Expression Levels Of MHC Class Research Articles

Abstract CT141: Combination of hormone therapy, GnRH agonist, and immunotherapy enhance immune activation in premenopausal ER+/HER2- metastatic breast cancer patients: Results of biomarker analysis from a pilot phase II study

Abstract Introduction: Immunotherapy has not been proved to be efficacious in ER+/HER2- metastatic breast cancer. Our team has reported an effective combination of exemestane, gonadotropin releasing hormone agonist, and pembrolizumab in premenopausal ER+/HER2- MBC. Here we report the updates of the final results and biomarker associates of the study. Methods: Premenopausal ER+/HER2- MBC patients who failed no more than 2 lines of hormone therapy without chemotherapy for MBC were enrolled in a tertiary medical center in Taiwan (NCT02990845). The primary endpoint was progression-free survival (PFS) rate at 8 months. The secondary endpoints included overall response rate, overall survival (OS), progression-free survival (PFS), and other biomarkers. Pre-treatment (N=12) and post-treatment (N=5) tumor tissue were collected for biomarker analysis, including tumor-infiltrating lymphocyte(TIL), PD-L1 expression by immunohistochemical staining, tumor mutational burden, RNAseq, and IO360 analysis. Results: A total of 15 patients were enrolled in the study with 14 evaluable patients. The progression-free survival rate at 8 months was 64.3%. The median PFS and OS were 10.34 months and 39.56 months respectively. The overall response was 35.7%. TIL, tumor mutational burden, or PD-L1 expression were not associated with the response to the treatment. In the NanoString IO-360 analysis, increased expression of genes in the immune related signature was noted in the post-treatment samples as compared to the pre-treatment samples. The expression levels of MHC class II and T cell-related genes were upregulated in the post-treatment samples. Most of the immune cell populations are increased after pembrolizumab/exemestane/leuprolide treatment. CD56 dim NK cells, Th1 cells, and NK cells are the top 3 populations that are increased after treatment. Using CIBERSORT algorithms to analyze RNAseq data from pretreatment samples, dendritic cells (p=0.039) and NK cells (p=0.082) were associated with the responders versus non-responders in the study. Conclusion: In this final analysis, pembrolizumab, exemestane, and leuprolide remain an effective treatment for premenopausal ER+/HER2- MBC. Pembrolizumab, exemestane, and leuprolide treatment is associated with immune system activation, turning the tumor microenvironment from cold to hot in luminal disease. Citation Format: I-Chun Chen, Ching-Hung Lin, Dwan-Ying Chang, Tom Wei-Wu Chen, Ming-Yang Wang, Wei-Li Ma, Yi-Ting Lin, Shu-Min Huang, Yen-Shen Lu. Combination of hormone therapy, GnRH agonist, and immunotherapy enhance immune activation in premenopausal ER+/HER2- metastatic breast cancer patients: Results of biomarker analysis from a pilot phase II study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT141.

Vaccination modulates pulmonary eosinophil subsets upon breakthrough influenza infection.

Abstract Eosinophils play a critical role in mucosal tissue homeostasis, anti-helminth responses, and allergy. Lung eosinophilia is associated with aberrant T cell responses to respiratory viral infection and is also a consequence of vaccine-associated enhanced respiratory disease (VAERD). A growing body of literature has demonstrated there are different phenotypic subsets of eosinophils and, depending on subset, can play a protective role and aid in lung recovery following infection. Previous work by Choi et al. showed recruitment of eosinophils to the lung in BALB/c mice vaccinated with alum-adjuvanted trivalent inactivated influenza vaccine (TIV) following a sub-lethal H1N1 (A/New Caledonia/20/1999; NC99) influenza challenge. In this study, flow cytometric analysis of the lung showed an increase in total lung eosinophil numbers in all NC99-challenged mice compared to controls. Furthermore, in TIV-vaccinated then challenged mice, flux of inflammatory eosinophil (iEos) and resident eosinophil (rEos) numbers at 7 days post-infection (DPI) resulted in iEos/rEos ratios similar to that of OVA-sensitized allergic control mice; this effect was independent of alum adjuvant. Treatment groups with increased lung eosinophil numbers did not show signs of enhanced morbidity or inflammatory cytokine signatures compared to unvaccinated mice. Unsupervised clustering using CATALYST confirmed findings from manual gating analysis and revealed differences in eosinophil activation status based on surface expression levels of MHC Class II. Our findings provide a starting point for more sophisticated phenotypic characterization of eosinophil subsets and their distinct functional contributions to vaccine- and infection-induced immunity. This research was partly funded by NIAID T32 Virus-Host Interactions Training Grant (5T32AI007647-22) and CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C).

Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro.

The dromedary camel (Camelus dromedarius) is well-adapted to the desert environment with the ability to tolerate increased internal body temperatures rising daily to 41–42°C during extreme hot. This study was undertaken to assess whether in vitro incubation of camel blood at 41°C, simulating conditions of heat stress, differently alters cell vitality, phenotype, and function of leukocytes, compared to incubation at 37°C (normothermia). Using flow cytometry, the cell vitality (necrosis and apoptosis), the expression of several cell markers and adhesion molecules, and the antimicrobial functions of camel leukocytes were analyzed in vitro. The fraction of apoptotic cells within the granulocytes, lymphocytes, and monocytes increased significantly after incubation of camel whole blood at 41°C for 4 h. The higher increase in apoptotic granulocytes and monocytes compared to lymphocytes suggests higher resistance of camel lymphocytes to heat stress. Functionally, incubation of camel blood at 41°C for 4 h enhanced the phagocytosis and ROS production activities of camel neutrophils and monocytes toward S. aureus. Monocytes from camel blood incubated at 41°C for 4 h significantly decreased their expression level of MHC class II molecules with no change in the abundance of CD163, resulting in a CD163high MHC-IIlow M2-like macrophage phenotype. In addition, heat stress treatment showed an inhibitory effect on the LPS-induced changes in camel monocytes phenotype. Furthermore, in vitro incubation of camel blood at 41°C reduced the expression of the cell adhesion molecules CD18 and CD11a on neutrophils and monocytes. Collectively, the present study identified some heat-stress-induced phenotypic and functional alterations in camel blood leukocytes, providing a paradigm for comparative immunology in the large animals. The clinical relevance of the observed changes in camel leukocytes for the adaptation of the camel immune response to heat stress conditions needs further in vitro and in vivo studies.

Study of the Effect of Linoleic Acid on the Expression Level of MicroRNA-106b and MicroRNA-20a and their Related Target MHC Class I Chain-related Protein A in Docetaxel-treated Gastric Cancer Cells

Background: MicroRNAs are involved in response to therapeutic agents and have the ability to regulate the expression level of the targets associated with cancer growth and progression. As a dangerous signal in tumor cells, increased expression level of MHC class I chain-related protein A (MICA) could activate the immune system and induce responses to tumor cells. We conducted the present research to study the effect of linoleic acid (LA) and docetaxel alone or in combination with miR-106b, miR-20a, and MICA expression level in metastatic gastric cancer (GC) cell line MKN-45. Method: The study was an in vitro study using the gastric cancer cell line MKN- 45, which was cultured and treated with docetaxel and LA. Subsequently, the expression level of miR-106b, miR-20a, and MICA were assessed with quantitative real-time polymerase chain reaction. Results: MiR-106b decreased in LA and LA/docetaxel (P < 0.0001 and P = 0.002),and increased in docetaxel alone (P = 0.01). Meanwhile, miR-20a significantly decreased in docetaxel and LA/docetaxel (P < 0.0001), increased in LA treatment (P = 0.02). Regarding MICA, it significantly decreased in all the treated cells (P < 0.0001, p <0.0001, and P=0.0002 for docetaxel, LA and docetaxel/LA, respectively) but with different reduction intensities. Conclusion: Using LA or docetaxel alone had a different effect on miR-106b, miR-20a, and MICA expression level, yet in a simultaneous treatment, their positive effects were intensified. LA enhanced the effect of docetaxel concerning the expression level of miR-106b, miR-20a, and MICA and vice versa, which suggested that LA could be employed as an effective complementary agent in GC along with docetaxel.

NF-κB-inducing kinase contributes to normal development of cortical thymic epithelial cells: its possible role in shaping a proper T-cell repertoire.

