• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1189-1197
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• 2017

Lichen-forming fungi are known to have various biological activities, such as antioxidant, antimicrobial, antitumor, antiviral, anti-inflammation, and anti proliferative effects. However, the immunosuppressive effects of Bryoria sp. extract (BSE) have not previously been investigated. In this study, the inhibitory activity of BSE on the proliferation of $CD8^+$ T cells and the mixed lymphocytes reaction (MLR) was evaluated in vitro. BSE was non-toxic in spleen cells and suppressed the growth of splenocytes induced by anti-CD3. The suppressed cell population in spleen cells consisted of $CD8^+$ T cells and their proliferation was inhibited by the treatment with BSE. This extract significantly suppressed the IL-2 associated with T cell growth and $IFN-{\gamma}$ as the $CD8^+$ T cell marker. Furthermore, BSE reduced the expression of the IL-2 receptor alpha chain ($IL-2R{\alpha}$) on $CD8^+$ T cells and CD86 on dendritic cells by acting as antigen-presenting cells. Finally, the MLR produced by the co-culture of C57BL/6 and MMC-treated BALB/c was suppressed by BSE. IL-2, $IFN-{\gamma}$, and CD69 on $CD8^+$ T cells in MLR condition were inhibited by BSE. These results indicate that BSE inhibits the MLR via the suppression of $IL-2R{\alpha}$ expression in $CD8^+$ T cells. BSE has the potential to be developed as an anti-immunosuppression agent for organ transplants.

• Journal of Veterinary Clinics
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• v.37 no.6
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• pp.350-354
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• 2020

Feline chronic lymphocytic leukemia (CLL) is a rare disease. Its diagnosis is not simple because of the absence of clinical signs and the presence of mature lymphocytosis. An 11-year-old female spayed Russian Blue cat was referred to the veterinary medical teaching hospital for lethargy, diarrhea, weight loss, and inappetence. Marked lymphocytic leukocytosis and a significantly increased number of small-to-intermediate-sized lymphocytes in the peripheral blood were found on hematological examination. The results of the feline leukemia virus and immunodeficiency virus test were negative. Further, mild splenomegaly was detected. Bone marrow aspirate analysis revealed mature lymphocytosis and a clonally rearranged T cell receptor gene with the polymerase chain reaction (PCR) for antigen receptor rearrangement assay. Flow cytometric immunophenotyping showed a homogeneous population of CD5+/CD21-T-cells in the peripheral blood and bone marrow. According to the results of the aforementioned examinations, CLL was diagnosed. Treatment was not initiated at the time of diagnosis because the clinical signs were mild and did not affect the quality of life. This report describes the clinical findings and use of advanced diagnostic tools such as molecular clonality analysis and immunophenotyping for the diagnosis of feline CLL.

• Tuberculosis and Respiratory Diseases
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• v.65 no.4
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• pp.343-350
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• 2008

PD-L1 is expressed in a variety of antigen-presenting cells and provides T cell tolerance via ligation with its receptor PD-1 and B7-1 on T cells. Stimulation with lipopolysaccharide (LPS) can increase the level of PD-L1 expression in B cells and macrophages, which suggests that this molecule plays a role in the immunosuppression observed in severe sepsis. The aim of this study was to identify which of the downstream pathways of TLR4 are involved in the up-regulation of PD-L1 by LPS in macrophages. Flow cytometry was used to examine the expression of PD-L1 in RAW 264.7 macrophages stimulated with LPS. The following chemical inhibitors were used to evaluate the role of each pathway: LY294002 for PI3K/Akt, SB202190 for p38 MAPK, and U0126 for MEK. LPS induced the expression of PD-L1 in a time- and dose-dependent manner. Transfection of siRNA for TLR4 suppressed the induction of PD-L1. Pretreatment with LY294002 and SB202190 decreased the level of PD-L1 expression but U0126 did not. Overall, the PI3K/Akt and p38 MAPK pathways are involved in the up-regulation of PD-L1 expression in RAW 264.7 macrophages stimulated with LPS.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.5
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• pp.303-310
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• 2002

