Tai-Gyu Kim

Association of HLA with Vogt-Koyanagi-Harada syndrome in Koreans

PURPOSE To study the distribution of human leukocyte antigen HLA-A/B antigens and HLA-DR/-DQ/-DP alleles and to investigate the immunogenetic background of Korean patients with Vogt-Koyanagi-Harada (VKH) syndrome and clinical course with different types of HLA. METHODS Human leukocyte antigen typings were performed in 18 Korean patients with VKH syndrome and in 128 healthy control subjects. HLA-A/B loci serologic typing was performed according to the standard microlymphocytotoxicity technique. DNA was extracted through the salting out method, and HLA-DR phenotyping and HLA-DR4, HLA-DQ, and HLA-DP subtyping were performed with the polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. RESULTS Among HLA-A/B antigens typed by the standard microlymphocytotoxicity method, the frequencies of HLA-A31 (RR = 6.1, P<1x10(-2)) and HLA-B55 (RR = 15.8, P<.05) were significantly increased in the patient group compared with the control group. Among HLA-DR/-DQ/-DP alleles subtyped by DNA methods, the frequencies of HLA-DRB1*04 (RR = 45.1, P<1x10(-7)) and HLA-DRB1*07 (RR = 3.2, P<.05) were significantly increased. However, significant decreases in HLA-DRB1*08 (RR = .1, P<.05), HLA-DRB1*13 (RR = .1, P<.05), and HLA-DRB1*14 (RR = .1, P<.05) frequencies were observed. The result of HLA-DR, HLA-DQ, and HLA-DP subtyping showed the significant increase in DRB1*0405 (RR = 45.1, P<1x10(-7)), DQA1*0302 (RR = 12.0, P<1x10(-4)), DQB1*0303 (RR = 5.0, P<1x10(-2)), DQB1*0401 (RR = 18.9, P<1x 10-6), and DPB1*0501 (RR = 3.8, P<.05). However, significant decreases in DQA1*0101 (RR = .1, P< .05), DQA10102 (RR = .1, P<1x10(-2)), DQA1*0103 (RR = .1, P<.05), DQA1*0501 (RR = .1, P<1x10(-2)), DQB1*0301 (RR = .1, P<.05), DQB1*0601 (RR = .1, P<.05), DPB1*0201 (RR = .3, P<.05), and DPB1*0401 (RR = .1, P<.05) frequencies were also observed. In patients with DRB1*0405 itself or HLA-DRB1*0405-DQA1*0302-DQB1*0401 haplotype, a reduction in visual acuity and ocular complications was common. CONCLUSIONS These results suggest that HLA-DRB1*0405 itself or HLA-DRB1*0405-DQA1*0302-DQB1*0401 haplotype is greatly increased and may play the most important role in the development and the clinical course of VKH syndrome in Korean patients.

Polymorphisms of IL-1B, IL-1RN, IL-2, IL-4, IL-6, IL-10, and IFN-γ genes in the Korean population

Cytokines play a crucial role in regulating the immune and inflammatory responses. The collective influence of several cytokines can regulate immune responses as complex as those underlying allograft rejections or autoimmune diseases. Polymorphisms in the regulatory regions of the cytokine genes may influence their expression. Therefore, the polymorphisms of cytokine genes are potentially important as genetic predictors of the disease susceptibility or clinical outcome. In 311 unrelated healthy Korean individuals, we investigated the polymorphisms of cytokine genes (interleukin-1 [IL-1], IL-2, IL-4, IL-6, IL-10, and interferon-gamma [IFN-gamma]), which had been previously reported to be associated with a number of immune diseases, transplant complications, and direct or indirect influences on the level of expression and production. And we also compared the results to those published for other populations. The genotype distributions were consistent with the assumption of the Hardy-Weinberg equilibrium, with the exceptions of IL-1B +3954 and IL-6-174 polymorphisms. The polymorphisms examined in this study were almost similar to that observed in Asian populations. There were significant differences of the polymorphisms, except for IL-4 receptor alpha +1902, between Korean and other populations. Comparing the alleles associated with higher level of expression and production, IL-1B +3954*T, IL-2-330*G, and IL-4-590*T alleles were significantly higher, and IL-1RN*A2, IL-10-1082*G, and IFN-gamma*2 alleles were lower in Koreans than other populations. Especially in IL-6 promoter -174 polymorphism, we found only the G allele associated with higher plasma IL-6 levels. In haplotype analysis of IL-10 promoter polymorphisms, the GCC haplotype, associated with higher expression of IL-10, was significantly lower in Koreans. These results may be helpful for understanding transplant-related complications, immune or autoimmune diseases, and malignant diseases in the Korean population.

