MHC Class I Polypeptide-related Sequence A Research Articles

CLPTM1L is a GPI-anchoring pathway component targeted by HCMV.

The GPI-anchoring pathway plays important roles in normal development and immune modulation. MHC Class I Polypeptide-related Sequence A (MICA) is a stress-induced ligand, downregulated by human cytomegalovirus (HCMV) to escape immune recognition. Its most prevalent allele, MICA*008, is GPI-anchored via an uncharacterized pathway. Here, we identify cleft lip and palate transmembrane protein 1-like protein (CLPTM1L) as a GPI-anchoring pathway component and show that during infection, the HCMV protein US9 downregulates MICA*008 via CLPTM1L. We show that the expression of some GPI-anchored proteins (CD109, CD59, and MELTF)-but not others (ULBP2, ULBP3)-is CLPTM1L-dependent, and further show that like MICA*008, MELTF is downregulated by US9 via CLPTM1L during infection. Mechanistically, we suggest that CLPTM1L's function depends on its interaction with a free form of PIG-T, normally a part of the GPI transamidase complex. We suggest that US9 inhibits this interaction and thereby downregulates the expression of CLPTM1L-dependent proteins. Altogether, we report on a new GPI-anchoring pathway component that is targeted by HCMV.

IMMU-16. SOMATIC MUTATIONS IN HUMAN LEUKOCYTE ANTIGEN GENES AND ANTIGEN PRESENTATION PATHWAY GENES IN MALIGNANT GLIOMA

Abstract Immunotherapeutic approaches in cancer aim to booster T cell mediated immune responses. Tumor-specific T cells recognize target cells via human leukocyte antigen (HLA) molecules before cell specific lysis is initiated. Malignant gliomas are known for their immunoevasive and immunosuppressive properties which impede effective tumor-specific immune responses, including clinical responses to immune checkpoint inhibition. Our aim was to investigate whether somatic mutation in HLA genes and antigen presentation pathway genes are present in malignant gliomas. METHODS We performed next generation sequencing in 138 patients, including matched tumor and blood samples from 100 IDHwt glioblastomas, 25 IDHmut astrocytomas and 13 IDHmut oligodendrogliomas. Sequencing was performed on tumor and peripheral blood for HLA class I (HLA-A, -B, -C), HLA class II (HLA-DR, -DP, -DQ), non-classical HLA molecules (HLA-E, -F, -G, -H), MICA/B, TAP1/2 and beta2 microglobulin (B2M). Identified calls were validated using targeted nanopore sequencing. RESULTS In total, we identified 28 mutations based on Illumina sequencing. Mutations were located in HLA-B (2 mutations), HLA-C (1), B2M (2), HLA-DQA1 (4), HLA-DQB1 (4), HLA-DPB1 (1), HLA-E (1), HLA-F (2), HLA-G (3), HLA-H (1), MICA (3), MICB (2), TAP1 (1) and TAP2 (1). No mutations were found in HLA-A and HLA-DRB1. A frequency cutoff of 5% was applied. Additional mutations (n = 46) below this cutoff were identified. The mutations were found in 30 patients (21.7%). Some patients harbored up to 7-8 mutations in multiple HLA and antigen presenting pathway genes. Most mutations were found in the MICA (MHC class I polypeptide–related sequence A) gene locus, which is engaged by the NKG2D ligand which is broadly expressed on NK cells, gd and ab T cells. CONCLUSION Our study demonstrates that somatic mutations in HLA and antigen presentation pathway genes are frequently present in malignant gliomas.

Expressional regulation of NKG2DLs is associated with the tumor development and shortened overall survival in lung adenocarcinoma