Nuclear factor (NF)-κB-inducing kinase (NIK) is known to be a critical regulator of multiple aspects of the immune response. Although the role of NIK in the development of medullary thymic epithelial cells (mTECs) has been well documented, the impact of NIK on the differentiation and function of cortical thymic epithelial cells (cTECs) remains ambiguous. To investigate the possible involvement of NIK in cTEC differentiation, we have compared the gene expression and function of cTECs from a NIK-mutant mouse, alymphoplasia (aly/aly) with those of cTECs from wild-type (WT) mice. Flow cytometric analyses revealed that expression levels of MHC class II, but not MHC class I or other TEC markers, were higher in aly/aly cells than in WT cells. Notably, the proportion of MHC class IIhi+ cTECs was elevated in aly/aly mice. We also demonstrated that expression of Ccl5 mRNA in the MHC class IIhi+ subset of aly/aly cTECs was decreased compared with that in WT cells, implying an abnormal pattern of gene expression in aly/aly cTECs. Analyses of bone marrow chimera using aly/aly or aly/+ mice as hosts suggested that Vβ usage and CD5 expression on WT T-cells were altered when they matured in aly/aly thymi. These results collectively indicate that NIK may be involved in controlling the function of cTEC in selecting a proper T-cell repertoire.

Lower protein expression levels of MHC class I chain-related gene A in hepatocellular carcinoma are at high risk of recurrence after surgical resection

MHC class I chain-related gene A (MICA) variants have been associated with hepatocellular carcinoma (HCC). Their association with MICA expression in cancer cells and cancer recurrence is unknown. SNP rs2596542 of MICA was tested in 193 HCC patients with surgical resection. The corresponding MICA expression in the cancer tissue was measured by immunochemistry microarray. Patients with the SNP rs2596542 A allele had significantly lower MICA expression in tumor tissue than did those with the GG genotype (24.7 ± 15.1% vs. 41.5 ± 23.4%, P < 0.001). Patients who had HCC recurrence had significantly lower MICA expression in tumor tissue (34.2 ± 21.8% vs. 24.0 ± 19.8%, P = 0.03). Cox regression analysis revealed that the factors independently predictive of HCC recurrence included low MICA expression (hazard ratio [HR]/95%confidence intervals [CI]: 2.77/1.07–7.14, P = 0.035) and tumor size (HR/CI: 5.22/2.11–12.96, P < 0.001). Compared to patients with tumors <5 cm and MICA expression >30%, patients with either one and both two risk factors had HCC HRs of 9.76 (C.I. 1.27–75.03, P = 0.03) and 27.30 (C.I. 3.46–215.6, P = 0.002), respectively. We concluded that low cellular MICA expressions were at a greater risk of HCC recurrence after curative treatment.

Dendritic cells pulsed with generated tumor cell lysate from Phyllanthus amarus Schum. & Thonn. induces anti-tumor immune response

BackgroundDendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors. In the present study, 80% ethanol extract of Phyllanthus amarus was used to generate tumor lysate (TLY) derived from HCT 116 and MCF-7 cancer cell lines via induction of apoptosis. Monocyte-derived DCs were generated ex vivo from the adherent population of peripheral blood mononuclear cells (PBMCs). The generated TLY were used to impulse DCs to investigate its effect on their cellular immune functions including antigen presentation capacity, phagocytic activity, chemotaxis capacity, T-cell proliferation and cytokines release.MethodsThe effect of P. amarus-generated TLY on DCs maturation was evaluated by determination of MHC class I, II and CD 11c expression as well as the co-stimulatory molecules CD 83 and 86 by using flow cytometry. The phagocytic capacity of TLY-pulsed DCs was investigated through FITC-dextran uptake by using flow cytometry. The effect on the cytokines release including IL-12, IL-6 and IL-10 was elucidated by using ELISA. The migration capacity and T cell proliferation activity of pulsed DCs were measured. The relative gene expression levels of cytokines were determined by using qRT-PCR. The major constituents of P. amarus extract were qualitatively and quantitatively analyzed by using validated reversed-phase high performance liquid chromatography (HPLC) methods.ResultsP. amarus-generated TLY significantly up-regulated the expression levels of MHC class I, CD 11 c, CD 83 and 86 in pulsed DCs. The release of interleukin IL-12 and IL-6 was enhanced by TLY-DCs at a ratio of 1 DC: 3 tumor apoptotic bodies (APO), however, the release of IL-10 was suppressed. The migration ability as well as allogeneic T-cell proliferation activities of loaded DCs were significantly enhanced, but their phagocytic capacity was highly attenuated. The gene expression profiles for IL-12 and IL-6 of DCs showed increase in their mRNA gene expression in TLY pulsed DCs versus unloaded and LPS-treated only DCs.ConclusionThe effect of P. amarus-generated TLY on the immune effector mechanisms of DCs verified its potential to induce an in vitro anti-tumor immune response against the recognized tumor antigen.

