• BMB Reports
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• v.30 no.6
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• pp.385-389
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• 1997

Many cell types are known to stimulate $CD8^+$ T cells in allogeneic recognition such as mixed lymphocyte reaction (MLR). Whereas dendritic cells are most potent among them. T cells are usually considered very poor in stimulating $CD8^+$ T cells although there are some tumor cells that are weakly stimulatory. T cells, as a stimulator, cultured in the presence of concanavalin A that were otherwise nonstimulatory to $CD8^+$ T cells appeared to stimulate $CD8^+$ T cells strongly when they were pretreated with neuraminidase. The enhancement of MLR by neuraminidase could be achieved by treating either the stimulators or responders with neuraminidase. Removal of negatively-charged sialic acid moieties from the cell surface, which reduced electrostatic repulsion between responders and stimulators to give better cell-cell contact might be responsible for the enhanced MLR. In addition, neuraminidase treatment also appeared to deliver activation signal to responding T cells since it could activate $CD8^+$ T cells in synergy with phorbol myristate acetate. The maximal responses were observed when both responders and stimulators were treated with neuraminidase.

• Archives of Pharmacal Research
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• v.26 no.3
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• pp.192-196
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• 2003

This study describes the synthesis of novel enol esters (3) and triketones (4) as analogues of succinylacetone (SA) (Ed- this abbreviation is introduced here based on your use of it in the body of the paper) and the evaluation on the mouse allogeneic mixed lymphocyte reaction (MLR) and the murine model of antigen-induced paw edema formation for immunosuppressive activity. Enol esters (3a-f) were about 2-4 fold more potent than SA in in vitro activity.

• YAKHAK HOEJI
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• v.49 no.1
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• pp.92-96
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• 2005

In order to investigate the effects of bisphenol A (BPA) on cell mediated immune reaction in vitro we examined the allogenic mixed lymphocyte reaction (MLR), splenocytes proliferation (SP) to T cell mitogens and IFN-${\gamma}\;production$. Splenocytes of Balb/c mice ($1.5{\times}10^5$ cells/well) were co-cultured with different numbers of mitomycin C-treated mature dentritic cells (DCs) in presence of BPA (25, 50, 100 ${\mu}M$) and $[^{3}H]$thymidine incorporation (cpm) was measured by scintilation counting. Splenocytes ($2{\times}10^6$ cells/well) were cultured with mitogens, Con A ($2\;{\mu}g/ml$), PHA ($5\;{\mu}g/ml$) and IL-2 ($0.1\;{\mu}g/ml$), or PMA ($5\;{\mu}g/ml$) and INO ($1\;{\mu}g/ml$) in presence of BPA (1, 10, 25, 50, 100 ${\mu}M$) and SP was assessed by MTT assay. $IFN-{\gamma}$ levels in culture supernant were determined by ELISA. At low concentration, BPA slightly increased MLR, SP and $IFN-{\gamma}$ levels, but at higher concentration it showed significant inhibitory effects on these immunological parameters. These results indicate that BPA is able to alternate cell-mediated immune reaction.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1066-1076
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• 2012

Previous observations demonstrated that various immunosuppressive agents and their combination therapies can increase allograft survival rates. However, these treatments may have serious side effects and cannot substantially improve or prolong graft survival in acute graft-versus-host disease (GVHD). To improve the therapeutic potency of divalent immunoadhesins, we have constructed and produced several tetravalent forms of immunoadhesins comprising each of cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), CD2, and lymphocyte activation gene-3 (LAG3). Flow cytometric and T cell proliferation analyses displayed that tetravalent immunoadhesins have a higher binding affinity and more potent efficacy than divalent immunoadhesins. Although all tetravalent immunoadhesins possess better efficacies, tetravalent forms of CTLA4-Ig and LAG3-Ig revealed higher inhibitory effects on T cell proliferation than tetravalent forms of TNFR2-Ig and CD2-Ig. In vitro mixed lymphocytes reaction (MLR) showed that combined treatment with tetravalent CTLA4-Ig and tetravalent LAG3-Ig was highly effective for inhibiting T cell proliferation in both human and murine allogeneic stimulation. In addition, both single tetravalent-form and combination treatments can prevent the lethality of murine acute GVHD. The results of this study demonstrated that co-blockade of the major histocompatibility complex class (MHC)II:T cell receptor (TCR) and CD28:B7 pathways by using tetravalent human LAG3-Ig and CTLA4-Ig synergistically prevented murine acute GVHD.