Natural killer group 2D ligands (NKG2DLs) are expressed on tumor cells as a ligand for Natural killer group 2D (NKG2D) receptors. NKG2DLs interact with NKG2D to induce immune cell-mediated cytotoxicity for eliminating tumors. Studies demonstrated that tumor cells can reduce NKG2DLs’ expression to escape from anti-tumor immunity, leading to an aggressive cancer phenotype and poor prognosis in some cancers. However, these studies are limited and there is no comprehensive work on the regulation of NKG2DLs in lung adenocarcinoma (LUAD) which is one of the deadliest cancers worldwide. Here, we conducted an in silico analysis to evaluate the changes in NKG2DLs in LUAD by analyzing The Cancer Genome Atlas and the Gene Expression Omnibus datasets including tumor vs. normal comparisons, TNM stages, survival and infiltrating immune estimation profile. Results indicated that some members of NKG2DL were downregulated in LUAD as compared to normal samples. We determined that MICA (MHC class I polypeptide-related sequence A) was the most and significantly downregulated ligand among others and the results were nearly consistent with the different datasets which we used. Furthermore, survival analysis revealed that down-regulated MICA transcript expression might be one of the prognostic indicators of LUAD. Interestingly, according to the immune cell infiltrating analysis, there wasn’t a direct correlation between the MICA transcript expression and immune cell infiltration, while for MICB there was. In addition, in genetic alteration, DNA methylation and miRNA analyses, we did not observe critical outcomes that would clarify the down-regulated MICA expression in detail. Regardless, this study is highly comprehensive and contributes valuable suggestions to further functional studies about the regulation of NKG2DLs and promising immunotherapeutic approaches in LUAD.

Baseline soluble MICA levels act as a predictive biomarker for the efficacy of regorafenib treatment in colorectal cancer

BackgroundTo evaluate the effect of regorafenib on soluble MHC class I polypeptide-related sequence A (MICA) (sMICA) level in vitro. In addition, we clinically examined whether its plasma levels were associated with regorafenib activity in terms of progression-free survival (PFS) in patients with CRC.MethodsHuman CRC cell line HCT116 and HT29 cells were treated with regorafenib and its pharmacologically active metabolites, M2 or M5 at the same concentrations as those in sera of patients. We also examined the sMICA levels and the area under the plasma concentration–time curve of regorafenib, M2 and M5.ResultsRegorafenib, M2, and M5 significantly suppressed shedding of MICA in human CRC cells without toxicity. This resulted in the reduced production of sMICA. In the clinical examination, patients with CRC who showed long median PFS (3.7 months) had significantly lower sMICA levels than those with shorter median PFS (1.2 months) (p = 0.045).ConclusionsMICA is an attractive agent for manipulating the immunological control of CRC and baseline sMICA levels could be a predictive biomarker for the efficacy of regorafenib treatment.

Expression profiles of Natural Killer Group 2D Ligands (NGK2DLs) in colorectal carcinoma and changes in response to chemotherapeutic agents.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Natural Killer Group 2D Receptor (NKG2D) and their ligands (NKG2DLs) play crucial roles in natural killer (NK) cell-mediated cytotoxicity. Tumorigeneses cause increased NKG2DLs expression on tumor cell surfaces, thereby these cells individually eliminated by NK cells. However, CRC cells can reduce their NKG2DL expression to escape from NK-mediated immune surveillance which is associated with poor prognosis. Therefore, previous studies suggest that up-regulation of NKG2DLs can contribute to promising NK cell-mediated immunotherapy strategies. We aimed to analyze NKG2DLs expression profiles in response to chemotherapeutic drugs and increased MHC class I polypeptide-related sequence A (MICA) expression, which is related to favorable prognosis in CRC, using low doses of bortezomib and epirubicin combination without causing direct cytotoxicity. Results showed that MICA expression sligthlyincreased following drug treatment in the CRC cells but not for the normal cells. Also, we enriched our study with Gene Expression Omnibus (GEO) datasets including expression profiles of various NKG2DLs using in silico analyses. Accordingly, NKG2DL expression in CRC was screened in proportion to other cancers, histologic subtypes, TNM stages and metastatic samples to compare with our data. Overall, the analyzed data showed that NKG2DLs demonstrate different expression profiles in response to chemotherapeutic agents and a combination of low-dose bortezomib and epirubicin slightly increased MICA mRNA expression in CRC cell lines. However, performing further analysis of the combination therapy for MICA protein expression and studying its interaction with NK cells will make the results more meaningful.

Retinoids Decrease Soluble MICA Concentration by Inhibiting the Enzymatic Activity of ADAM9 and ADAM10.

