Association of polymorphic extracellular domains of MICA with cervical cancer in northeastern Thai population

Cancer of the cervix is one of the common cancers among women worldwide. The primary risk factor of cervical cancer is the high-risk group human papillomavirus infection. Host genetic factors should also be involved. Major histocompatibility complex class I chain related A (MICA), a ligand to the natural killer cell receptor group (NKG)2D receptor relevant to immune surveillance, was investigated as a potential candidate. MICA is highly polymorphic. Although the data were limited regarding functional polymorphism, it is conceivable that polymorphism of MICA may contribute to different degree of immune activation caused by different NKG2D-binding affinity, acting as a susceptible factor for development of cervical cancer. In this study, we have developed a polymerase chain reaction-sequence-specific primer technique defining most of MICA alleles with a total of 41 primer mixes. This set of primers could especially discriminate MICA*045 (formerly 052), a common allele in northeastern Thai population, from MICA*00701, a common allele in Caucasian population. Based on the distribution of MICA in northeastern Thai population, only 27 primer mixes were required to screen the MICA polymorphisms in this population. This set of primers was used for MICA typing of 100 samples of cervical cancer compared with 94 samples of healthy northeastern Thai females (NETF). Thirteen alleles or groups of alleles were identified in these samples. Common alleles in our population were MICA*00801(027,048)/0803, MICA*010 and MICA*00201(020, 023, 050)/30/41. Statistically significant differences were not observed in the distributions of MICA alleles between different stages of patients and the control group. However, there were particular residues that were negatively associated with cervical cancer, suggesting active MICA motifs in immune activation.     

High resolution molecular phototyping of MICA and MICB alleles using sequence specific primers

Major histocompatibility complex (MHC) class I chain-related genes, MICA and MICB, are located centromeric to human leukocyte antigen B (HLA-B) on chromosome 6. In response to stress stimuli, MIC is expressed on epithelial, endothelial and fibroblast cells, but not lymphocytes and has been demonstrated to ligate the natural killer (NK) cell receptor, NKG2D. Nucleotide sequences of MICA and MICB are highly polymorphic and several methods have been established to identify these polymorphisms, including sequence-based typing and sequence-specific oligonucleotide probing. In this study we have developed a high-resolution polymerase chain reaction-sequence-specific primer (PCR-SSP) phototyping scheme that detects all WHO-recognized MICA alleles and all 12 MICB alleles. Our method will also recognize a MICA deletion haplotype and distinguish between MICA alleles with different binding affinities for NKG2D, encoded by a non-synonymous nucleotide substitution in codon 129. Furthermore, our scheme targets almost 90% of the dimorphic codon positions in exons 2, 3, and 4, which result in non-synonymous amino acid changes. This method can be used to determine MIC allele frequencies within different populations, as well as investigate MIC associations in cohorts of patients with autoimmune and infectious diseases and explore the impact of MIC on the survival of solid organ and stem cell transplants.     

High resolution MIC genotyping: design and application to the investigation of inflammatory bowel disease susceptibility

The highly polymorphic nonclassical MHC class I chain-related (MIC) genes MICA and MICB encode stress inducible glycoproteins expressed on a variety of epithelial cells including intestinal cells. Interaction with the receptor NKG2D is likely to provide an important costimulatory signal for activation and proliferation of NK cells, activated macrophages and CD8 alphabeta and gammadelta T cells. Fifty-four MICA and 17 MICB alleles have been described to date. Although the functional significance of this polymorphism is not known, the high degree of nonconservative substitution, concentration to the putative ligand-binding site and recent observation that different MICA alleles bind to NKG2D with varying affinity has generated much interest. The MIC genes are attractive functional and positional candidate genes for inflammatory bowel disease susceptibility as a consequence of their position in the HLA region and expression on the gastrointestinal epithelium. We developed a robust, high-resolution PCR-SSP genotyping method that can be incorporated into the standard 'Phototyping' system and which effectively identifies 46 of 54 MICA alleles, and all 17 MICB alleles. We applied this system in combination with microsatellite genotyping of the exon 5 variable number of tandem repeats (VNTR) to the investigation of genetic susceptibility to the inflammatory bowel diseases, ulcerative colitis and Crohn's disease. We studied 248 patients with Crohn's disease, 329 with ulcerative colitis and 354 ethnically matched controls. Linkage disequilibrium patterns between HLA-B, MICA and MICB are presented. Analysis by individual allele or by multilocus haplotype failed to identify any significant disease associations.     

