MHC Class I-related Chain A Research Articles

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Aim Natural killer (NK) cells are key players in immune surveillance against malignancies. Major histocompatibility complex (MHC) class I related chain A (MICA) is a cognate ligand for the activating receptor NKG2D expressed on the surface of NK, NKT, CD8+ and TCR γδ+ T cells. Allelic variants of MICA due to a single amino acid substitution at position 129 with a methionine (M) or valine (V) in the alpha2 domain have been reported to result in large differences in NKG2D binding, which may be related to acute and chronic GvHD. The utility of MICA-129 genotype as a genetic marker in association with clinical outcomes is linked to whether a sizable proportion of the population is represented in different genotypes. Methods We investigated the MICA 129 genotype distribution among two cohorts. Cohort I consisted of 388 bone marrow and solid organ donors (351 Cauc and 37 AF) as a random sample of the population. Cohort II consisted of 530 sequential BMT recipients (507 Cauc & 23 AF). The genotype distribution of both cohorts is shown in Table 1 , Table 2 . Results Our analysis indicated that at least 8% of cohorts analyzed represented the least frequent genotype (MM) and a racial difference in genotype distribution among Cauc and AF in cohort I (p-value Conclusions These results indicate a sizable representation of MICA-129 genotypes in different populations which increases its clinical utility as a candidate biomarker. Observed differences among Cauc vs. AF and HSCT recipients vs. random population samples warrant further investigation on larger cohorts including other races.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Human Leukocyte Antigen and Major Histocompatibility Complex Class I-Related Chain A Antibodies After Kidney Transplantation in Turkish Renal Transplant Recipients

BackgroundThis study was designed to determine whether human leukocyte antigen (HLA) and major histocompatibility complex class I chain-related A (MICA) antibody (Ab) production during the first 6 months posttransplantation correlated with long-term graft survival and rejection rate. The study group included 147 first transplantations from either living related (LRDs) or deceased donors (DDs) who were divided into two subgroups: rejection (RG, n = 28) and nonrejection (NRG, n = 119). Serum samples (n = 441) collected from each patient on posttransplant days 30, 90, and 180 were tested for HLA and MICA Ab using the Luminex technique. ResultsAmong 82 Ab-positive patients (55.8%), 40 had both HLA and MICA, 33 only HLA, and 9 only MICA Ab in the posttransplant period. The rates of HLA class I, class II, or both Ab positivities were greater in the RG than the NRG (P = .011, .037, and .0275, respectively). At 180 days posttransplant, 64.3% of patients in the RG had Ab and 41.2% in the NRG (P = .0349). The data for the LRD (n = 116) group were similar to those for the entire group; whereas there was no significant difference in Ab positivity between RG and NRG patients who received organs from DDs. There was no significant difference with respect to HLA class II and/or MICA Ab positivity between RG and NRG among patients who lacked HLA class I Ab. DiscussionWe confirmed that HLA and MICA Ab may be harmful posttransplant, promoting rejection processes and representing an important cause of graft failure. HLA class II and MICA Ab positivities were only important predictors of graft failure when present together with HLA class I positivity. Patients who developed HLA alone or both HLA and MICA Ab rejected their grafts more frequently than Ab-negative recipients.

Histamine reduces susceptibility to natural killer cells via down‐regulation of NKG2D ligands on human monocytic leukaemia THP‐1 cells