The association between MHC class I polypeptide-related sequence A (MICA) and hepatocellular carcinoma (HCC) development was identified in our previous genome-wide association study. Decreasing soluble MICA (sMICA) through MICA sheddases suppression facilitates natural killer (NK) cell-mediated cytotoxicity. The expression of ADAM9 in HCC has been correlated with poor prognosis, and our recent study showed that its suppression contributes to cancer elimination by decreasing sMICA. Human HCC cell line PLC/PRF/5 and HepG2 cells were used. sMICA levels were measured by ELISA. Expression of retinoid X receptors (RXRs) and retinoic acid receptors (RARs) was knocked down by siRNA. In our screening of FDA-approved drugs in vitro, retinoids were found to be efficient ADAM9 and ADAM10 inhibitors. Treatment with retinoids reduced sMICA levels in human HCC cells. Interestingly, the effects were abrogated by depletion of the retinoid receptor RXRα. Retinoids can be potential novel agents for HCC treatment.

Type 1 Diabetes and Autoimmune Thyroid Disease-The Genetic Link.

Type 1 diabetes (T1D) and autoimmune thyroid disease (AITD) are the most frequent chronic autoimmune diseases worldwide. Several autoimmune endocrine and non-endocrine disorders tend to occur together. T1D and AITD often cluster in individuals and families, seen in the formation of autoimmune polyendocrinopathy (AP). The close relationship between these two diseases is largely explained by sharing a common genetic background. The HLA antigens DQ2 (DQA1*0501-DQB1*0201) and DQ8 (DQA1*0301-DQB1*0302), tightly linked with DR3 and DR4, are the major common genetic predisposition. Moreover, functional single nucleotide polymorphisms (or rare variants) of various genes, such as the cytotoxic T-lymphocyte- associated antigen (CTLA4), the protein tyrosine phosphatase non-receptor type 22 (PTPN22), the interleukin-2 Receptor (IL2Ra), the Vitamin D receptor (VDR), and the tumor-necrosis-factor-α (TNF) that are involved in immune regulation have been identified to confer susceptibility to both T1D and AITD. Other genes including cluster of differentiation 40 (CD40), the forkhead box P3 (FOXP3), the MHC Class I Polypeptide-Related Sequence A (MICA), insulin variable number of tandem repeats (INS-VNTR), the C-Type Lectin Domain Containing 16A (CLEC16A), the Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3) gene, the interferon-induced helicase C domain-containing protein 1 (IFIH1), and various cytokine genes are also under suspicion to increase susceptibility to T1D and AITD. Further, BTB domain and CNC homolog 2 (BACH2), C-C motif chemokine receptor 5 (CCR5), SH2B adaptor protein 3 (SH2B3), and Rac family small GTPase 2 (RAC2) are found to be associated with T1D and AITD by various independent genome wide association studies and overlap in our list, indicating a strong common genetic link for T1D and AITD. As several susceptibility genes and environmental factors contribute to the disease aetiology of both T1D and AITD and/or AP subtype III variant (T1D+AITD) simultaneously, all patients with T1D should be screened for AITD, and vice versa.

Expression of MHC class I polypeptide-related sequence A (MICA) in colorectal cancer.

Background: The major histocompatibility complex class I polypeptide-related sequence A (MICA) is one of the ligands of the natural killer group 2D (NKG2D) activating receptor. MICA stimulates NKG2D, which further triggers activation of natural killer cells and leads to killing of infected target cells. To subvert the biological function of NKG2D, tumor cells utilize an escape strategy by shedding overexpressed MICA. In this study, we determined the levels of MICA in colorectal cancers (CRCs). Additionally, we established correlations between MICA expression and clinical characteristics. Publicly available data and bioinformatics tools were used for validation purposes. Methods: We determined the MICA RNA expression levels and assessed their correlation with clinicopathological parameters in CRC using the UALCAN web-portal. We performed immunohistochemical analysis on tissue microarrays having 192 samples, acquired from 96 CRC patients, to validate the expression of MICA in CRC and adjacent uninvolved tissue and investigated its prognostic significance by Kaplan-Meier and proportional hazards methods. Results: Bioinformatics and immunohistochemical analyses showed that MICA expression was significantly upregulated in CRCs as compared to uninvolved tissue, and the overexpression of MICA was independent of pathologic stage, histotype, nodal metastasis status, p53-status, as well as patient's race, age and gender. Moreover, PROGgeneV2 survival analysis of two cohorts showed a poor prognosis for CRC patients exhibiting high MICA expression. Conclusions: Overall, our findings for CRC patients demonstrate generally high expression of MICA, and suggest that a poor prognosis relates to high MICA expression. These results can be further explored due to their potential to provide clues to the contribution of the tumor microenvironment to the progression of CRC.