• IMMUNE NETWORK
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• v.2 no.3
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• pp.133-136
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• 2002

Background: The costimulatory molecule 4-1BB, a member of nerve growth factor receptor/tumor necrosis factor (NGFR/TNFR) super family, is involved in cell survival and death. Methods: In this study, female C57BL/6 ($H-2^b$) mice were used as a recipient, and DBA/2 ($H-2^d$) as a donor to assess a mixed lymphocyte reaction (MLR) and CTL response in vitro, and skin graft survival. IL-2, IFN level was measured by ELISA. Results: Mixed lymphocyte reaction (MLR) analysis showed that 4-1BB-deficient responder cells showed enhanced cellular proliferation over littermate controls. In contrast, IL-2 production was diminished only in 4-1BB knockout cultures. The IFN expression, on the other hand, was comparable between the groups. When female C57BL/6 ($H-2^b$) mice were grafted with the trunk skin of DBA/2 ($H-2^d$) mice, the in vivo tissue destruction of 4-1BB-deficient mice was not distinct from the normal littermates. Conclusion: These data suggest that 4-1BB is critical for the induction of alloreactive responses in vitro but 4-1BB alone could not change the course of skin rejection in vivo.

• Toxicological Research
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• v.11 no.1
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• pp.15-21
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• 1995

Several populations of lymphocytes possess receptors for autonomic neurotransmitter, which make lymphocytes susceptible to autonomic stimulation. This study was to evaluate the functional alternation of effector cells of the immune system. Female Balb/C mice, 15-20 g, were injected with MA subcutaneously under various conditions. Mixed lymphocyte reaction (MLR) showed certain T cell subsets were affected by MA. The level of interleukin-2 (IL-2) production was inhibited due to a defect in expression of the IL-2 receptor. In mice injected with 20 mg MA/kg, 1 day before assay, phagocytosis of peritoneal macrophages showed $14.07\pm3%$, which was similar degree to 5 mg MA/kg treatment for 4 consecutive days. Phagocytosis was almost recovered to that of control after 4 day in 20 mg/kg injected mice. Maximum inhibition of plaque forming cell (PFC) occurred when MA was given early, indicating the inductive time point of antibody production was affected. The cortisol level increased in the MA treated group (0.05, 0.20, and $0.08{\mu}g$/dl for control, low, and high dose-MA treated mice, respectively). Based on these results, MA has general suppression effects on the immune systems by functional alteration of effector cells. Considering the increment of serum cortisol levels, MA partially impacts the neuroendocrine system to lead to failure of immune response.

• IMMUNE NETWORK
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• v.13 no.1
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• pp.16-24
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• 2013

CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.

• IMMUNE NETWORK
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• v.3 no.4
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• pp.302-309
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• 2003

Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-$dhfr^-$ cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.15-21
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• 2004

The immunomodulating activities and chemical characteristics of a water-soluble exopolymer from submerged mycelial culture of Phellinus pini were studied. Anticomplementary activity of this polymer was found to be $73.2\%$, and its activation system occurred through both classical and alternative pathways, where the classical pathway was detected to be the major one by crossed immunoelectrophoresis. Nitric oxide (NO) release ability and acid phosphatase activity of macrophage were increased by 1.6-fold ($100{\mu}g/ml$) and 3.4-fold ($500{\mu}g/ml$), respectively, and splenocyte proliferation in mixed lymphocyte reaction (MLR) was also increased by 2.6-fold ($200{\mu}g/ml$), compared to the control. The molecular weight of this polymer, determined by HPLC, was under 5 kDa. Total sugar and protein contents were 89.7 and 10.3%, respectively. Both sugar and amino acid compositions of the exopolymer were also analyzed.

• Archives of Plastic Surgery
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• v.32 no.4
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• pp.533-538
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• 2005

Due to its safety and softness, autologous fat transplantation has been commonly performed for soft tissue correction. However, the injected fat is absorbed resulting in the reduction of volume of the graft by 40-60% within a few months. Thus, there was an attempt to use adipocytes differentiated from preadipocytes in vitro for transplantation. Differentiated adipocytes were biocompatible and matured with gradual volume increase at transplantation site in clinical study(unpublished data). In addition, they did not induce immune rejection in response to nonself lymphocytes in a mixed lymphocyte reaction(MLR)(unpublished data). The purpose of this study is to differentiate mouse preadipocytes following labeling into adipocytes to establish an animal model for allogenic transplantation. Preadipocytes isolated from inguinal and retroperitoneal fat pad of C57BL/6 mice were proliferated with growth medium by passage 3 and differentiated into adipocytes with different culture conditions after labeled with BrdU. At most suitable conditions, above 90% of preadipocytes were differentiated and BrdU labeling did not affect differentiation rate and function of differentiated adipocytes. These results demonstrate that BrdU-labeled adipocytes resulting from this in vitro differentiation protocol are useful for allogenic transplantation study.