Natural killer (NK) group 2D (NKG2D) is a key activating receptor expressed on NK cells, whose interaction with ligands on target cells plays an important role in tumorigenesis. However, the effect of histamine on NKG2D ligands on tumour cells is unclear. Here we showed that human monocytic leukaemia THP-1 cells constitutively express MHC class I-related chain A (MICA) and UL16-binding protein 1 on their surface, and incubation with histamine reduced the expression in a dose-dependent and time-dependent manner as assessed by flow cytometry. Interferon-γ augmented the surface expression of the NKG2D ligands, and this augmentation was significantly attenuated by histamine. The histamine H1 receptor (H1R) agonist 2-pyridylethylamine and H2R agonist dimaprit down-regulated the expression of NKG2D ligands, and activation of H1R and H2R signalling by A23187 and forskolin, respectively, had the same effect, indicating that the histamine-induced down-regulation of NKG2D ligands is mediated by H1R and H2R. Quantitative reverse transcription-PCR showed that mRNA levels of the NKG2D ligands and relevant microRNAs were not significantly changed by histamine. Histamine down-regulated the surface expression of endoplasmic reticulum protein 5, and inhibition of matrix metalloproteinases did not impair this down-regulation, indicating that proteolytic shedding was not involved. Instead, pharmacological inhibition of protein transport and proteasome abrogated it, and histamine enhanced ubiquitination of MICA. Furthermore, histamine treatment significantly reduced susceptibility to NK cell-mediated cytotoxicity. These results suggest that histamine down-regulates NKG2D ligands through the activation of an H1R- and H2R-mediated ubiquitin-proteasome pathway and consequently reduces susceptibility to NK cells.

549 BLADDER CANCER CELLS EVADE NK CELL IMMUNITY BY USING CELL-SURFACE O-GLYCANS

INTRODUCTION AND OBJECTIVES: To explore the detailed molecular mechanisms underlying bladder cancer metastasis, we focused on the role of cell-surface carbohydrates. We immunohistochemically analyzed paraffin-embedded bladder cancer specimens for the expression of a glycosyltransferase, core2 -1,6-N-acetylglucosminyltransferase (C2GnT) which forms core2 branch on O-glycans (A). We discovered that C2GnT expression was closely related with highly metastatic phenotypes of bladder cancer and that C2GnT-expressing cancer cells possessed high ability to evade NK cell immunity. We then aimed to elucidate their evasion mechanisms. There are two major ways of NK attacking cancer cells. Cancer cell-expressing ligands bind NK-activating receptors, thereby activating NK cells to attack cancer cells. NK cell-expressing ligands such as TNF-related apoptosis inducing ligand (TRAIL) bind death receptors in tumor cells, thereby inducing apoptosis of tumor cells. An NK-activating receptor, natural killer group 2 member D (NKG2D) and TRAIL play a major role in tumor rejection (B). METHODS: A primary ligand for NKG2D in cancer cells is MHC class I-related chain A (MICA). We analyzed O-glycans of MICA and MUC1 (a cancer cell-surface major mucin) by Western blotting and lectin column chromatography. We then examined the roles of Oglycans in evasion from NK cell immunity by tumor formation assay and cytotoxicity assay. RESULTS: In C2GnT-expressing cancer cells, poly N-acetyllactosamine was present on core2 O-glycans on MICA and MUC1, and galectin-3 bound MICA and MUC1 through this poly N-acetyllactosamine. We discovered that C2GnT-expressing bladder cancer cells took two different strategies to evade NK cell attack. One is that modification of MICA with poly N-acetyllactosamine and galectin-3 reduces the affinity of MICA for NKG2D, thereby severely impairing NK cell activation (C) (Tsuboi, S. et al. 2011 EMBO J 30:3173-3185). Another one is that modification of MUC1 with poly N-acetyllactosamine and galectin-3 reduces the accessibility of NK cells to the cancer cell surface, thereby shielding cancer cells from NK cell attack by TRAIL (D). CONCLUSIONS: The immunoevasion mechanisms using core2 O-glycans (C and D) allow bladder cancer cells to survive longer in the circulation, resulting in metastasis. Source of Funding: None

112 Identification of Non-HLA Antibodies in Ventricular Assist Device Recipients

t is known that patients bridged to heart transplantation with ventricular assist device (VAD) have a higher incidence to develop antibodies directed against human leukocyte (HLA). Both HLA antibodies and non-HLA antibodies like major histocompatibility complex class I-related chain A (MICA) and functional autoantibodies against angiotensin type 1 receptor (AT1R) and endothelin receptor A (ETAR) are implicated in pathogenesis of acute rejection (AR) and cardiac allograft vasculopathy (CAV). Therefore, in this study we monitored HLA and non-HLA antibodies in VAD recipients (VADR) during the first year after VAD-implantation.