A Therapy Modality Using Recombinant IL-12 Adenovirus plus E7 Protein in a Human Papillomavirus 16 E6/E7-Associated Cervical Cancer Animal Model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.

Enhanced antitumour immunity by combined use of temozolomide and TAT-survivin pulsed dendritic cells in a murine glioma

Although chemotherapy remains among the best treatment options for most cancers, adjuvant therapies such as dendritic cell (DC)‐based immunotherapy have been added to treatment protocols to destroy residual tumour cells. Combination treatment with low‐dose temozolomide (TMZ) chemotherapy followed by vaccination with TAT‐survivin‐pulsed DCs enhanced T‐cell responses specific for survivin and improved survival rate, as compared with DC alone or TMZ alone. Moreover, antigen‐specific immunity appears to be mediated by CD8+ T cells, as determined by in vitro T‐cell subset depletion. These studies demonstrated that a combination of low‐dose TMZ chemotherapy and TAT‐based DC immunotherapy may be a novel strategy for safe and effective treatment of malignant gliomas.

Immunological Factors Relating to the Antitumor Effect of Temozolomide Chemoimmunotherapy in a Murine Glioma Model

ABSTRACT In this study, we investigated the potential of combined treatment with temozolomide (TMZ) chemotherapy and tumor antigen-pulsed dendritic cells (DCs) and the underlying immunological factors of TMZ chemoimmunotherapy with an intracranial GL26 glioma animal model. The combined treatment enhanced the tumor-specific immune responses and prolonged the survival more effectively than either single therapy in GL26 tumor-bearing animals. Apoptosis was induced in the tumors of the animals by the treatment with TMZ. Calreticulin (CRT) surface exposure was detected by immunofluorescence staining of TMZ-treated GL26 cells. TMZ chemotherapy increased tumor antigen cross-priming from tumor cells, leading to cross-priming of tumor antigen-specific CD4+ T cells and CD8+ T cells. This chemotherapy appeared to suppress the frequency of CD4+ CD25+ regulatory T cells (Treg). Moreover, this combined therapy resulted in an increase in the tumor infiltration of CD4+ and CD8+ T cells. Collectively, the findings of this study provide evidence that the combination of TMZ chemotherapy and treatment with DC-based vaccines leads to the enhancement of antitumor immunity through increased tumor-specific immune responses via the cross-priming of apoptotic tumor cell death mediated by CRT exposure and, in part, the suppression of Treg. Therefore, CRT exposure, regulatory T cells, and cross-priming by TMZ chemotherapy may be immunological factors related to the enhancement of the antitumor effects of chemoimmunotherapy in an experimental brain tumor model.

CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model.

We previously reported that both E7 and CpG‐oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7‐associated tumour challenge. Here we investigate dendritic cells (DC)‐based approach in this protection. In the study, we isolated bone marrow‐derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin‐12, as compared to that with E7 or ODN alone. Further injection with E7+ODN‐stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7‐specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon‐γ (IFN‐γ) production from CD4+ T cells and a more significant production of IFN‐γ from CD8+ T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN‐γ staining levels of CD8+ T cells. Tumour protection further appeared to be mediated by CD8+ T cells, as determined by in vivo T‐cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7‐associated tumour challenge.

In vitro induction of carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes by dendritic cells transduced with recombinant adenoviruses

Carcinoembryonic antigen (CEA), which is expressed in several cancer types, is a potential target for specific immunotherapy. In this study, the feasibility of using dendrite cells (DCs) for tumor immunotherapy after transduction with a recombinant adenovirus containing CEA gene (AdVCEA) was investigated. The recombinant AdV provided a highly efficient reproducible gene transfer into monocyte-derived DCs and its efficiency was increased in a multiplicity of infection (MOI)-dependent manner. As consequence of AdVCEA infection, the level of surface CEA on DCs was slightly increased and the dose (MOI) of AdVCEA had no effect on the surface CEA expression. However, the intracellular CEA expression was impressively increased in an MOI-dependent manner. Moreover, the AdVCEA infection had no appreciable effect on apoptosis of DCs compared with that of mock-infected and actinomycin D (AcD)-treated DCs. The AdVCEA-infected DCs-induced CEA-specific proliferative responses and it was higher than that of peptide-loaded DCs. The T-cell lines, primed by the recombinant AdVCEA-infected DCs in vitro, not only recognized CEA peptide-loaded target cells but also CEA-expressing tumor cell lines in a human leukocyte antigen (HLA) class I-restricted manner. Cytotoxic activity toward target cells was found to be mediated primarily by CD8(+) T-cells, although both CD8(+) cells and CD4(+) cells were able to lyse CEA peptide-loaded target cells. These preliminary results suggest that DCs, transduced with AdV encoding CEA, may be used for the development of adoptive cellular immunotherapy and DC-based cancer vaccine for the treatment of CEA-expressing tumors.

Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML

Abstract: The reverse transcriptase‐polymerase chain reaction (RT‐PCR) was compared with fluorescence in situ hybridization (FISH) and real‐time quantitative RT‐PCR (RQ‐PCR) for minimal residual disease (MRD) monitoring in 266 post‐transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR‐ABL positive samples determined by first‐round (1st) RT‐PCR, second‐round (2nd) RT‐PCR, and RQ‐PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR‐ABL/ABL ratio by RQ‐PCR had a mean of 0.000 13 in the 1st RT‐PCR‐negative samples and 1.42 in the 1st RT‐PCR‐positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT‐PCR‐negative and ‐positive samples (P< 0.001). The mean ratios of BCR‐ABL/ABL by RQ‐PCR were significantly different in N/N (1st/2nd RT‐PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ‐PCR is confined to the 1st RT‐PCR level. In this respect, monitoring of the 1st RT‐PCR might be useful for estimating normalized BCR‐ABL levels after transplantation. Nested RT‐PCR was of limited use, as RQ‐PCR quantified the BCR‐ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT‐PCR. FISH was significantly correlated with RQ‐PCR in FISH‐positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ‐PCR in a few cases with molecular relapse. By analyzing a large number of samples post‐transplant, we found that RQ‐PCR might be the most useful assay for MRD monitoring; however, FISH and RT‐PCR were found to be useful complementary tools.

Adoptive Transfer of Epstein-Barr Virus-Specific Cytotoxic T-Lymphocytes for the Treatment of Angiocentric Lymphomas

Angiocentric lymphoma, known as natural killer (NK)/T-cell non-Hodgkin’s lymphoma, has been reported to be associated with the Epstein-Barr virus (EBV). We performed adoptive transfer of EBV-specific polyclonal T-cell lines in 3 patients with extranodal NK/T-cell lymphoma, nasal type, and evaluated the treatment for safety, immunologic reconstitution, and clinical outcomes. The tissue samples collected from the 3 patients were confirmed by polymerase chain reaction analysis to be EBV positive. In the cases of the first and second patients, EBV-transformed B-lymphoblastoid cell lines (LCLs) and T-cell lines were generated from peripheral lymphocytes of HLA-matched sibling donors. The third patient’s T-cell lines were induced with autologous lymphocytes. Polyclonal T-cell infusion was carried out after high-dose radiotherapy because active relapsed disease remained in all of the patients. The first patient received 4 weekly infusions of 2 X 107 cells/m2, and the second and third patients underwent treatment with 2 cycles of infusions of the same dosage. All T-cell lines showed >60% NK activity, cytotoxic T-lymphocyte (CTL) responses of >40% against autologous LCLs, and no CTL activity against patient-derived lym-phoblasts. The level of cytotoxicity increased substantially in all patients after cell infusion. The 2 patients who received T-cell therapy twice had stabilized disease for more than 3 years. These safe treatments exhibited no severe inflammatory response, and no serious toxicity developed during T-cell therapy. Our findings demonstrate that adoptively transferred cells may provide reconstitution of EBV-specific CTL responses in patients with active relapsed angiocentric lymphoma. These results provide a rationale for the immunotherapy of angiocentric lymphoma.

Polymorphisms of tumor necrosis factor (TNF) α and β genes in Korean patients with psoriasis

To evaluate the association of TNF-α (TNFA) and TNF-β (TNFB) polymorphisms with psoriasis in the Korean population, we investigated TNF-α −238 and −308 promoter region and TNF-β NcoI polymorphism using PCR-RFLP in 103 Korean psoriasis patients and 125 normal controls. The carriage and allele frequencies of TNFB*2 were significantly increased in patients with psoriasis compared with normal controls. However, TNFB*1/1 homozygote and TNFB*1 allele were significantly decreased in the patients. There were no significant differences in the polymorphism of TNF-α promoter −238 and −308 between the patients and controls. We also analyzed the frequencies of TNFB alleles according to the clinical characteristics of the psoriasis patients, but no significant differences were found. However, female patients with early-onset psoriasis showed an association with the TNFB*2 allele. In conclusion, our results suggest that polymorphisms of the TNFB gene may contribute to a predisposition to psoriasis in the Korean population.