with the duration of culture time, while the amount of IL-4 production did not show significant changes. The SSCP band patterns of patients' T-cells appear as discrete bands unlike the smeary streak produced from normal samples. Some SSCP bands, each representing selected expansion of a TCR containing certain subtype of $V_{\beta}$ peptides, appeared to be identical in more than one patients. Among these, the expansion of SSCP band representing the $V_{\beta}$ 14 CDR3 region persisted after switching the antigen to the immunogenic human peptide (256-270). Conclusion: CII-reactive T-cells expressing distinct CDR3 motifs are selectively expanded in the peripheral blood and synovial fluid of RA patients, and their persistent proliferation upon CII stimulation, as well as the production Th 1-type cytokines, may play pivotal roles in RA pathogenesis.

• Journal of Ginseng Research
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• v.40 no.1
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• pp.18-27
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• 2016

Background: It is not clear whether ginseng affects cyclosporine A (CsA)-induced desirable immunosuppressive action. In this study, we evaluated the immunological influence of combined treatment of ginseng with CsA. Methods: Using CD4+ T cells from mouse spleens stimulated with the T cell receptor (TCR) or allogeneic antigen-presenting cells (APCs), we examined the differentiation of naïve T cells into T helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs), and their cytokine production during treatment by Korean Red Ginseng extract (KRGE) and/or CsA. The influence of KRGE on the allogeneic T cell response was evaluated by mixed lymphocyte reaction (MLR). We also evaluated whether signal transducer and activator of transcription 3 (STAT3) and STAT5 are implicated in this regulation. Results: Under TCR stimulation, KRGE treatment did not affect the population of CD4+interferon gamma ($IFN{\gamma}$)+ and CD4+interleukin (IL)-4+ cells and their cytokine production compared with CsA alone. Under the Th17-polarizing condition, KRGE significantly reduced the number of CD4+IL-17+ cells and CD4+/phosphorylated STAT3 (p-STAT3)+ cells, but increased the number of CD4+CD25+forkhead box P3 (Foxp3)+ cells and CD4+/p-STAT5+ cells compared with CsA alone. In allogeneic APCs-stimulated CD4+ T cells, KRGE significantly decreased total allogeneic T cell proliferation. Consistent with the effects of TCR stimulation, KRGE reduced the number of CD4+IL-17+ cells and increased the number of CD4+CD25+Foxp3+ cells under the Th17-polarizing condition. Conclusion: KRGE has immunological benefits through the reciprocal regulation of Th17 and Treg cells during CsA-induced immunosuppression.