297 Non-HLA Antibody Screening after Heart Transplantation Identifies High Risk for Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy (CAV) after heart transplantation (HTx) is a major therapeutic challenge, occuring in over 50% of HTx recipients in the first years after transplantation. Antibodies against human leukocyte antigens (HLA) or non-HLA antigens like major histocompatibility complex class I-related chain A (MICA), angiotensin type 1 receptor (AT1R) or endothelin receptor A (ETAR) gain in importance as modulators of allograft function and survival.

Potential therapeutic applications of phosphodiesterase inhibition in prostate cancer

Phosphodiesterases (PDEs) play a role in controlling cyclic nucleotide action, including cyclic guanosine monophosphate (cGMP). Previous studies have ascribed a protective role of cGMP signaling on hypoxia-mediated cancer progression. Herein, we determine their potential role in hypoxia-mediated chemoresistance and immune escape. Phosphodiesterase assays were used to measure PDE activity in prostate cancer cell lines (DU145, PC3). Immunoblots were performed to determine the presence of PDEs in human prostate tissue samples. The effect of PDE inhibition on hypoxia-induced chemoresistance (compared to normoxic controls, 20% O2) was determined using clonogenic assays. Flow cytometry was used to determine the effects of PDE inhibition on surface MHC class I-related chain A (MICA), a natural killer (NK) cell-activating ligand. A mouse model was used to evaluate the in vivo effects of PDE inhibition on the growth of human prostate cancer cells. PDE5 and PDE11 were the most prominent PDEs in the cell lines, representing between 86 and 95% of the total cGMP-specific PDE activity. Treatment of DU-145 cells with a PDE inhibitor significantly reduced the hypoxia-associated acquisition of resistance to doxorubicin, with a mean 51% reduction in surviving fraction compared to controls (p < 0.001, ANOVA). As well, PDE inhibition completely reversed (p = 0.02, ANOVA) hypoxia-induced shedding of the immune stimulatory molecule, MICA, and attenuated the growth of human prostate tumor xenografts in an NK cell-competent murine model (p = 0.03, Wilcoxon, Mann-Whitney). These results suggest a rationale for future studies on the potential therapeutic applications of PDE inhibitors in men with prostate cancer.

Association of MICA gene polymorphisms with liver fibrosis in schistosomiasis patients in the Dongting Lake region

Major histocompatibility complex class I chain-related A (MICA) is a highly polymorphic gene located within the MHC class I region of the human genome. Expressed as a cell surface glycoprotein, MICA modulates immune surveillance by binding to its cognate receptor on natural killer cells, NKG2D, and its genetic polymorphisms have been recently associated with susceptibility to some infectious diseases. We determined whether MICA polymorphisms were associated with the high rate of Schistosoma parasitic worm infection or severity of disease outcome in the Dongting Lake region of Hunan Province, China. Polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (SBT) were applied for high-resolution allele typing of schistosomiasis cases (N = 103, age range = 36.2-80.5 years, 64 males and 39 females) and healthy controls (N = 141, age range = 28.6-73.3 years, 73 males and 68 females). Fourteen MICA alleles and five short-tandem repeat (STR) alleles were identified among the two populations. Three (MICA*012:01/02, MICA*017 and MICA*027) showed a higher frequency in healthy controls than in schistosomiasis patients, but the difference was not significantly correlated with susceptibility to S. japonicum infection (Pc > 0.05). In contrast, higher MICA*A5 allele frequency was significantly correlated with advanced liver fibrosis (Pc < 0.05). Furthermore, the distribution profile of MICA alleles in this Hunan Han population was significantly different from those published for Korean, Thai, American-Caucasian, and Afro-American populations (P < 0.01), but similar to other Han populations within China (P > 0.05). This study provides the initial evidence that MICA genetic polymorphisms may underlie the severity of liver fibrosis occurring in schistosomiasis patients from the Dongting Lake region.