The intrinsic pro-tumorigenic role of IRF1

Abstract Interferon regulatory factor 1 (IRF1) is a nuclear transcription factor which appears to have anti-tumor activity in human and mice. IRF1 can be induced by interferon-gamma (IFNγ). Although recent studies indicated that the IFNγ-PD-L1 axis played an important role during T cell exhaustion, and promoted tumorigenesis, the cell intrinsic role of IRF1 is not completely clear in this context. In this study, we investigated the intrinsic role of IRF1 during tumor progression. We generated several IRF1-deficient (IRF1-KO) murine tumor cell lines (CT26, MC38 and B16-F10) via CRISPR/Cas9-mediated genome editing, and compared their growth rates with wild type (WT) cells both in vitro and in vivo using syngeneic mouse tumor models. We found that the loss of IRF1 did not affect cell growth rates in vitro. However, the IRF1-KO cells showed significantly slower tumor growth than WT cells in mice models. Depletion of CD8+ T cell using antibody rescued the ability of IRF1-KO cells to grow tumors. Furthermore, analysis of tumor infiltrating T cells showed more activated CD8+ T cells and less exhausted T cells in IRF1-KO induced tumors than those induced by WT cells. To confirm whether these results were caused by IFNγ-IRF1-PD-L1 axis, we evaluated IFNγ-induced inhibitory ligands on tumor cells in both WT and IRF1 KO cell lines. We found that the expression levels of MHC class I were not affected by loss of IRF1, whereas, the induction of PD-L1 by IFNγ was abolished in IRF1 KO cells. In conclusion, intrinsic loss of IRF1 in tumor cells significantly reduces tumorigenesis in solid tumors. This phenomenon depends on the regulation of PD-L1 expression on tumor cells via IFNγ-IRF1-PD-L1 axis, and eventually affecting host anti-tumor immune responses, such as CD8+ T cells.

Cancer-Associated Oxidase ERO1-α Regulates the Expression of MHC Class I Molecule via Oxidative Folding.

ERO1-α is an oxidizing enzyme that exists in the endoplasmic reticulum and is induced under hypoxia. It reoxidizes the reduced form of protein disulfide isomerase that has oxidized target proteins. We found that ERO1-α is overexpressed in a variety of tumor types. MHC class I H chain (HC) has two disulfide bonds in the α2 and α3 domains. MHC class I HC folding is linked to the assembly of MHC class I molecules because only fully disulfide-bonded class I HCs efficiently assemble with β2-microglobulin. In this study, we show that ERO1-α associates with protein disulfide isomerase, calnexin, and immature MHC class I before being incorporated into the TAP-1-associated peptide-loading complex. Importantly, ERO1-α regulates the redox state as well as cell surface expression of MHC class I, leading to alteration of susceptibility by CD8(+) T cells. Similarly, the ERO1-α expression within cancer cells was associated with the expression level of MHC class I in colon cancer tissues. Thus, the cancer-associated ERO1-α regulates the expression of the MHC class I molecule via oxidative folding.