Cell therapy applied to wound healing or tissue regeneration presents a revolutionary realm to which principles of gene engineering and delivery may be applied. One promising application is the transplantation of cells into the wounded tissue to help the tissue repair. However, when cells are transplanted from in vitro to in vivo, immune rejection occurs due to the immune response triggered by the activation of T-cell, and the transplanted cells are destroyed by the attack of activated T-cell and lose their function. Immune suppressant such as FK506 is commonly used to suppress immune rejection during transplantation. However, such kind of immune suppressants not only suppresses immune rejection in the periphery of transplanted cells but also suppresses whole immune response system against pathogenic infection. In order to solve this problem, we developed a method to protect the desired cells from immune rejection without impairing whole immune system during cell transplantation. Previously, we reported the success of constructing glomerular epithelial cells for removal of immune complex, in which complement receptor of type 1 (CR1) was over-expressed on the membrane of renal glomerular epithelial cells and could bind immune complex of DNA/anti-DNA-antibody to remove immune complex through phagocy-tosis [1]. Attempting to apply the CR1-expressing cells to cell therapy and evade immune rejection during cell transplantation, we constructed three plasmids containing genes encoding a soluble fusion protein of cytolytic T lymphocyte associated antigen-4 (CTLA4Ig) and an enhanced green fluorescent protein (EGFP). The plasmids were transfected to the above-mentioned glomerular epithelial cells to express both genes simultaneously. Using the clone cells for cell transplantation showed that mice with autoimmune disease prolonged their life significantly as compared with the control mice, and two injections of the cells at the beginning of two weeks resulted in remarkable survivability, whereas it requires half a year and 50 administrations of proteins purified from the same amount of cells to achieve the same effect.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2184-2191
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• 2016

The time-consuming and high-cost preparation of soluble peptide-major histocompatibility complexes (pMHC) currently limits their wide uses in monitoring antigen-specific T cells. The single-chain trimer (SCT) of peptide-${\beta}2m$-MHC class I heavy chain was developed as an alternative strategy, but its gene fusion is hindered in many cases owing to the incompatibility between the multiple restriction enzymes and the restriction endonuclease sites of plasmid vectors. In this study, overlap extension PCR and one-step cloning were adopted to overcome this restriction. The SCT gene of the $OVA_{257-264}$ peptide-$(GS_4)_3-{\beta}2m-(GS_4)_4-H-2K^b$ heavy chain was constructed and inserted into plasmid pET28a by overlap extension PCR and one-step cloning, without the requirement of restriction enzymes. The SCT protein was expressed in Escherichia coli, and then purified and refolded. The resulting $H-2K^b/OVA_{257-264}$ complex showed the correct structural conformation and capability to bind with $OVA_{257-264}$-specific T-cell receptor. The overlap extension PCR and one-step cloning ensure the construction of single-chain MHC class I molecules associated with random epitopes, and will facilitate the preparation of soluble pMHC multimers.

• Journal of Microbiology and Biotechnology
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• v.23 no.7
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• pp.1023-1030
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• 2013

Lipoteichoic acid (LTA), uniquely expressed on gram-positive bacteria, is recognized by Toll-like receptor 2 (TLR2) on not only antigen-presenting cells but also activated T cells. Therefore, it is reasonable to assume that LTA is acting on T cells. However, little is known about the effect of LTA on T-cell regulation. In the present study, we investigated the immunomodulatory effects of LTA on $CD4^+$ T cells. Effector $CD4^+$ T cells, induced after co-culture with S. aureus-pulsed dendritic cells, produced high levels of interferon-${\gamma}$, CD25, CD69, and TLRs 2 and 4. When effector $CD4^+$ T cells were treated with LTA, the expressions of the membrane-bound form of transforming growth factor (TGF)-${\beta}$ and forkhead box P3 increased. Coincidently, the proliferation of effector $CD4^+$ T cells was declined after LTA treatment. When TGF-${\beta}$ signaling was blocked by the TGF-${\beta}$ receptor 1 kinase inhibitor, LTA failed to suppress the proliferation of effector $CD4^+$ T cells. Therefore, the present results suggest that LTA suppresses the activity of effector $CD4^+$ T cells by enhancing TGF-${\beta}$ production.