MICA-STR A.4 Is Associated With Slower Hearing Loss Progression in Patients With Ménière’s Disease

Immune response may influence hearing outcome in Ménière's disease (MD). Major histocompatibility complex class I chain-related A (MICA) encodes a highly polymorphic stress-inducible protein, which interacts with NKGD2 receptor on the surface of NK, γδ T cells and T CD8 lymphocytes. We investigated the association of MICA gene with hearing outcome in MD and its linkage disequilibrium (LD) with human leukocyte antigen (HLA)-B. MICA short tandem repeat polymorphism (MICA-STR) was genotyped using a polymerase chain reaction-based method in a total of 302 Spanish patients with MD and 420 healthy controls. Genotyping of HLA-B was performed using polymerase chain reaction and detected with reverse sequence-specific oligonucleotide probe system in 292 patients and 1,014 controls. Hearing loss was associated with the duration of MD (p = 0.001). We found that MICA*A5 alelle was significantly associated in the Mediterranean set (Pc = 0.04, odds ratio = 0.51 [95% confidence interval, 0.30-0.84]), but this finding was not replicated in the Galicia population. However, median time to develop hearing loss greater than 40 dB was 16 years (95% confidence interval, 9-23) for patients with the MICA*A.4 allele and 10 years (95% confidence interval, 9-11) for patients with another MICA-STR allele (log-rank test, p = 0.0038). We did not find statistical differences in the distribution of B locus between the MD and the control group. In the LD analysis, MICA*A5.1-HLA-B*07 (8.8%), MICA*A6-HLA-B*44 (8.3%), and MICA*A6-HLA-B*51 (8.3%) were the most common haplotypes, and the stronger LD was found for haplotypes MICA*A.4-HLA-B*18 (r2 = 0.41) and MICA*A.4-HLA-B*27(r2 = 0.29). The allelic variant MICA*A.4 is significantly associated with slower progression of hearing loss in patients with MD. This suggests that the immune response influence hearing level in MD.

Interleukin-1β enhances the production of soluble MICA in human hepatocellular carcinoma.

The production of soluble major histocompatibility complex class I-related chain A (MICA) is thought to antagonize NKG2D-mediated immunosurveillance. Interleukin-1β (IL-1β) is elevated in patients with chronic hepatitis C (CH), and this might contribute to the escape of hepatocellular carcinoma (HCC) cells from innate immunity. In this study, we investigated the immunoregulatory role of IL-1β in the production of soluble MICA of HCC cells. First, we investigated the correlation between the serum IL-1β levels and soluble MICA in CH patients. Serum IL-1β levels were associated with soluble MICA levels in CH patients. The serum IL-1β levels of CH patients with the HCC occurrence were significantly higher than those of CH patients without HCC. We next examined the MICA production of IL-1β-treated HCC cells. Addition of IL-1β resulted in significant increase in the production of soluble MICA in HepG2 and PLC/PRF/5 cells, human HCC cells. But soluble MICA was not detected in both non-treated and IL-1β-treated normal hepatocytes. Addition of IL-1β did not increase the expressions of membrane-bound MICA on HCC cells. These were observed similarly in various cancer cells including a gastric cancer (MKN1), two colon cancers (HCT116 and HT29) and a cervical cancer (HeLa). Addition of IL-1β also increased the expression of a disintegrin and metalloproteinase (ADAM)9 in HCC cells, and the knockdown of ADAM9 in IL-1β-treated HCC cells resulted in the decrease in the production of soluble MICA of HCC cells. These findings indicate that IL-1β might enhance the production of soluble MICA by activating ADAM9 in human HCC.

