• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.611-616
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• 2014

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associated antigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whether transduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigen-specific cytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocyte-derived DCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated target of the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCs was measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry. DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag). Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulated by LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methyl thiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfully constructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83 than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstrated good stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumor effects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have an enhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.

• Asian Pacific Journal of Cancer Prevention
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• 제17권11호
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• pp.4907-4911
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• 2016

Glioblastoma multiforme (GBM) is the most aggressive of the gliomas, a collection of tumors arising from glia in the central nervous system. Possible associations between the human cytomegalovirus (HCMV) and the JC virus with GBM are now attracting interest. Our present aim was to investigate the prevalence of the two viruses in Iranian patients from Kerman's cities in the south of Iran. In addition, the expression rates of pp65, large T antigen and p53 proteins were assessed and their relation with GBM evaluated using reverse transcription real time PCR (rReal Time PCR). A total of 199 patients with GBM cancer were enrolled, with $mean{\pm}SD$ ages of $50.0{\pm}19.5$ and $50.7{\pm}19.6$ years for males and females, respectively. The P53 rate was dramatically low suggesting an aetiological role,. Large T antigen expression was found in JC positive samples, while the PP65 antigen was observed in patients positive for CMV and JC. HCMV products and JC virus with oncogenic potential may induce the development of various tumors including glioblastomas. The JC virus produces an early gene product, T-antigen, which has the ability to associate with and functionally inactivate well-studied tumor suppressor proteins including p53 and pRB.

• IMMUNE NETWORK
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• 제7권4호
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• pp.173-178
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• 2007

Background: We studied the role for expression of CD40 and CD40L by CD4 and CD8 T cells in the generation of CD8 T cell response to minor histocompatibility antigen, H60. H60 is a cellular antigen to which CD8 responses require CD4 T cell help. Methods: CD40- or CD40L-deficient mice were adoptively transferred with normal CD4 or CD8 T cells or with memory CD4 or CD8 T cells, and were immunized with male H60 congenic splenocytes to induce CD8 T cell response to H60. Peripheral blood CD8 T cell from the immunized mice were stained with the H60 tetramer. Results: CD8 T cell response to H60 was not induced in both CD40- and CD40L-deficient mice. Adoptive transfer of $CD40^{+/+}$ CD8 T cells into CD40-deficient mice did not compensate the defect in inducing CD8 T cell response to H60, while the H60-specific CD8 T cells were activated in the CD40-deficient mice that were adoptively transferred with $CD40^{+/+}$ CD4 T cells. Adoptive transfer of $CD40L^{+/+}$ CD4 T cells into CD40L-deficient mice induced primary CD8 T cell response for H60 and the presence of $CD40L^{+/+}$ CD4 T cells was required even for memory CD8 T cells response to H60. Conclusion: Our results suggest that the CD40-CD40L interaction mediates the delivery of CD4 T cell help to naive and memory H60-specific CD8 T cells. While the expression of CD40L by CD4 T cells is essential, signaling through CD40 on CD8 T cells is not required for the induction of CD8 T cell response to H60.

• Asian Pacific Journal of Cancer Prevention
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• 제14권4호
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• pp.2447-2451
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• 2013

Objective: To study changes of tumor associated carbohydrate antigen (TACAs) expression and mRNA levels for tumor associated glycosyltransferases, and assess subcellular localizations of N-acetyl galactosyltransferases (GalNAc-Ts) in the K562 leukemia cell line after imatinib treatment. Methods: RT-PCR was performed to analyze the expression of glycosyltransferases which synthesize O-glycan in tumor-associated carbohydrate antigens (TCTAs). The expression of Tn antigen, T antigen and sialyl T antigen on K562 cell membranes was measured by flow cytometry after treatment with different concentrations of imatinib. Co-localization of GalNAc-Ts and ER (endoplasmic reticulum) was determined by confocal laser scanning microcopy. Results: Transcript expression levels of several glycosyltransferases related to TCTAs were decreased after imatinib ($0-0.3{\mu}M$) treatment. Expression of Tn antigen and T antigen was increased while that of sialyl T antigen was decreased. Co-localization of GalNAc-Ts and ER was reduced by $0.2{\mu}M$ of imatinib. Conclusion: Imatinib inhibited the expression of O-glycan related TACAs and several related glycosyltransferases, while decreasing the co-localization of GalNAc-Ts and ER and normalizing O-glycosylation in the K562 human leukemia cell.

• IMMUNE NETWORK
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• 제14권6호
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• pp.328-332
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• 2014

Dexamethasone (Dex) was shown to inhibit the differentiation, maturation, and antigen-presenting function of dendritic cells (DC) when added during DC generation or maturation stages. Here, we examined the direct effects of Dex on MHC-restricted antigen processing. Macrophages were incubated with microencapsulated ovalbumin (OVA) in the presence of different concentrations of Dex for 2 h, and the efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 T cells. Dex inhibited both class I- and class II-restricted presentation of OVA to T cells; this inhibitory effect on antigen presentation was much more potent in immature macrophages than in mature macrophages. The presentation of the exogenously added OVA peptide SIINFEKL was not blocked by Dex. In addition, short-term treatment of macrophages with Dex had no discernible effects on the phagocytic activity, total expression levels of MHC molecules or co-stimulatory molecules. These results demonstrate that Dex inhibits intracellular processing events of phagocytosed antigens in macrophages.

