• IMMUNE NETWORK
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• 제10권6호
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• pp.206-211
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• 2010

Background: Dendritic cell (DC)-based tumor vaccine is an attractive modality for the treatment of colon cancer because it has been recurred and produced few side effects in patients. Secretory glycoprotein 90K has been found at elevated level in various cancer tissues and sera. We investigated to establish a more effective DC vaccine for the treatment of colon cancer in which the levels of 90K are elevated. Methods: We obtained the concentrated 90K from 293T cells stably expressing 90K. DCs were cultured from peripheral blood monocytes, and a DC vaccine pulsed with tumor lysate was compared with a DC vaccine pulsed with 90K. We measured the functional activity for CTLs by using IFN-${\gamma}$-enzyme linked immunoabsorbent spot (ELISPOT) assay. Results: DCs pulsed with tumor lysate+90K exhibited the enhanced T cell stimulation, polarization of $\ddot{i}$ T cell toward Th1. The CTLs generated by DCs pulsed with 90K efficiently lysed HCT116 cells. The results indicate that 90K-speicifc-CTLs can recognize 90K proteins naturally presented by colon cancer cells. Conclusion: Our study suggests that 90K-specific CTLs generated by 90K-pulsed DCs could be useful effector cells for immunotherapy in colon cancer.

• IMMUNE NETWORK
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• 제13권1호
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• pp.10-15
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• 2013

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and $GLT{\gamma}1$ expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.

• IMMUNE NETWORK
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• 제11권3호
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• pp.182-189
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• 2011

Background: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-${\gamma}$ ELISPOT assay, cytotoxicity assay and tetramer staining. Results: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of $CD8^+$ and $CD4^+$ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

• IMMUNE NETWORK
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• 제9권6호
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• pp.265-273
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• 2009

Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

• IMMUNE NETWORK
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• 제3권4호
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• pp.295-301
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• 2003

Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. Methods: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard $^{51}Cr$-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with $mIFN-{\gamma}$ELISPOT. Results: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of $IFN-{\gamma}$ secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. Conclusion: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies.

• Asian Pacific Journal of Cancer Prevention
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• 제16권10호
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• pp.4329-4333
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• 2015

Background: To investigate the change of frequency and immuno-inhibitory function of myeloid-derived suppressor cells (MDSCs) after treatment of cisplatin (DDP) in A375 human melanoma model. Materials and Methods: BALB/c nude mice were inoculated with A375 cells to establish the human melanoma model and randomly divided into control group given normal saline (NS) and experimental group treated with DDP (5 mg/kg). The percentages of MDSCs in the tumor tissue and peripheral blood after DDP treatment were detected by flow cytometry. The proliferation and interferon-${\gamma}$ (IFN-${\gamma}$) secretion of T cells co-cultured with MDSCs were analyzed through carboxyfluorescein succinimidyl ester (CFSE) labeling assay and enzyme-linked immunospot (ELISPOT) assay, respectively. Results: In A375 human melanoma model, DDP treatment could significantly decrease the percentage of MDSCs in the tumor tissue, but exerted no effect on the level of MDSCs in peripheral blood. Moreover, DDP treatment could attenuate the immuno-inhibitory function of MDSCs. T cells co-cultured with DDP-treated MDSCs could dramatically elevate the proliferation and production of INF-${\gamma}$. Conclusions: DDP can decrease the frequency and attenuate immuno-inhibitory function of MDSCs in A375 melanoma model, suggesting a potential strategy to augment the efficacy of combined immunotherapy.

• IMMUNE NETWORK
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• 제7권2호
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• pp.80-86
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• 2007

Background: CD40-activated B (CD40-B) cells might be an attractive source of autologous antigen-presenting cells (APCs) for immunotherapy due to the convenience to obtain from peripheral blood and expand in vitro. Moreover, CD40-B cells were found to be comparable with DCs in their capacity to raise antigen-specific CD8+ T cells. Here, we have established K562 cells expressing CD40L to expand CD40-activated B cells used for APCs. Methods: After activation of B cell by K562/CD40L, CD40-B cells were examined by counting B cell numbers. Surface expression of CD54, CD80, CD86 and HLA class II was measured by flow cytometry. The CD40-B cells were tested for its function as APC by mixed lymphocyte reactions (MLR) and by induction of T cell responses specific for pp65 peptide in vitro. Results: The expansion of B cells by K562/CD40L increased about 6-folds compared with anti-CD40 or K562. Furthermore, the expression of CD54, CD80, CD86 and HLA class II was up-regulated by K562/CD40L. B cells by K562/CD40L showed comparable antigen presentation activity with mature DCs as shown in MLR, INF-${\gamma}$ ELISPOT assay. Conclusion: These results suggest that K562/CD40L could be used to generate activated B cells as potent APCs which could be useful for cellular vaccination and adoptive immunotherapy.