Interferon-γ Up-Regulates Major-Histocompatibility-Complex Class I-Related Chain A Expression and Enhances Major-Histocompatibility-Complex Class I-Related Chain A-Mediated Cytolysis of Human Corneal Epithelium by Natural Killer CellsIn Vitro

Natural killer (NK) cells are important in the ocular surface innate response against viral and bacterial infection. Major-histocompatibility-complex class I-related chain A (MICA) antigens are ligands of natural killer group 2D, an activating or coactivating receptor expressed on NK cells. Recent studies demonstrated that interferon-gamma (IFN-γ) could modulate MICA expression in tumor cells. However, little is known about MICA expression and regulation in human corneal epithelium. Our study assessed whether the proinflammatory cytokine, IFN-γ, affects MICA expression in human corneal epithelium. We identified low levels of surface MICA expression in corneal epithelium using flow cytometry. IFN-γ promoted surface MICA expression in corneal epithelium and increased soluble MICA levels in a dose-dependent manner. IFN-γ also enhanced NK cell-mediated cytotoxicity against the corneal epithelium. Anti-MICA antibodies could further block this process. In summary, we describe a novel IFN-γ function in the regulation of the innate response in ocular surfaces.

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O-glycans.

The O-glycan branching enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT), forms O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (core2 O-glycans) on cell-surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT-expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT-expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK-activating receptor, NKG2D, by its tumour-associated ligand, Major histocompatibility complex class I-related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT-expressing bladder tumour cells, poly-N-acetyllactosamine was present on core2 O-glycans on MICA, and galectin-3 bound the NKG2D-binding site of MICA through this poly-N-acetyllactosamine. Galectin-3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT-expressing bladder tumour cells, resulting in tumour metastasis.

Differential major histocompatibility complex class I chain‐related A allele associations with skin and joint manifestations of psoriatic disease

About 30% of patients with psoriasis have psoriatic arthritis (PsA), an inflammatory arthritis that can affect both axial and peripheral joints. Major histocompatibility complex class I chain-related A (MICA) alleles have previously been shown to be associated with PsA; however it is unclear whether there is a differential association of MICA alleles with skin and joint manifestations of PsA. Here, we describe a case-control study that aims to validate previously reported MICA allele associations with PsA and determine whether MICA alleles differentiate patients with PsA from those with psoriasis without PsA. Two hundred forty-nine unrelated Caucasian PsA patients, 243 psoriasis patients without arthritis, and 248 healthy controls were genotyped for 55 MICA alleles using PCR-SSP, and for human leucocyte antigen (HLA)-B and HLA-C alleles by PCR-SSO reverse line blot. Allele frequencies were calculated and logistic regressions were performed, adjusting for HLA-B and HLA-C alleles previously shown to be associated with psoriasis and/or PsA. Several MICA alleles were associated with psoriatic disease, PsA, and psoriasis compared with controls, and PsA compared with psoriasis in univariate analyses. Haplotype analysis showed evidence of strong linkage disequilibrium (LD) between PsA and psoriasis risk alleles of HLA-C, HLA-B, and MICA. After adjusting for significant HLA-B and HLA-C alleles in multivariate analyses, MICA*016 remained significantly associated with psoriasis [odds ratio (OR) = 5.5, P = 0.008]. MICA*00801 homozygosity was associated with susceptibility to PsA when compared with patients with psoriasis alone (OR = 2.26, P = 0.009). We conclude that most MICA allele associations with psoriasis and PsA are dependent on LD with HLA-B and HLA-C risk alleles. Independent of HLA, only MICA*016 influences the risk of developing psoriasis without arthritis, and homozygosity for MICA*00801 increases the risk of developing PsA in patients with psoriasis.