Donor specific anti-HLA antibodies and cardiac allograft vasculopathy: A prospective study using highly automated 3-D optical coherence tomography analysis

IntroductionRecent studies suggested potential positive correlations between HLA-specific antibodies and development of cardiac allograft vasculopathy (CAV). MethodsThis prospective two-center study investigated early progression of CAV by coronary optical coherence tomography in 1 month and 12 months after heart transplantation (HTx) in 104 patients. Detection and characterization of donor specific (DSA) and MHC class-I polypeptide-related sequence A (MICA) antibodies were performed before, 1, 6 and 12 months after transplantation. ResultsDuring the first post-HTx year, we observed a significant reduction in the mean coronary luminal area (P < .001), and progression in mean intimal thickness (IT) (P < .001). DSA and anti-MICA occurred in 17% of all patients, but no significant relationship was observed between presence of DSA/anti-MICA and IT progression within 12 months after HTx. In contrast, we observed significant association between presence of DSA (p=0.031), de-novo DSA (p=0.031), HLA Class II DSA (p=0.017) and media thickness (MT) progression. ConclusionResults of our study did not identify a direct association between presence of DSA/anti-MICA and intimal thickness progression in an early period after HTx. However, we found significant relationships between DSA and media thickness progression that may identify a newly recognized immune-pathological aspect of CAV.

Association of MICA gene polymorphisms with thionamide-induced agranulocytosis.

Thionamide-induced agranulocytosis (TIA), namely antithyroid drug (ATD)-induced agranulocytosis, is one of the most feared adverse effect of ATDs. It is defined as a granulocyte count of less than 0.5 × 109/L after ATD administration. Several studies reported that TIA is associated with human leukocyte antigen (HLA) and nearby genes. Our previous study found that the susceptibility genes of TIA are similar in north China and European populations. We evaluated the associations of 23 candidate single nucleotide polymorphisms (SNPs) in 37 patients with TIA and 254 patients with Graves' disease (GD) as controls by iPLEX MassARRAY system. Five SNPs in the MHC class I polypeptide-related sequence A(MICA) genes [rs4349859 (p = 1.43E-7); rs145575084 (p = 5.79E-6); rs116135464 (p = 3.70E-5); rs148015908 (p = 3.79E-5) and rs189600525 (p = 2.15E-4)] were found to be significantly associated with TIA after Bonferroni correction. After combining with previous data of rs4349859 and HLA-B*27:05, the haplotype analysis showed that patients carrying P-A-C-A-T-T-A haplotype have a higher risk of TIA (p = 9.76E-7; OR = 14.85, 95% CI 3.63-60.77). Our findings suggest that five high linked SNPs of MICA gene are significantly associated with susceptibility to TIA.

Abstract C038: MHC class I polypeptide related sequence A (MICA) race-related differential expression in prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in American men, with higher incidence and death rates in African Americans (AA) relative to Caucasian Americans (CA); nevertheless, these disparities are still poorly understood. MHC class I polypeptide related sequence A (MICA) is a cell surface protein able to promote activation of natural killer cells. Cleavage of MICA and generation of its soluble form (sMICA) has been described as an immunoevasion mechanism presented by different types of aggressive tumors. In agreement with the role of MICA on tumor immunoevasion, recent studies correlated better prognosis for patients expressing higher levels of MICA in different types of cancer. We speculated that MICA expression would be different between AA and CA PCa tumors. To access MICA expression in PCa patients, we stained a TMA containing 30 AA and 27 CA tumor cores. Our results showed that CA PCa tumor cores had higher expression of MICA than the AA (p=0.0019). In order to establish an in vitro system to study the biologic effects of MICA in the context of disparity in PCa, we assessed the expression of surface MICA and release rate of sMICA in different PCa cell lines: MDA-PCa-2b and E006AA-hT, derived from AA, and DU-145 and LNCaP, derived from CA. Flow cytometry analysis showed baseline cell fraction expression positive for surface MICA of 3% in MDA-PCa-2b, 18% in E006AA-hT, 54% in DU-145, and 67% in LNCaP, with significant difference between each cell line (p&amp;lt;0.0001). Baseline release rate of sMICA was nondetectable for MDA-PCa-2b, 5pg/ml/106 cells in E006AA-hT, 120pg/ml/106 cells in DU-145, and 54pg/ml/106 cells for LNCaP cells. Overall, our results show that MICA expression is race related. Further studies are necessary to address the biologic function of the expression and the impact in the immune system. Citation Format: Charles R. Pound, Marcelo J. Sakiyama, Amit Reddy, Ingrid Espinoza, Eldrin Bhanat, Krista Syrigos, Roya Gordji, Deepak Kumar, Jack Lewin, Xinchun Zhou, Jesus Monico, Christian Gomez. MHC class I polypeptide related sequence A (MICA) race-related differential expression in prostate cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C038.