C형 간염 바이러스 감염 간암 세포주와 T 림프구의 상호작용에 대한 연구

Recently, Hepatitis C virus (HCV) replication system has been established using human hepatoma cells (huh cell) and a variety of HCV clones. In this study, we established an infectious HCV replication system using huh7.5 cells and J6/JFH1 clone (genotype 2a). In addition, we investigated the antigen presentation capability of HCV-infected huh7.5 cells to HCV-specific T cells. Interestingly, HCV-infected huh7.5 cells were not capable of activating HCV-specific T cells. However, huh7.5 cells stimulated by exogenous HCV peptide were able to activate HCV-specific T cells, which was shown to produce TNF-α and IFN-γ. We further examined if HCV infection has an inhibitory effect on the expression of MHC class I molecule of huh7.5 cells. We found that HCV infection did not change the expression level of MHC class I molecule on huh7.5 cells.

IL-17 regulates Toll-like receptor-mediated activation of dendritic cells (TUM4P.905)

Abstract We have proposed that IL-17 produced by γδ T cells promoted angiogenesis and subsequent tumor progression in tumor microenvironment. However, the precise effect of IL-17 on immune system was remained unclear. Since dendritic cells (DCs) play a crucial role in immune regulation, we investigated the impact of IL-17 on DC function. Bone marrow-derived DCs were treated with IL-17 for 16 h and then stimulated with various toll-like receptor (TLR) ligands. IL-17 treatment significantly reduced IL-12, TNF-α, or IL-6 production by the DCs in a dose dependent manner. Interestingly, we found that IL-12 production was most clearly decreased compared to the other cytokines. In addition, IL-17 markedly suppressed the upregulation of MHC class II, CD40, and CD86 expressions on DCs after the stimulation. To evaluate effects of IL-17 on T cell-stimulating activity, IL-17-treated DCs were co-cultured with OVA antigen-specific T cells in the presence of OVA peptides or OVA proteins. We confirmed that IFN-γ production and T cell proliferation were suppressed by the IL-17-treatment. To determine effects of IL-17 on in vivo DC function, we established a novel tumor-bearing model using IL-17-producing cancer cells. As a result, expression levels of MHC class II and costimulatoly molecules on DCs were reduced in this model. These findings clearly indicated that IL-17 would inhibit activation of DCs including cytokine production and T cell activation in tumor microenvironment.

Lectin-like Receptor Ly49s3 on Dendritic Cells Contributes to the Differentiation of Regulatory T Cells in the Rat Thymus

Naturally occurring regulatory T cells (nTregs), important for immune regulation and the maintenance of self-tolerance, develop in the thymus. The Hirosaki hairless rat (HHR), derived from the Sprague-Dawley rat (SDR), was shown to have decreased peripheral lymphocyte number, small thymus, and leukocyte infiltration in its dermis. In the HHR thymus, the medulla was underdeveloped and nTreg number was decreased. Array comparative genome hybridization revealed the deletion of an NK cell lectin-like receptor gene, Ly49s3, detecting MHC class I molecules on target cells, in the chromosome 4q42 region in HHRs. The gene was expressed in thymic conventional dendritic cells (cDCs) in SDRs, but not in HHRs. When CD4-single-positive or CD4(+)CD8(-)CD25(-) thymocytes were cultured with thymic cDCs, the expression of nTreg marker genes was lower when these cells were from HHRs than from SDRs, suggesting that HHR cDCs are deficient in the ability to induce and maintain nTreg differentiation. Expression of the genes was recovered when Ly49s3 was expressed on HHR thymic cDCs. Expression levels of MHC class II genes, presumably from cDCs, were parallel to those of nTreg marker genes in mixed-cell cultures. However, in the presence of an anti-MHC class I Ab, blocking interaction between Ly49s3 and MHC class I molecules, the expression of the former genes was upregulated, whereas the latter was downregulated. These results suggest that Ly49s3 contributes to nTreg regulation along with MHC class II molecules, whose effects alone are insufficient, and loss of Ly49s3 from thymic cDCs is the reason for the nTreg deficiency in HHRs.

