• Molecules and Cells
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• 제45권8호
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• pp.513-521
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• 2022

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an immune checkpoint molecule that is mainly expressed on activated T cells and regulatory T (Treg) cells that inhibits T-cell activation and regulates immune homeostasis. Due to the crucial functions of CTLA-4 in T-cell biology, CTLA-4-targeted immunotherapies have been developed for autoimmune disease as well as cancers. CTLA-4 is known to compete with CD28 to interact with B7, but some studies have revealed that its downstream signaling is independent of its ligand interaction. As a signaling domain of CTLA-4, the tyrosine motif plays a role in inhibiting T-cell activation. Recently, the lysine motif has been shown to be required for the function of Treg cells, emphasizing the importance of CTLA-4 signaling. In this review, we summarize the current understanding of CTLA-4 biology and molecular signaling events and discuss strategies to target CTLA-4 signaling for immune modulation and disease therapy.

• IMMUNE NETWORK
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• 제2권2호
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• pp.72-78
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• 2002

Background: The precise mechanism by which CTLA-4 regulates T cell immune responses is still not fully understood. Previously we proposed that CTLA-4 could downregulate T cell function by modulating a signaling cascade initiated from the T cell receptor complex. The evidence for this notion comes from our findings that CTLA-4 associated with the T cell receptor zeta (TCR zeta) chain, and hence regulated TCR zeta phosphorylation by co-associated SHP-2 tyrosine phosphatase (1). In this report, we investigated whether any other signaling molecules could be involved in the CTLA-4 signaling pathway. Methods: We have taken biochemical approaches, such as immunoprecipitation followed by autoradiography or immunoblotting, to identify the molecules associated with CTLA-4. To perform these assays, we used activated primary T cells and ectopically transfected 293 cells. Various truncation mutants of CTLA-4 were used to map the interaction site on CTLA-4. Results: We found that in addition to TCR zeta and SHP-2, a recently cloned small adaptor molecule, SAP (SLAM-associated protein), was also able to associate with CTLA-4. We identified the domain of SAP association in CTLA-4 being a motif involving GVYVKM. This motif has been previously found to bind SHP-2 through its phosphorylated tyrosine interaction with SH-2 domain of SHP-2. Indeed, co-expression of SAP and SHP-2 reduced their binding to CTLA-4 significantly, suggesting that SAP and SHP-2 compete for the common binding site, GVYVKM. Thus, by blocking SHP-2 recruitment SAP could function as a negative regulator of CTLA-4. Conclusion: Taken together, our data suggest the existence of complicate signaling cascade in regulating CTLA-4 function, and further provide evidence that SAP can act either as a positive or negative regulator depending on the nature of the associating receptors.

• IMMUNE NETWORK
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• 제13권1호
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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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• 제3권4호
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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.

• KSBB Journal
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• 제24권3호
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• pp.246-252
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• 2009

본 연구에서는 형질전환된 벼 세포를 이용하여 자가면역 질환의 치료제인 human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig)을 생산하였고, RAmy3D promoter와 RAmy1A signal pertide를 사용하여 당 고갈시에 목적 단백질의 발현과 배지로의 분비를 유도하였다. 이 시스템의 문제점은 당 고갈에 의해 목적 단백질의 생산이 유도되기 때문에 에너지원이 없어 세포의 사멸, 이로 인한 배지내로의 protease 분비를 유발하게 된다. 이것은 목적 단백질의 안정성 저해와 궁극적으로는 생산성의 손실을 일으킨다. 따라서 세포를 보호하여 사멸을 막는 효과가 있음이 보고된 proline을 첨가하여 생산성 증진 효과를 확인하고자 하였다. 4 mM의 proline을 첨가하여 배양하였을 때, 세포의 사멸 및 pretense의 분비를 줄일 수 있었고 이는 결과적으로 hCTLA4Ig의 생산성 증진으로 이어졌다. 또한 단백질 안정제를 통하여 배지내로 분비된 hCTLA4Ig의 안정화를 이루어 생산성을 높이고자 하였다. 0.01 g/L의 gelatin을 적용하여 생산성 증진 효과를 확인하였으며, 이는 hCTLA4Ig의 안정화에 의한 것임을 알 수 있었다. Pretense의 분비를 줄이기 위한 세포 보호와 pretense의 공격을 막기 위한 hCTLA4Ig 보호를 통하여 형질전환된 벼 세포를 이용한 hCTLA4Ig의 안정성 및 생산성을 증대시킬 수 있음을 확인하였다.