N-Glycans Mediate the Ebola Virus-GP1 Shielding of Ligands to Immune Receptors and Immune Evasion.

The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A*0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A*0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins.

Association of MICA-129Met/Val polymorphism with clinical outcome of anti-TNF therapy and MICA serum levels in patients with rheumatoid arthritis

MHC class I polypeptide-related sequence A (MICA) is a stress-induced protein involved in activation of NK and T cells through interaction with NKG2D receptor. These molecules are atypically expressed in synovium of patients diagnosed with rheumatoid arthritis (RA). A total of 279 patients with RA, qualified to TNF-blockade therapy, were genotyped for MICA rs1051792 SNP. The effectiveness of anti-TNF agents was assessed with European League Against Rheumatism criteria. Significant relationship between MICA rs1051792 and outcome of TNF-blockade therapy has been found. The MICA rs1051792 GG genotype was overrepresented in patients non-responsive to anti-TNF drugs in comparison with other genotypes (p = 0.010). On the other hand, beneficial therapeutic response was more frequently detected among RA subjects possessing heterozygous genotype than those with homozygous genotypes (p = 0.003). Furthermore, increased MICA concentrations in serum were observed in patients possessing MICA rs1051792 GG genotype as compared with those with GA or AA genotypes (p = 1.8 × 10−5). The results from this study indicate the potential influence of MICA rs1051792 polymorphism on modulation of therapeutic response to TNF-blockade treatment in RA.

MHC Class I Polypeptide-Related Sequence A (MICA) Polymorphism Association with Ankylosing Spondylitis in Iraqi Patients

MHC Class I Polypeptide-Related Sequence A (MICA) Polymorphism Association with Ankylosing Spondylitis in Iraqi Patients

Race-associated expression of MHC class I polypeptide-related sequence A (MICA) in prostate cancer

Despite the lack of a complete understanding of the disparities involved, prostate cancer (PCa) has both higher incidence and death rates in African American Men (AAM) relative to those of Caucasian American Men (CAM). MHC class I polypeptide related sequence A (MICA) is an innate immunity protein involved in tumor immunoevasion. Due to a lack of reports of race-specific expression of MICA in PCa, we evaluated MICA expression in patients' tumors and in cell lines from a racially diverse origin. Immunohistochemistry was done on a tissue microarray (TMA) with antibodies against MICA. Tumor MICA mRNA was assessed by data mining using Oncomine and PROGeneV2. Surface MICA and release rate of soluble (s) MICA was evaluated in PCa cell lines originally derived from African American (MDA-PCa-2b) or Caucasian (LNCaP and DU-145) PCa patients. Prostate tumor tissue had a 1.7-fold higher MICA expression relative to normal tissue (p < .0001). MICA immunoreactivity in PCa tissue from AAM was 24% lower (p = .002) compared to CAM. Survival analysis revealed a marginal association of low MICA with poor overall survival (OS) (p = .058). By data mining analysis, a 2.9-fold higher level of MICA mRNA was evidenced in tumor compared to normal tissue (p < .0001). Tumors from AAM had 24% lower levels of MICA mRNA compared to tumors from CAM (p = .038), and poor prognosis was found for patients with lower MICA mRNA (p = .028). By flow cytometry analysis, cell fraction positive for surface MICA was of 3% in MDA-PCa-2b cells, 54% in DU-145 cells, and 67% in LNCaP cells (p < .0001). sMICA was detected in DU-145 and LNCaP cells, but was not detected in MDA-PCa-2b cells. Both LNCaP and DU-145 cells were sensitive to cytolysis mediated by Natural killer (NK) cells. MDA-PCa-2b cells, however were between 1.3-fold at 10:1 Effector:Target (E:T) ratio (p < .0001) and 2-fold at 50:1 E:T ratio (p < .0001) more resistant to NK-mediated cytolysis relative to cells from Caucasian origin.These results suggest that MICA expression may be related to the aggressive nature of PCa. Our findings also demonstrate for the first time that there are variations in MICA expression in the context of racial differences. This study establishes a rationale for further investigation of MICA as a potential race-specific prognostic marker in PCa.