Estrogen upregulates MICA/B expression in human non-small cell lung cancer through the regulation of ADAM17

Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.     

Associations of major histocompatibility complex class I chain-related molecule polymorphisms with Behcet's disease in Caucasian patients

HLA-B*51 is known to be associated with Behcet's disease (BD) in many ethnic groups. The pathogenic gene, however, may lie close to the HLA-B locus and therefore be in linkage disequilibrium with HLA-B*51. On the basis of the proximity of MIC genes to HLA-B, their expression pattern and their affinity for the activating NKG2D receptor on natural killer (NK) cells and gammadelta T cells, these molecules have been postulated as susceptibility factors in BD. DNA from 56 western European Caucasians with BD and 90 Caucasian controls were analysed by polymerase chain reaction using allele-specific primers for MICA and MICB alleles. An increased allele frequency of MICA*009 was found in the BD patient group (25.0%) when compared with the controls (7.2%). This was associated with a corresponding decrease in MICA*008 in the BD patients (36.6%) compared with the controls (46.7%), which was not significant. MICA*009 was strongly associated with the presence of HLA-B*51 in patients and controls. No significant difference in frequency of MICB alleles was found between patients and controls. Both HLA-B*51 and MICA*009 are strongly associated with BD in a pure Caucasian BD patient group, and the two alleles are in linkage disequilibrium. No MICB allele was found to associate significantly with the disease, an unexpected finding considering the close proximity of the MICA and MICB loci. Our results suggest that while MICB does not influence the development of BD, polymorphisms in MICA may be pathogenic, perhaps through the interaction with NK and gammadelta T cells.     

NKG2D engagement of colorectal cancer-specific T cells strengthens TCR-mediated antigen stimulation and elicits TCR independent anti-tumor activity

The NKG2D receptor is expressed by human NK, gammadelta T and alpha/beta T lymphocytes and its engagement results in the stimulation of effector cells. We evaluated the role of NKG2D receptor in anti-colorectal cancer (CRC) immune response. The cell surface expression of stress-inducible NKG2D ligands MICA/B (MHC class I-related chain molecules A/B) and ULBP (UL16 binding protein) by a panel of CRC lines was evaluated by flow cytometry. MICA and ULBP2/3 were widely expressed by the analyzed lines, with a minority of them being also ULBP-1+, whereas MICB was undetectable. CD8+ and CD4+ HLA-restricted anti-tumor T cell clones of a CRC patient were used to evaluate whether NKG2D engagement could mediate tumor recognition. Three out of four CD8+ T cell clones recognized the autologous tumor with a marginal NKG2D engagement, a finding that was correlated with the weak expression of NKG2D ligands by the autologous tumor. On the contrary, NKG2D triggering of these CD8+ T cell clones induced recognition of allogeneic CRC lines showing high expression of MICA and ULBP. A costimulatory role of NKG2D was observed with one CD4+/NKG2D+ T cell clone when stimulated by tumors sharing the HLA class II alleles and expressing NKG2D ligands. Taken together these data indicate that the engagement of NKG2D, depending on the expression of its ligands by target cells, can influence the pattern of anti-tumor reactivity by T lymphocytes.     

Potentiation of NK cell-mediated cytotoxicity in human lung adenocarcinoma: role of NKG2D-dependent pathway

Natural cytotoxicity receptors and NKG2D correspond to major activating receptors involved in triggering of tumor cell lysis by human NK cells. In this report, we investigated the expression of NKG2D ligands (NKG2DLs), MHC class I-related chain (MIC) A, MICB and UL16-binding proteins 1, 2 and 3, on a panel of human non-small-cell lung carcinoma cell lines, and we analyzed their role in tumor cell susceptibility to NK cell lysis. Although adenocarcinoma (ADC) cells expressed heterogeneous levels of NKG2DLs, they were often resistant to NK cell-mediated killing. Resistance of a selected cell line, ADC-Coco, to allogeneic polyclonal NK cells and autologous NK cell clones correlated with shedding of NKG2DLs resulting from a matrix metalloproteinase (MMP) production. Treatment of ADC-Coco cells with a MMP inhibitor (MMPI) combined with IL-15 stimulation of autologous NK cell clones lead to a potentiation of NK cell-mediated cytotoxicity. This lysis is mainly NKG2D mediated, since it is abrogated by anti-NKG2D-neutralizing mAb. These results suggest that MMPIs, in combination with IL-15, may be useful for overcoming tumor cell escape from the innate immune response.     

Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3 K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCRgammadelta(+) and TCRalphabeta(+)CD8(+) T lymphocytes, but it has been also detected in CD4(+) T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4(+) T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4(+)NKG2D(+) T lymphocytes that coexpressed perforin. NKG2D was detected in CD28(-) and CD28(dull )subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4(+) T cells, triggering proliferation and cytokine production (i.e. IFN-gamma and TNF-alpha). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4(+) T lymphocytes and thus may have a role in the response against infected HLA class II(+) cells displaying NKG2D ligands.     

Eight novel MICB alleles, including a null allele, identified in gastric MALT lymphoma patients

MICA and MICB, as members of the major histocompatibility complex (MHC) class I-chain-related genes (MIC), encode stress-inducible glycoproteins that act as activating ligands for NKG2D and gammadelta T-cell receptor-bearing cells. We here describe the identification of eight novel MICB variants, including a null allele, which were identified in peripheral blood leukocytes of gastric MALT lymphoma patients. Only two of the novel alleles are characterized by point mutations, whereas the other variants display a recombination of known exonic MICB sequences that may be best explained by intragenic conversions. The novel MICB null allele is characterized by a Cytosin (C) deletion in a stretch of four Cs beginning from nucleotide 135 of exon 2 that leads to a premature stop codon (TGA) at codon 66.     

Release of MICB molecules by tumor cells: mechanism and soluble MICB in sera of cancer patients

MICA, a ligand of the activating immunoreceptor NKG2D, is released by tumor cells in a soluble form and can be detected in sera of tumor patients at significant levels. Soluble MICA has been proposed to counteract NKG2D-mediated immunosurveillance of tumors. Here, we report that MICB, the second member of the human MIC protein family, is likewise shed by metalloproteases from tumor cells and is present in sera of patients with gastrointestinal tumors. While cell-bound MICB causes downregulation of surface NKG2D, soluble MICB did not alter NKG2D expression on NK cells in vitro. Thus, proteolytic shedding of MICB by tumor cells may impair immunogenicity of tumors primarily by reducing NKG2D-ligand densities on malignant cells.     

Palmitoylation of MICA, a ligand for NKG2D, mediates its recruitment to membrane microdomains and promotes its shedding

MICA and MICB (MHC-class-I-related chain A/B) are transmembrane proteins expressed in pathological conditions that are ligands for NKG2D, an activating receptor found on cytotoxic lymphocytes. The recognition on target cells of NKG2D ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells. Besides being expressed at the cell surface, MICA/B can be released as soluble proteins. Soluble NKG2D ligands downmodulate expression of the NKG2D receptor on lymphocytes, leading to a diminished cytotoxic response. Prior studies suggested that recruitment of MICA/B molecules to cholesterol-enriched microdomains was an important factor regulating the proteolytic release of these molecules. We now show that recruitment of MICA to these microdomains depends on palmitoylation of two cysteine residues that allow MICA molecules to reside in the membrane in the same domains as caveolin-1. Compared with WT molecules, nonpalmitoylated mutant MICA molecules were shed to the supernatant with low efficiency; however, both WT and mutant MICA were able to trigger NK cell cytotoxicity. These data suggest that the presence of NKG2D ligands at the plasma membrane is sufficient to activate cytotoxicity and reflect the need of different ligands to exploit different cellular pathways to reach the cell surface upon different stress situations.     

Lymphocyte activation via NKG2D: towards a new paradigm in immune recognition?

NKG2D is an activating cell surface receptor expressed on a wide range of immune effector cells including NK cells, NKT cells, gammadelta T cells as well as CD8(+) alphabeta T cells. Recent data indicate two major features: first, that human (MICA, MICB and ULBP) and mouse (Rae1 and H60) NKG2D ligands can be induced and/or upregulated upon cellular distress; and second, that on T cells NKG2D serves as a co-stimulation molecule for TCR triggering, whereas on NK cells NKG2D may act as a primary recognition structure.     

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production.Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells.ULBP2 expression was however linked to expression of mature CD57⁺ NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated “mature” NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.     

ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor

The human cytomegalovirus glycoprotein, UL16, binds to two members of a novel family of molecules, the ULBPs, and to the MHC class I homolog, MICB. The ULBPs are GPI-linked glycoproteins belonging to the extended MHC class I family but are only distantly related to MICB. The ULBP and MICB molecules are ligands for the activating receptor, NKG2D/DAP10, and this interaction is blocked by a soluble form of UL16. The ULBPs stimulate cytokine and chemokine production from NK cells, and expression of ULBPs in NK cell-resistant target cells confers susceptibility to NK cell cytotoxicity. Masking of NK cell recognition of ULBP or MIC antigens by UL16 provides a potential mechanism by which human cytomegalovirus-infected cells might evade attack by the immune system.     