• 한국식품영양과학회지
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• 제33권7호
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• pp.1085-1091
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• 2004

먼저 OVA항원에 대해 특이적으로 증식반응을 나타내는 T세포주를 수립하였고, 수림된 세포주는 세포 표면 단백질이 CD4$^{＋}$CD8$^{-}$이며 IL-2와 IFN-${\gamma}$를 분비하는 Type I에 속하는 보조 T세포(Thl)인 것을 확인하였다. 보중익기탕의 total 분획은 OVA항원에 대해 특이적으로 반응하는 Thl세포의 증식반응을 증가시키는 효과를 나타내지 않았으며 고농도에서 오히려 증식반응을 억제하였다. 그러나, 보중익기탕의 polysaccharide 분획은 전반적 인 농도에서 T세포의 증식반응을 유의하게 증가시키는 효과가 있는 것으로 나타났다. 보중익기탕의 polysaccharide 분획을 첨가하였을 때 T세포의 IL-2 분비량은 대조군보다 약간 적었지만, IFN-${\gamma}$ 분비량은 대조군보다 증가하였다. 그리고, 분비된 IL-2와 결합하는 T세포의 IL-3 수용체 발현양도 증가하는 것으로 나타났다. 또한, 항원제시세포의 MHC class II의 발현양도 증가시켰다. 이상의 결과로 보중익기탕의 polysaccharide분획은 T세포의 IL-2수용체 발현양을 증가시키고, 항원제시세포의 MHC classs II의 발현양을 증가시켜서 T세포의 증식반응을 증가시키는것으로 생각된다 그리고 보중익기탕이 생체 면역반응에 미치는 보다 정확한 효과를 평가하기 위해서는 직접 살아있는 실험동물에 투여하는 in vivo 실험이 필요하다.

• BMB Reports
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• 제47권5호
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• pp.241-248
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• 2014

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to ${\alpha}{\beta}$ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of ${\alpha}{\beta}$ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant $V{\alpha}14-J{\alpha}18$ TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.

• Molecules and Cells
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• 제42권3호
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• pp.228-236
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• 2019

CD4 T cells differentiate into $ROR{\gamma}t/IL$-17A-expressing cells in the small intestine following colonization by segmented filamentous bacteria (SFB). However, it remains unclear whether SFB-specific CD4 T cells can differentiate directly from naïve precursors, and whether their effector differentiation is solely directed towards the Th17 lineage. In this study, we used adoptive T cell transfer experiments and showed that naïve CD4 T cells can migrate to the small intestinal lamina propria (sLP) and differentiate into effector T cells that synthesize IL-17A in response to SFB colonization. Using single cell RT-PCR analysis, we showed that the progenies of SFB responding T cells are not uniform but composed of transcriptionally divergent populations including Th1, Th17 and follicular helper T cells. We further confirmed this finding using in vitro culture of SFB specific intestinal CD4 T cells in the presence of cognate antigens, which also generated heterogeneous population with similar features. Collectively, these findings indicate that a single species of intestinal bacteria can generate a divergent population of antigen-specific effector CD4 T cells, rather than it provides a cytokine milieu for the development of a particular effector T cell subset.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2003년도 2003 Annual Meeting, BioExhibition and International Symposium
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• pp.55-62
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• 2003

The mucosal immune system provides a first line of defense against invasion of infectious agents via inhalation, ingestion and sexual contact. For the induction of protective immunity at these invasion sites, one must consider the use of the CMIS, which interconnects inductive tissues, including PP and NALT, and effector tissues of the intestinal, respiratory and genitourinary tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant or use of mucosal antigen delivery vehicle has been shown to be essential. When vaccine antigen is administered via oral or nasal route, antigen-specific Th 1 and Th2 cells, cytotoxic T lymphocytes(CTLs) and IgA B cell responses are effectively induced by the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium(FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying DCs for the generation of antigen-specific T cells and IgA committed B cells. These antigen-specific lymphocytes are then home to the distant mucosal effector tissues for the induction of antigen-specific humoral(e.g., IgA) and cell-mediated (e.g., CTL and Th1) immune responses in order to form the first line of defense. Elucidation of the molecular/cellular characteristics of the immunological sequence of mucosal immune response beginning from the antigen sampling and processing/presentation by M cells and mucosal DCs followed by the effector phase with antigen-specific lymphocytes will greatly facilitate the design of a new generation of effective mucosal antigen-specific lymphocytes will greatly facilitate the design of a new generation of a new generation of effective mucosal adjuvants and of a vaccine deliver vehicle that maximizes the use of the CMIS.

• IMMUNE NETWORK
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• 제6권2호
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• pp.102-110
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• 2006

Background: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective anti-tumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific $CD8^+$ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. Methods: Bone marrow-derived DC (EM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of ${\beta}$-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using ${\beta}$-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. Results: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. Conclusion: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I- restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.