• Biomolecules & Therapeutics
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• v.30 no.5
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• pp.418-426
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• 2022

Chimeric antigen receptor T (CAR-T) cell therapy is one of the promising anticancer treatments. It shows a high overall response rate with complete response to blood cancer. However, there is a limitation to solid tumor treatment. Additionally, this currently approved therapy exhibits side effects such as cytokine release syndrome and neurotoxicity. Alternatively, bispecific antibody is an innovative therapeutic tool that simultaneously engages specific immune cells to disease-related target cells. Since programmed death ligand 1 (PD-L1) is an immune checkpoint molecule highly expressed in some cancer cells, in the current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) which can engage T cells to PD-L1⁺ cancer cells. We observed that the BiTE-bound OT-1 T cells effectively killed cancer cells in vitro and in vivo. They substantially increased the recruitment of effector memory CD8⁺ T cells having CD8⁺CD44⁺CD62L^low phenotype in tumor. Interestingly, we also observed that BiTE-bound polyclonal T cells showed highly efficacious tumor killing activity in vivo in comparison with the direct intravenous treatment of bispecific antibody, suggesting that PD-L1-directed migration and engagement of activated T cells might increase cancer cell killing. Additionally, BiTE-bound CAR-T cells which targets human Her-2/neu exhibited enhanced killing effect on Her-2-expressing cancer cells in vivo, suggesting that this could be a novel therapeutic regimen. Collectively, our results suggested that engaging activated T cells with cancer cells using αCD3xαPD-L1 BiTE could be an innovative next generation anticancer therapy which exerts simultaneous inhibitory functions on PD-L1 as well as increasing the infiltration of activated T cells having effector memory phenotype in tumor site.

• BMB Reports
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• v.47 no.2
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• pp.115-120
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• 2014

In this study, we show that Mycobacterium avium subsp. paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-${\alpha}$, and IL-$1{\beta}$) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naive T cells to polarized $CD4^+$ and $CD8^+$ T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. paratuberculosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of $CD4^+$ and $CD8^+$ T cells.

• Tuberculosis and Respiratory Diseases
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• v.42 no.6
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• pp.823-830
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• 1995

Background: It has been found that Helper T cells in the peripheral blood are decreased in the cell mediated immunity in the pulmonary tuberculosis. But it has not been confirmed yet that only decrease in number of cells which has phenotype in the peripheral blood is defined to decrease in cell mediated immunity. The immunocytochemical study was performed to observe the change of the percentage of T-lymphocytes with their subsets and activated T cells in the peripheral blood of pulmonary tuberculosis and to know how many T cells would be activated, relative to resting cells in the peripheral blood. Methods: The peripheral blood obtained from twenty two patients and ten healthy controls were smeared on the gelatin coated slide glass prepared for of mononuclear cells. The double bridge technique of alkaline phosphatase-antialkaline phosphatase(APAAP) method was used. As the primary antibodies, $T_1$(anti-human T cell), $T_4$(anti-human helper/inducer T cells) and $T_8$(anti-human supressor/cytotoxic T cell) antibodies and interleukin-2 receptor (for early activated T cell), very late activation antigen (for activated cytotoxic T cell), T cell lineage specific activation antigen monoclonal actibodies were used. Results: 1) There were significantly decrease in the absolute number of $T_4$(+) cells but significantly increase of $T_8$(+) cells in the peripheral blood of pulmonary tuberculosis (p<0.05). 2) The percentage of $T_4$(+) cells showed significantly decrease in pulmonary tuberculosis but $T_8$(+)cells significantly increase(p<0.05). $T_4(+)/T_8(+)$ ratio showed significantly decrease in the peripheral blood of the pulmonary tuberculosis(p<0.05). 3) There were significantly increase in the absolute number of variable stages of activated T cells in the peripheral blood of the pulmonary tuberculosis(p<0.05). 4) The percentage of IL-2R, VLA-1, TLiSA were 6.45+1.56%, $7.64+1.34^*$, 10.45+1.16% in order which showed significantly increase in the peripheral blood of the pulmonary tuberculosis(p<0.05). Conclusion: We speculate that only a few percentage of T lymphocyte is activated in cell mediated immunity in pulmonary tuberculosis.

• 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.