• 대한의생명과학회지
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• 제13권4호
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• pp.325-332
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• 2007

Cell-mediated immune response (CMI) is a major immune protective mechanism against tuberculosis (TB) infection. Among several components involved in CMI, recent studies suggest that CD8+ T cells are important in controlling TB infection. In our previous report, we defined four Mycobacterium tuberculosis (MTB) derived epiotpe peptides specific for HLA-A*0201-restricted CD8+ T cells. These four peptides are $PstAl_{75-83}$, $ThyA_{30-38}$, $RpoB_{127-135}$ and $85B_{15-23}$. In this study, these epitope peptides specific CD8+ T cell responses in tuberculous pleurisy were investigated using ex vivo $IFN-\gamma$ elispot assay and intracellular $IFN-\gamma$ staining method. As a result, we observed these epitope peptide specific CD8+ T cell responses are induced in all three patients with tuberculous pleurisy suggesting that CD8+ T cells are involved in protective immune mechanism against MTB infection in tuberculous pleurisy. However, the CMI to mitogens and MTB antigens from pleural fluids of patients with tuberculous pleurisy does not seem to correlate with that from peripheral blood, although the sample size is too small to make any conclusion. In sum, the MHC I restricted CD8+ T cell responses seem to be induced efficiently in the pleural fluids, at the site of TB infection, in which the CMI is actively induced. In addition, these experiments suggest that MHC I restricted CD8+ T cell mediated immune responses are also involved in protective mechanism against MTB infection in extra-pulmonary TB.

• 한국어병학회지
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• 제21권3호
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• pp.201-208
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• 2008

We compared the pathogenicity and antigenicity of two different Edwardsiella tarda (E. tarda) strains KFE and Edk-2 isolated in Korea and Japan respectively. In the pathogenicity analysis with challenge test against loach, E. tarda KFE isolate showed stronger pathogenicity compared to that of E. tarda Edk-2 isolate. The differences were also confirmed by the comparison of OMP (outer membrane protein) in SDSPAGE which showed three major bands, 41kDa, 37kDa and 30kDa, for E. tarda KFE isolates and two major bands, 41kDa and 30kDa, for E. tarda Edk-2 isolates. On the base of these results, we tried to determine the differences of antigenicities of these two isolates in loach which is one of the important species in freshwater aquaculture in Korea. Numbers of specific antibody secreting cells (SASC), appeared to be higher in loach immunized with FKC of E. tarda Edk-2 than loach immunized with FKC of E. tarda KFE. ELISPOT-assay for the comparison of antigenicity showed relatively high percentage of cross-reaction and implied the presence of some common epitopes in the antigens of these two E. tarda isolates.

• 대한수의학회지
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• 제53권2호
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• pp.95-101
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• 2013

The study was carried out to evaluate and compare the immune responses in mice experimentally infected with either wild-type or isogenic mutants of Salmonella enterica serovars Enteritidis (SE), Salmonella Typhimurium (ST) and Gallinarum (SG). The mutant strains were constructed by allelic replacement of some virulence-associated genes in the wild-type strains. Seven-week-old female BALB/c mice were orally or intraperitoneally inoculated by injecting bacterial suspension. To evaluate the immune responses, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay were conducted with serum and fecal samples. As a result, the mice group infected orally with the SE mutant strain showed the highest level of specific IgA-secreting splenocytes, compared to the other groups. The peritoneally injected groups showed the greater levels of IgG1 than the orally injected groups, which was in a good agreement with the previous studies. In addition, the mutant infected groups had the similar secretion levels of antibodies with the wild-type infected groups. These results demonstrated that the SE mutant strain elicited humoral immune response as much as wild-type, implying that it can be useful as a delivery vehicle as well as a candidate of a live attenuated vaccine.