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Immunomodulatory Effect of Marine Cembrane-Type Diterpenoids on Dendritic Cells

Dendritic cells (DCs) are antigen presenting cells, which can present antigens to T-cells and play an important role in linking innate and adaptive immunity. DC maturation can be induced by many stimuli, including pro-inflammatory cytokines and bacterial products, such as lipopolysaccharides (LPS). Here, we examined the immunomodulatory effects of marine cembrane compounds, (9E,13E)-5-acetoxy-6-hydroxy-9,13-dimethyl-3-methylene-3,3a,4,5,6,7,8,11,12,14a-decahydro-2H-cyclotrideca[b]furan-2-one (1), (9E,13E)-5-acetoxy-6-acetyl-9,13-dimethyl-3-methylene-3,3a,4,5,6,7,8,11,12,14a-decahydro-2H-cyclotrideca[b]furan-2-one (2), lobocrassin B (3), (−)14-deoxycrassin (4), cembranolide B (5) and 13-acetoxysarcocrassolide (6) isolated from a soft coral, Lobophytum crassum, on mouse bone marrow-derived dendritic cells (BMDCs). The results revealed that cembrane-type diterpenoids, especially lobocrassin B, effectively inhibited LPS-induced BMDC activation by inhibiting the production of TNF-α. Pre-treatment of BMDCs with Lobocrassin B for 1 h is essential to prohibit the following activation induced by various toll-like receptor (TLR) agonists, such as LPS, zymosan, lipoteichoic acid (LTA) and Pam2CSK4. Inhibition of NF-κB nuclear translocation by lobocrassin B, which is a key transcription factor for cytokine production in TLR signaling, was evident as assayed by high-content image analysis. Lobocrassin B attenuated DC maturation and endocytosis as the expression levels of MHC class II and the co-stimulatory molecules were downregulated, which may affect the function of DCs to initiate the T-cell responses. Thus, lobocrassin B may have the potential in treatment of immune dysregulated diseases in the future.

Characteristics of Porcine Induced Pluripotent Stem Cells in their MHC Protein Expression

Induced pluripotent stem cells (iPSCs) are taught to be highly beneficial in the field of regenerative medicine and believed to overcome immunogenic barrier for cell transplantation. However, there still have been remaining concerns regarding their safety and efficiency for actual use. Moreover, there have been some recent reversal reports that autogenic iPSCs could also be a target of autologous immune system. For promoting beneficial impact of transplanted cells, in-depth analysis using appropriate animal models, such as porcine, are required. Despite latest efforts to generate porcine iPSCs, there has been no investigation of their immunologic properties yet. We generated porcine induced pluripotent cells from somatic cells by retro-mono vector containing Yamanaka 4 factors (Sox2, Klf4, Oct4 and c-Myc). To examine the possibilities of autologous immune rejection after transplantation, we measured the level of MHC class I and II proteins expression on cell surface. Porcine iPSCs showed only low expression level of MHC class I and moderately increased the level on their differentiated derivates. However, MHC class II proteins were rarely expressed. In the presence of IFN-γ, the expression level of MHC class I protein on the differentiated cells elevated. Our report would suggest tremendous potentials of porcine iPSCs as alternative tools of human iPSCs for preclinical evaluation of the safety, immune response and drug screening and metabolism. This study was funded by the Animal Plant and Fisheries Quarantine and Inspection Agency, Ministry of Agriculture and Forestry, Republic of Korea. This work was also supported by a grant from the Next-Generation BioGreen 21 program (No.P J008030, J008032), Rural Development Administration, Republic of Korea.

Altered expression levels of HLA class II and costimulatory molecules on circulating monocytes from patients with cervical intraepithelial neoplasia and squamous cervical cancer

The aim of this study was to investigate the role of the cell surface expression levels of HLA class I and II molecules, the costimulatory molecules CD80/B7-1 and CD86/B7-2, and the adhesion molecules CD54 and CD58 during cervical carcinogenesis. The expression levels of MHC classIandII molecules, the costimulatory molecules CD80/B7-1 and CD86/B7-2 and the adhesion molecules CD54 and CD58 on CD14+ peripheral blood monocytes (PBMs) from 21cases of cervical intraepithelial neoplasia (CIN) II-III, 51 squamous cervical carcinomas (SCCs) and 18healthy controls were analyzed using flow cytometry analysis. We found increased expression levels of HLA-DR (p=0.000), HLA-DQ (p=0.000), CD86/B7-2 (p=0.002) and CD58 (p=0.000) on PBMs from patients with SCC and CINII-III, compared with healthy control subjects, whereas no significant difference existed in the expression levels of HLA class I antigens, HLA-DP CD80/B7-1 and CD54. Upregulated expression levels of HLA-DR, HLA-DQ, CD86/B7-2 and CD58 were associated with disease progression, indicating that an increased expression of HLA-DR, HLA-DQ, CD86/B7-2 and CD58 on PBMs may be correlated with the evolution of cervical cancer.