MICA Gene Polymorphism in Kidney Allografts and Possible Impact of Functionally Relevant Variants

It is likely that the polymorphic MICA (MHC class I related chain A) molecules on graft endothelial cells (ECs) may be a target for specific antibodies and T cells directed against solid organ grafts. Although there is evidence for a role of MICA in vascular and transplant biology, genotyping is not performed routinely, and thus there are few correlations between polymorphism and endothelial phenotype. The present study examined the frequency of the various alleles for the nonclassical MHCI MICA gene among a cohort of kidney transplant donors, particularly MICA genetic variants (MICA A5.1 and MICA-129) that may affect MICA expression and function on graft EC. Genotyping was performed on genomic DNA derived from primary cultures of EC established from transplant donors at the time of transplantation. Herein we have reported that among 106 alleles analyzed, 28/69 MICA alleles were distributed among 7 major variants (*00804, *00801, *004, *00201, *00901*, *011, *010), representing 70% of the donors. MICA*008 the most abundant allele (31.1%) was associated with the MICA A5.1 mutation. The majority of donors (52.8%) had at least one MICA A5.l allele, with 13.2% homozygous for this mutation. The MICA-129 val/val genotype, which encodes a low-affinity ligand, was predominant (49%), while the MICA-129 met/met, corresponding to a high-affinity ligand, was observed in 11.3% of the transplants. Our findings highlighted the MICA gene polymorphism that produces functional diversity in transplant recipients with variable interactions between MICA and its receptor NKG2D.

The role of HIF-1 in up-regulating MICA expression on human renal proximal tubular epithelial cells during hypoxia/reoxygenation

BackgroundHuman major histocompatibility complex class I-related chain A (MICA) plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI) could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity.ResultsWe find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2) cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased.ConclusionsThese results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is through a HIF-1 pathway. The increased IFNgamma secretion and enhanced NK cell cytotoxicity was mainly due to the surface expression of MICA induced by over-expression of HIF-1alpha. This study enhances our understanding of MICA activation mechanisms during kidney transplantation and provides insights into how IRI can influence transplant outcome. Moreover, these findings might be also important for developing strategies to reduce the effect of MICA in kidney transplant outcomes in the future.

Discrepant effects of Chlamydia trachomatis infection on MICA expression of HeLa and U373 cells

Natural killer (NK) cells are an important component of the host immune defense against Chlamydia trachomatis (CT). In this study we investigated the effects of CT on the expression of two forms of MHC class I-related chain A (MICA). One form, a truncated variant that has a partial transmembrane region and no cytoplasmic tail, was expressed in HeLa cells. The other, a full-length variant, was expressed in astrocytoma U373 cells. CT infection had little effect on the expression of the truncated form, which was constitutively expressed in HeLa cells, but expression of the full-length MICA was down-regulated in CT-infected U373 cells. Down-regulation of MICA after CT infection protected U373 cells from NK cell lysis, whereas HeLa cells expressing the truncated form were killed. This study shows that the constitutive expression of the truncated from of MICA prevents CT from evading immune recognition by NK cells.

Expression of CD133 confers malignant potential by regulating metalloproteinases in human hepatocellular carcinoma

Although CD133 expression is identified as a cancer stem cell marker of hepatocellular carcinoma (HCC), the detailed characteristics of HCC cells expressing CD133 remain unclear. We examined the malignant characteristics of CD133-expressing HCC cells. CD133-expressing cells could be detected with low frequency in 5 HCC tissues. We derived two different HCC cell lines by (1) transfection of CD133 siRNA in PLC/PRF/5 cells in (CD133si-PLC/PRF/5), and (2) by a magnetic cell sorting method that allowed to divide Huh7 cells into two CD133 positive (+) and negative (-) groups. CD133 knockdown in PLC/PRF/5 cells resulted in a decrease of the mRNA and protein expressions of matrix metalloproteinase (MMP)-2 and a disintegrin and metalloproteinase (ADAM)9. We next examined the malignant characteristics related to decreasing MMP-2 and ADAM9 in HCC cells. In CD133si-PLC/PRF/5 cells and CD133- Huh7 cells, invasiveness and vascular endothelial growth factor (VEGF) production, which are both related to the activity of MMP-2, were inhibited compared CD133-expressing HCC cells. We previously demonstrated that ADAM9 protease plays critical roles in the shedding of MHC class I-related chain A (MICA) which regulates the sensitivity of tumor cells to natural killer cells (NK). Decreasing ADAM9 expression in CD133si-PLC/PRF/5 cells and CD133- Huh7 cells resulted in an increase in membrane-bound MICA and a decrease in soluble MICA production. Both CD133si-PLC/PRF/5 cells and CD133- Huh7 cells were susceptible to NK activity, depending on the expression levels of membrane-bound MICA, but CD133-expressing HCC cells were not. These results demonstrate that CD133 expression in HCC cells confers malignant potential which may contribute to the survival of HCC cells.