• The Korean Journal of Physiology and Pharmacology
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• 제16권5호
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• pp.349-353
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• 2012

Activated T cells express inhibitory receptors such as CTLA-4 that can downregulate immune responses. Blockade of or genetic deficiency in CTLA-4 can result in autoimmunity. Therefore, strategies to increase the inhibitory function of CTLA-4 may be attractive in settings of undesirable T cell responses such as autoimmunity or transplant rejection. We have tested the hypothesis that transgenic constitutive expression of CTLA-4 can further attenuate immune responses when compared with normal inducible expression. Our results indicate that transgenic expression of CTLA-4 in mouse T cells (CTLA-4-Tg T cells) results in reduced cell cycle progression and increased apoptosis of TCR-stimulated T cells. CTLA-4-Tg T cells display reduced T cell proliferation in an in vivo model of graft versus host disease (GVHD). These results further our understanding of how CTLA-4 can be manipulated to inhibit immune responses and may help development of new therapeutic strategies for clinical settings of autoimmunity and transplantation.

• Clinical and Experimental Pediatrics
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• 제49권5호
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• pp.545-551
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• 2006

목 적 : Cytotoxic T lymphocyte-associated antigen-4(CTLA-4)를 표현하는 유전자는 IgE 조절과 T 세포의 활성에 중요한 역할을 하는 후보유전자로 알려져 있다. 본 연구에서는 CTLA-4 exon 1(＋49 position)과 promoter 유전자(-318 position)의 다형성과 한국 소아의 아토피피부염의 유전적 감수성 및 아토피피부염의 임상양상의 관계에 대해 알아보고자 하였다. 방 법 : 아토피성 습진 145명, 비아토피성 습진 69명과 대조군 96명을 대상으로 restriction fragment length polymorphism방법으로 CTLA-4 promoter(-318 T/C)와 exon 1(＋49 A/G)의 다형성을 조사하였다. 결 과 : CTLA-4 exon 1과 promoter의 유전자 다형성이 아토피성 습진 환아군이나 비아토피성 습진 환아군에서 대조군에 비해 의의있는 분포의 차이를 보이지 않았으며, 아토피피부염의 중증도, IgE 농도, 호산구의 수와도 유의한 관계가 없없다. 결 론 : 본 연구에서는 CTLA-4 유전자 다형성이 한국 소아의 아토피피부염의 유전적 감수성 및 아토피피부염의 임상양상에 관여하지 않는 것으로 생각된다.

• KSBB Journal
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• 제31권4호
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• pp.277-283
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• 2016

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.

• KSBB Journal
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• 제20권4호
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• pp.278-284
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• 2005

식물세포의 느린 생장과 낮은 생산량의 이유로 지금까지 는 주로 미생물이나 동물세포에서 유전자 재조합 단백질을 생산하여 왔다. 그러나 저렴한 배지 가격, 동물 유래 바이러스 감염 위험성으로부터의 안정성, glycosylation 등의 post-translational modification이 가능하다는 장점들로 인하여 최근 들어 식물세포배양은 생물학적 활성을 가진 고부가가치의 단백질을 생산하는데 많이 이용되고 있다. 본 연구에서는 생장배지에 첨가했던 sucrose의 소비와 induction 배지로의 교환에서 오는 배지내의 삼투압을 조절하여 hCTLA4-Ig의 생산성을 높이고자 하였다. 다양한 삼투압 조절제 첨가 실험을 통해 sorbitol을 선별하고, 40 mM의 sorbitol 첨가에서 상대적으로 높은 생존도와 induction 후 7일째 대조구보다 1.7배 높은 생산성을 확인하였다. 또한, 저농도의 glucose 첨가를 통한 생산성 증대에 있어서는 8 mM glucose에서 induction 이후에도 높은 세포농도를 유지하면서 최대 37.3 mg/L까지 hCTLA4-Ig 생산량을 증가시켰다. 5-L bioreactor에서 회분식 배양과 induction시의 hCTLA4-Ig 생산량을 비교한 결과 induction시 배양 18일째 최고 45.3 mg/L까지 높일 수 있었으며, 회분식 배양에 비해 2.1배 증가됨을 확인하였다.

• IMMUNE NETWORK
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• 제2권2호
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• pp.102-108
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• 2002

Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.