Decreased expression of MHC class II and cathepsin E in dendritic cells might contribute to impaired induction of antigen-specific T cell response in NC/Nga mice

NC/Nga (NC) mice are an animal model for human atopic dermatitis. We found that induction of antigen (Ag)-specific T cell response is diminished in ovalbumin (OVA)-immunized NC mice. Ability of Ag presentation in NC mouse dendritic cells (DCs) was significantly weaker than that in BALB/c and DBA/2 mouse DCs. Expression levels of MHC class II molecules and cathepsin E in NC mouse DCs were significantly lower that those in BALB/c and DBA/2 mouse DCs. These results indicate that low expression levels of MHC class II and cathepsin E might contribute to the defect in induction of Ag-specific T cells in NC mice.

Human NK Cells Differ More in Their KIR2DL1-Dependent Thresholds for HLA-Cw6-Mediated Inhibition than in Their Maximal Killing Capacity

In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.

Intracellular estrogen receptor-binding fragment-associated antigen 9 exerts in vivo tumor-promoting effects via its coiled-coil region

Estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is a tumor-promoting factor of largely unknown function. To assess a causative role of EBAG9 in advanced malignancies, we generated the EG7-OVA and MethA murine tumor cell lines that stably express full-length or truncated EBAG9 protein, using retroviral-mediated gene transduction. Upon subcutaneous inoculation into immunocompetent mice, both cell lines showed marked acceleration of in vivo tumor growth when full-length EBAG9 was overexpressed. Interestingly, deletion of the coiled-coil region, thereby producing truncated EBAG9 protein, abolished the tumor-acceleration effect, establishing the importance of this domain in EBAG9-mediated tumor promotion. However, there was no alteration in in vitro cell proliferation or expression levels of MHC class I and co-stimulatory molecules believed to play a role in immune evasion of tumor cells in these tumor cell lines expressing full-length or truncated EBAG9 protein. Furthermore, both full-length and truncated EBAG9 proteins showed a predominantly cytoplasmic localization in the tumor cells. Collectively, these results suggest that EBAG9 overexpression can be causative in enhancing the malignant properties of tumor cells, and that tumor promotion likely requires EBAG9 intracellular association with as yet unidentified binding partners via the coiled-coil region.

Listeria monocytogenes Infection Affects a Subset of Ly49-Expressing NK Cells in the Rat

NK cells are protective against certain bacterial and viral infections, and their production of IFN-γ is important for the early innate immune defence against L. monocytogenes. We have previously shown that depletion of NK cells in rats leads to increased bacterial burden upon L. monocytogenes infection, and that a subset of NK cells encompassing the majority of Ly49 receptors (Ly49s3+ NK cells) contributed to this effect. In this study, we have further investigated how the Ly49s3+ NK cell subset is affected by L. monocytogenes infection. We observed an increased percentage of Ly49s3+ NK cells in the spleen and a reduction in the bone marrow within the first 48 hrs of L. monocytogenes infection. Concomitantly, we observed increased expression levels of the inflammatory chemokine receptors CCR5 and CXCR3 by Ly49s3+ bone marrow NK cells, as compared to Ly49s3− NK cells, suggesting involvement of Ly49s3+ NK cells in the early phase of infection. However, NK cell production of IFN-γ was independent of Ly49 receptor expression. Furthermore, we observed increased expression levels of MHC class I molecules on both macrophages and NK cells during the first 48 hrs of infection, paralleled by a reduction in the surface expression of Ly49s3 on NK cells. In conclusion, L. monocytogenes infection modulates the tissue distribution of Ly49s3+ NK cells, and induces increased MHC class I expression and hence reduced surface expression of Ly49 receptors on NK cells. These changes indicate that L. monocytogenes infection may have multiple effects on NK cells in vivo, and suggests the involvement of Ly49-expressing NK cells in the immune responses towards L. monocytogenes.