MICA-129Met/Val Polymorphism Is Associated with Early-Onset Breast Cancer Risk

The major histocompatibility complex class I-related chain A (MICA), expressed on cell surface, plays an important role in the elimination of both virus-infected cells and tumor through the activation of the natural killer (NK) receptor NKG2D. A polymorphic change from methionine (Met) to valine (Val) at amino acid position 129 categorizes MICA alleles into strong and weak binders for the NKG2D receptor and has been found in a variety of immune-related disorders. In this study, we investigated the potential interaction between genetic polymorphism of MICA and the development of breast cancer. We recruited 192 unrelated Tunisian women affected by breast cancer and 205 controls age-matched women, all genotyped for MICA-129 Met/Val (rs 1051792). A significant association was found between the Val allele and Val/Val genotype and the risk of breast cancer (p = 0.002, OR = 1.64, 95% CI = [1.17–2.27]; p = 0.002, OR = 1.88, 95% CI = [1.24–2.87], respectively). After stratification with clinical-pathology parameters, we found that 71% of women aged lower than 40 years had a Val/Val genotype versus 49% (p = 0.014). About 72% of these patients having a family history of cancers had a Val/Val genotype (p = 0.04). These results suggest that tumor escape mechanism because of failure in order to activate NK cells by MICA-129 Val allele may play a role in individual susceptibility for breast cancer development in Tunisian women.     

Estradiol regulates MICA expression in human endometrial cells

The human endometrium undergoes cyclical changes regulated by sex hormones. Evidence suggests that sex hormones regulate NK cell recruitment into the uterus in large numbers. NKG2D is an activating receptor expressed on human NK cells, gammadelta and CD8 T cells. NKG2D ligands are known to be sensors of cellular "stress". In this study, we investigated whether sex hormones directly regulate expression of NKG2D ligands in the human uterus. Estradiol increased MICA expression on uterine epithelial cells; regulation was estrogen receptor-dependent. Real-time PCR analysis showed that NKG2D ligands MICA and MICB were expressed in the human endometrium. MICA protein was detected primarily on epithelial cells, and greater expression was observed in immunohistochemical analysis of tissues from patients in the secretory phase of the menstrual cycle. Thus, estrogens regulate expression of MICA. These data suggest hormonal regulation of innate immunity and NKG2D-mediated recognition in other tissues and diseases where estrogen may be involved.     

Association of MICA-TM and MICB C1_2_A Microsatellite Polymorphisms with Tumor Progression in Patients with Colorectal Cancer

Purpose

The major histocompatibility complex class I related A (MICA) and MICB molecules are ligands of NKG2D receptors on natural killer cells, gamma/delta T cells, and CD8aß T cells that mediate host antitumor immune response. The role of MICA-TM and MICB C1_2_A alleles in patients with colorectal cancer has not yet been investigated.

Methods

We have analyzed the MICA-TM and MICB C1_2_A polymorphisms in colorectal cancer patients (n?=?79) by polymerase chain reaction amplification, subsequent electrophoresis, and sequencing in comparison to a previously analyzed cohort of healthy controls (n?=?306). Allele frequencies obtained for MICA-TM and MICB C1_2_A were compared to histopathological data regarding tumor invasion, disease progression, microsatellite instability, and the presence of KRAS mutations (codon 12) and analyzed for possible impact on tumor-related survival (n?=?61).

Results

Allele frequencies of MICA-TM and MICB C1_2_A polymorphisms were not different in patients with colorectal cancer in comparison to normal controls. In colorectal cancer patients, MICA-TM A4 allele was directly and MICA-TM A5 allele was inversely associated with lymph node involvement and advanced UICC stages. Tumor-related survival in colorectal cancer patients was significantly reduced in the presence of the MICA-TM A4 allele (p?=?0.015). In patients with microsatellite stable tumors, survival was reduced in association with the MICA-TM A4 allele (p?=?0.006) and MICA-TM A9 allele (p?=?0.034), but increased in patients showing the MICA-TM A5 allele (p?=?0.042).

Conclusions

Specific MICA-TM alleles seem to influence tumor progression and midterm survival of patients with colorectal cancer, indicating an important role of host innate immune predisposition involving NKG2D mediated antitumor response.