Pomalidomide increases immune surface marker expression and immune recognition of oncovirus-infected cells

ABSTRACTMost chronic viruses evade T-cell and natural killer (NK) immunity through downregulation of immune surface markers. Previously we showed that Pomalidomide (Pom) increases surface expression of major histocompatibility complex class I (MHC-I) in Kaposi sarcoma-associated herpesvirus-infected latent and lytic cells and restores ICAM-1 and B7-2 in latent cells. We explored the ability of Pom to increase immune surface marker expression in cells infected by other chronic viruses, including human T-cell leukemia virus type-1 (HTLV-1), Epstein-Barr virus (EBV), human papilloma virus (HPV), Merkel cell polyoma virus (MCV), and human immunodeficiency virus type-1 (HIV-1). Pom increased MHC-1, ICAM-1, and B7-2/CD86 in immortalized T-cell lines productively infected with HTLV-1 and also significantly increased their susceptibility to NK cell-mediated cytotoxicity. Pom enhancement of MHC-I and ICAM-1 in primary cells infected with HTLV-1 was abrogated by knockout of HTLV-1 orf-1. Pom increased expression of ICAM-1, B7-2 and MHC class I polypeptide related sequence A (MICA) surface expression in the EBV-infected Daudi cells and increased their T-cell activation and susceptibility to NK cells. Moreover, Pom increased expression of certain of these surface markers on Akata, Raji, and EBV lymphoblastic cell lines. The increased expression of immune surface markers in these virus-infected lines was generally associated with a decrease in IRF4 expression. By contrast, Pom treatment of HPV, MCV and HIV-1 infected cells did not increase these immune surface markers. Pom and related drugs may be clinically beneficial for the treatment of HTLV-1 and EBV-induced tumors by rendering infected cells more susceptible to both innate and adaptive host immune responses.

Enzymatic inhibition of MICA sheddase ADAM17 by lomofungin in hepatocellular carcinoma cells.

In our previous study on hepatocellular carcinoma (HCC) susceptibility genes in chronic hepatitis patients, we identified the MHC class I polypeptide-related sequence A (MICA). Natural killer cells eliminate various cancer cells, including HCC, by suppressing MICA shedding. Therefore, we investigated MICA sheddases and inhibitors for HCC immunotherapy. In this study, HepG2, PLC/PRF/5, and Hep3B were treated with the siRNA of a disintegrin and metalloproteases (ADAMs) and matrix metalloproteases to measure the concentration of soluble MICA (sMICA) by ELISA to detect the therapeutic target. Furthermore, an FDA-approved drug library was tested for the enzymatic inhibition of the targeted enzyme in an in vitro drug screening assay system. ADAM17 knockdown reduced sMICA levels and increased membrane-bound MICA (mMICA) expression in HCC cells. In an in vitro drug screen using an FDA-approved drug library, lomofungin, an antifungal drug, was found to strongly decrease ADAM17 activity. In HCC cells, mMICA expression was induced and sMICA production was inhibited in a dose-dependent manner. These effects were cancelled upon ADAM17 knockdown, suggesting that lomofungin targeted ADAM17. Analysis of lomofungin analogs revealed the responsible functional groups. In summary, we suggest lomofungin to be an attractive agent for the immunological control of HCC, via the suppression of ADAM17.

Association of MICA Alleles and Human Leukocyte Antigen B in Turkish Patients Diagnosed With Behçet's Disease

