• BMB Reports
• /
• 제30권6호
• /
• pp.385-389
• /
• 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.

• 한국임상수의학회지
• /
• 제28권4호
• /
• pp.399-402
• /
• 2011

In recent years, the mesenchymal stem cells (MSC) derived from various tissues have been widely tested for developing cell therapies, tissue repair and transplantation. Although there has been much interest in the immunomodulatory properties of MSC and their immunologic reactions following autologous, allogeneic and xenogenic transplantation of MSC in vivo, up to date, the expression of immunogenic markers, such as class I and II human leukocyte antigens (HLA), after differentiation of human umbilical cord blood (hUCB)-derived MSC has been poorly investigated and require extensive in vitro and in vivo testing. In this experiment, the expression of the HLA-ABC and HLA-DR on hUCB-derived MSC have been tested by immunocytochemical staining. The undifferentiated MSC were moderately stained for HLA-ABC but very weakly for HLA-DR. In order to investigate the inhibitory effect of allogeneic lymphocytes on proliferation of MSC, the MSC were cultured in the presence or absence of peripheral allogeneic lymphocytes stimulated with concanavalin A. The allogeneic lymphocytes did not significantly inhibit MSC proliferation. We conclude that hUCB-MSC expressed moderately class I HLA antigen while almost negatively class II HLA antigen. The MSC have an immunomodulatory effect which can suppress the allogeneic response of lymphocytes. These in vitro data suggest that allogeneic MSC derived from cord blood can be useful candidate for allogeneic cell therapy and transplantation without a major risk of rejection.

• Journal of Microbiology and Biotechnology
• /
• 제22권8호
• /
• pp.1066-1076
• /
• 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.

• Archives of Pharmacal Research
• /
• 제26권3호
• /
• pp.192-196
• /
• 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.

• IMMUNE NETWORK
• /
• 제13권1호
• /
• pp.16-24
• /
• 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.

• Journal of Ginseng Research
• /
• 제40권1호
• /
• pp.18-27
• /
• 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.

• Archives of Pharmacal Research
• /
• 제25권3호
• /
• pp.364-369
• /
• 2002

Hepatitis C virus (HCV) is remarkably efficient at establishing chronic infection. One of the reasons for this appears to be the suppression of the accessory cell function of professional antigen presenting cells. In the present study, the immunosuppressive activity of HCV protein was examined on dendritic cells (DCs) generated from mouse bone marrow progenitor cells in vitro. We found that the DCs forced to express HCV protein have defective allostimulatory ability. DCs expressing HCV protein were phenotypically indistinguishable from normal DCs. However, they were unable to produce IL-12 effectively when stimulated with lipopolysaccharide. The functional domain of the HCV protein essential for immunosuppression was determined using a series of ${NH_2}-and$ C-terminal deletion mutants of HCV core protein. We found that amino acid residues residing between the 21 st and the 40th residues from the ${NH_2}-terminus$ of HCV core protein are required for immunosuppression. These findings suggest that HCV core protein suppresses the elicitation of protective Th1 responses by the inhibition of IL-12 production by DCs.

• 대한핵의학회지
• /
• 제39권1호
• /
• pp.26-33
• /
• 2005

목적 : 이 연구는 $^{99m}Tc$-HMPAO에 표지된 미성숙 또는 성숙 수지상 세포의 마우스 생체 내 분포와 이동 양상에 대해 알아보고자 하였다. 대상 및 방법: 마우스의 대퇴골과 경골의 골수로부터 수지상 세포를 배양하고 미성숙, 성숙 수지상세포를 $^{99m}Tc$-HMPAO로 표지하였다. 방사성 표지 전후에 수지상 세포의 기능 및 표현형의 변화 유무를 알기 위해 동종 혼합 림프구 반응 (allogeneic mixed lymphocyte reaction)과 형광 활성 세포 선별 (fluorescence-activated cell sorting)을 시행하였다. 정맥 주사된 수지상 세포의 생체 내 이동은 감마 카메라 영상과 생체 분포 실험을 통하여 평가하였고, 피하 종양 마우스 모델과 대조군에서 비교하였다. 폐, 간, 비장, 신장, 종양 등 조직에서 그램 당 주사량의 백분율(%ID/g)을 계산하였다. 결과: 미성숙, 성숙 수지상 세포의 표지 효율은 각각 $60.4{\pm}5.4%$와 $61.8{\pm}6.7%$ 였다. 수지상 세포의 정맥주사 후 방사능은 폐에서 가장 먼저 관찰되었고, 이후 간과 비장에 분포되었다. 성숙 수지상 세포가 미성숙 수지상 세포에 비해 비장으로 더 많이 이동하였다(대조군; $38.3{\pm}4.0%\;vs.\;32.2{\pm}4.1%$, 종양이식 군: $40.4{\pm}4.1%\;vs.\;35.9{\pm}3.8%$, p<0.05). 종양으로의 이동 역시 성숙 수지상 세포가 미성숙 수지상 세포에 비해 더 많은 비율을 보였다($2.4{\pm}0.3%\;vs\;1.7{\pm}0.2%$; p=0.034). 결론: $^{99m}Tc$-HMPAO 에 표지된 수지상 세포를 이용하여 마우스 생체 내 이동을 실시간 영상화 할 수 있었다. 마우스 정맥에 주사되었을 때, 더 많은 비율의 성숙 수지상 세포가 미성숙 수지상 세포에 비해서 비장이나 종양으로 이동함을 알 수 있었다.

• IMMUNE NETWORK
• /
• 제3권4호
• /
• pp.302-309
• /
• 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.

• IMMUNE NETWORK
• /
• 제8권1호
• /
• pp.21-28
• /
• 2008

Background: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. Methods: Balb/c $(H-2^d)$ mice were fed with dendritic cell line, DC2.4 $(H-2^d)$ every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. Results: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-${\gamma}$, IL-5, and TNF-${\alpha}$. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. Conclusion: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.