Phenotype markers and cytokine intracellular production by CD8+ γδ T lymphocytes do not support a regulatory T profile in Behçet's disease patients and healthy controls

γδ T lymphocytes (GD) have been suggested as one of the causes of cytokine dysregulation that results in neutrophils hyperactivation in Behçet's disease (BD) patients. In addition, GD can provoke cytotoxic lesions in autoimmune diseases by interaction with MICA (MHC class I chain-related A) molecules, through NKG2D receptor on its surface. In contrast, the CD8+ subset of γδ T lymphocytes (GDCD8+) has been related to regulatory T activity. The aim of this study was to determine the phenotype and the intracellular cytokine profile in GD from peripheral blood, to discern if they were skewed to an effector or regulatory pattern in BD. We performed phenotype analysis, by three-colour flow cytometry, in 28 BD, 15 healthy controls (HC) and 14 patients with recurrent bucal ulcers (RBU). We studied intracellular cytokine production in 10 BD and 14 HC, after polyclonal stimulation. In addition, we analysed serum IL-15 and soluble MICA, by ELISA, in 27 BD, 21 HC and 40 rheumatoid arthritis patients. The hallmark in BD was a specific increase in CD8 expression by GD, and in GDCD8+ absolute numbers. Most of GDCD8+ presented CD8 αα homodimers and were negative for CD103, Foxp3 and CTLA-4. GDCD8+ and GDCD8− were high IFNγ−, but poor IL-2, IL-10, TGFβ and IL-4-producing cells, with no differences between BD and HC. NKG2D expression on CD8+ T cells, serum IL-15 and soluble MICA were not significantly increased in BD. Our results do not suggest a T regulatory profile for GDCD8+ neither in HC, nor in BD. We cannot rule out other suppression mechanisms or some heterogeneity within this subset that could contribute to regulatory function.

Heat shock protein expression is low in intestinal mucosa in juvenile idiopathic arthritis: a defect in immunoregulation?

Objectives: Heat shock proteins (HSPs) are involved in the regulation of inflammation and in the maintenance of mucosal integrity. Their altered expression may be a marker of mucosal inflammation and also contribute to tissue injury. The small intestinal mucosa in children with juvenile idiopathic arthritis (JIA) shows signs of intestinal immune activation, such as increased intraepithelial cytotoxic lymphocyte counts. To further evaluate the characteristics of this immune activation in JIA, we have studied the expression of several HSPs, major histocompatibility complex (MHC) class I-related chain A (MICA), and the heat shock transcription factor 1 (HSF1) in intestinal biopsies from children with JIA.Methods: We studied 15 patients with JIA. Controls included 13 children without JIA, studied for various gastrointestinal (GI) symptoms, but eventually shown not to have any GI disease. The subjects were examined by endoscopy. The expression of HSP60, HSP70, MICA, and HSF1 was analysed in ileal and duodenal biopsies by using immunohistochemistry.Results: The expression levels of HSP60, MICA, and HSF1 were significantly lower in the duodenal epithelium in the JIA patients compared to the controls. MICA and HSF1 also showed lower expression in the ileal epithelium. The expression of HSP70 did not differ between the groups.Conclusions: The downregulation of HSP60, MICA, and HSF1 in small intestinal mucosa may indicate that intestinal epithelial cells show immune aberration in JIA. We speculate that the low heat shock response may play a role in the pathogenesis of JIA, interfering with mucosal integrity and local intestinal immunoregulation.