This study aims to investigate whether or not MHC class I polypeptide-related sequence A (MICA) polymorphisms are associated with the susceptibility to Behçet's disease (BD) in a Turkish population. The study included 38 Turkish BD patients (20 males, 18 females; mean age 34±10.9 years) and 51 ethnically matched healthy controls (30 males, 21 females; mean age 36±12.8 years). MICA and human leukocyte antigen B (HLA-B) alleles were determined in all subjects by using the Luminex technology. LABType sequence-specific oligonucleotide MICA test (One Lambda) and sequence-specific oligonucleotide B locus tests (Gene-Probe) were used for the typing studies. A total of 16 MICA alleles were found in BD patients as well as in control subjects. The gene frequency of MICA*006 was significantly higher in the BD patients compared to controls (14.5% vs. 0.9%; odds ratio [OR]: 17.092 95% confidence interval [CI] [2.155~135.554]; p<0.05). When haplotypes were evaluated, an association was found between the haplotypes HLA-B*51-MICA*006 (11.8% and 0.9%; OR: 13.567 95% CI [1.679~109.577]; p<0.001) and HLA-B*51-MICA*009 (27.6% and 13.7%; OR: 2.4 95% CI [1.127~5.109]; p<0.05). Frequencies of HLA-B*49-MICA*004 (0% and 6.8%) and HLA-B*52- MICA*009 (0% and 10.7%) were significantly higher in controls compared to BD patients (p<0.05). Our study shows that the MICA*006 (MICA-A6) and the MICA*009 alleles are associated with BD susceptibility in HLA-B*51 positive Turkish population, particularly in HLA-B*51 patients with MICA*006, which might be considered as a diagnostic biomarker for BD in the future.

Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia.

Interactions between immune and tumor cells in the tumor microenvironment (TME) often impact patient outcome, yet remain poorly understood. In addition, the effects of biophysical features such as hypoxia [low oxygen (O2)] on cells within the TME may lead to tumor evasion. Gamma delta T cells (γδTcs) naturally kill transformed cells and are therefore under development as immunotherapy for various cancers. Clinical trials have proven the safety of γδTc immunotherapy and increased circulating γδTc levels correlate with improved patient outcome. Yet, the function of γδTc tumor infiltrating lymphocytes in human breast cancer remains controversial. Breast tumors can be highly hypoxic, thus therapy must be effective under low O2 conditions. We have found increased infiltration of γδTc in areas of hypoxia in a small cohort of breast tumors; considering their inherent plasticity, it is important to understand how hypoxia influences γδTc function. In vitro, the cell density of expanded primary healthy donor blood-derived human γδTc decreased in response to hypoxia (2% O2) compared to normoxia (20% O2). However, the secretion of macrophage inflammatory protein 1α (MIP1α)/MIP1β, regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L by γδTc were increased after 40 h in hypoxia compared to normoxia concomitant with the stabilization of hypoxia inducible factor 1-alpha protein. Mechanistically, we determined that natural killer group 2, member D (NKG2D) on γδTc and the NKG2D ligand MHC class I polypeptide-related sequence A (MICA)/B on MCF-7 and T47D breast cancer cell lines are important for γδTc cytotoxicity, but that MIP1α, RANTES, and CD40L do not play a direct role in cytotoxicity. Hypoxia appeared to enhance the cytotoxicity of γδTc such that exposure for 48 h increased cytotoxicity of γδTc against breast cancer cells that were maintained in normoxia; conversely, breast cancer lines incubated in hypoxia for 48 h prior to the assay were largely resistant to γδTc cytotoxicity. MICA/B surface expression on both MCF-7 and T47D remained unchanged upon exposure to hypoxia; however, ELISAs revealed increased MICA shedding by MCF-7 under hypoxia, potentially explaining resistance to γδTc cytotoxicity. Despite enhanced γδTc cytotoxicity upon pre-incubation in hypoxia, these cells were unable to overcome hypoxia-induced resistance of MCF-7. Thus, such resistance mechanisms employed by breast cancer targets must be overcome to develop more effective γδTc immunotherapies.

Leinal polypeptide promotes NK cells to suppress PCa survival in vitro.

As an important component in the innate immune system, natural killer (NK) cells have been demonstrated to be clinically associated with prostate cancer (PCa) progression and castration resistance. Therefore, the development of novel agents that may enhance the cytotoxicity of NK cells possesses promising therapeutic applications. In the present study, leinal polypeptide (LP) solution was supplemented into a co-culture system of NK and PCa cells, as it was previously demonstrated that LP are able to activate NK cells, which kill PCa cells based on an MTT cell viability assay. Mechanistic dissection demonstrated that LP enhanced androgen receptor degradation, which resulted in an upregulation of MHC class I polypeptide-related sequence A (MICA) and MICB. In turn, the induced expression of MICA and MICB was able to further trigger NK cell activation, forming a positive loop between NK cells and PCa cells in the presence of LP solution.