Expression of HLA class I antigens in transporter associated with antigen processing (TAP)-deficient mutant cell lines

Abstract: Cells lacking expression of the transporter associated with antigen processing (TAP) are deficient in surface HLA class I, yet express reduced levels of HLA-A2 antigen through TAP-independent processing pathways. We have analysed the expression of HLA-A, -B and -C antigens on the 721.174 and T2 TAP-deficient mutant cell lines using a panel of monoclonal antibodies specific for the HLA antigens encoded by the genotype of these cells. Our study has shown the constitutive expression of HLA-Cwl molecules on the cell surface of both T2 and 721.174 cells and has confirmed that HLA-A2 and HLA-B51 are expressed at low levels. Transfection of 721.174 cells with cDNAs encoding TAP1 and TAP2 proteins did not fully restore HLA class I antigen expression on these cells, which appeared to be mainly due to a deficiency in expression of the HLA-B51-associ-ated Bw4 epitope. This suggests that additional antigen-processing genes may be required for optimal generation of HLA-B-binding peptides. Our results indicate that TAP-independent pathways of antigen-processing provide peptides for functional expression of all three classical HLA class I molecules.     

Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways

BACKGROUND: 10% of sporadic colorectal cancers are characterised by a low level of microsatellite instability (MSI-L). These are not thought to differ substantially from microsatelite-stable (MSS) cancers, but MSI-L and MSS cancers are distinguished clinicopathologically and in their spectrum of genetic alterations from cancers showing high level microsatellite instability (MSI-H). AIMS: To study the distribution of molecular alterations in a series of colorectal cancers stratified by DNA microsatellite instability. METHODS: A subset of an unselected series of colorectal cancers was grouped by the finding of DNA MSI at 0 loci (MSS) (n = 51), 1-2 loci (MSI-L) (n = 38) and 3-6 loci (MSI-H) (n = 25). The frequency of K-ras mutation, loss of heterozygosity (LOH) at 5q, 17p and 18q, and patterns of p53 and beta catenin immunohistochemistry was determined in the three groups. RESULTS: MSI-H cancers had a low frequency of K-ras mutation (7%), LOH on chromosomes 5q (0%), 17p (0%) and 18q (12.5%), and a normal pattern of immunostaining for p53 and beta catenin. MSI-L cancers differed from MSS cancers in terms of a higher frequency of K-ras mutation (54% v 27%) (p = 0.01) and lower frequency of 5q LOH (23% v 48%) (p = 0.047). Whereas aberrant beta catenin expression and 5q LOH were concordant (both present or both absent) in 57% of MSS cancers, concordance was observed in only 20% of MSI-L cancers (p = 0.01). CONCLUSIONS: MSI-L colorectal cancers are distinct from both MSI-H and MSS cancers. This subset combines features of the suppressor and mutator pathways, may be more dependent on K-ras than on the APC gene in the early stages of neoplastic evolution, and a proportion may be related histogenetically to the serrated (hyperplastic) polyp.     

Development and characterization of mouse anti-human LMP2, LMP7, TAP1 and TAP2 monoclonal antibodies

Abstract: Low molecular mass polypeptides (LMP) 2 and LMP7 and transporter associated with antigen processing (TAP) subunits TAP1 and TAP2 play a crucial role in antigen processing and cell surface expression of HLA class I molecules. Since monoclonal antibodies (mAb) to these molecules will facilitate the analysis of their expression, structure and function in normal and transformed cells, in the present study we have developed these reagents. Specifically anti-LMP2 and LMP7 mAb were generated from BALB/c mice immunized with specific peptides, and anti-TAPl and TAP2 mAb from BALB/c mice immunized with respective recombinant proteins. mAb VF101–39F7 and VF1O1–39G5 were shown to be specific for LMP2, mAb VF103–5D5 and VF103–8C2 for LMP7, mAb VF108–1B3 and VF108–12D6 for TAP1 and mAb VF118–1E4 and VF118–2C5 for TAP2, since they reacted specifically with the corresponding immunogens in ELISA and with the corresponding LMP and TAP subunits when tested in Western blotting with human lymphoid cell extracts. Furthermore, the mAb immunoprecipitated components with the characteristic electrophoretic mobility from lymphoid cells. Both anti-LMP and anti-TAP mAb stained keratinocytes and infiltrating lymphocytes in frozen and formalin-fixed, paraffin embedded sections of normal skin in indirect immunoperoxidase reactions. Furthermore, all the mAb except mAb VF103–5D5 stained the cytoplasm of lymphoid cells in an intracytoplasmic staining reaction. The specificity and reactivity pattern of the mAb we have characterized indicate that they will be valuable reagents to analyze the cellular expression and tissue distribution of LMP and TAP subunits.     

Genetic variation of antigen processing machinery components and association with cervical carcinoma

The antigen processing machinery (APM) plays an important role in immune recognition of virally infected and transformed cells. Defective expression of several APM components is associated with progression and clinical outcome in cervical carcinoma. Genetic variation in the genes encoding APM components is known to be associated with risk of occurrence of several malignancies. However, only limited evidence exists supporting the role of single nucleotide polymorphisms (SNPs) in APM components in cervical carcinoma. We have therefore investigated the occurrence of APM component SNP genotypes and haplotypes in cervical carcinoma. Thirteen coding SNPs in the LMP2, LMP7, TAP1, TAP2, and ERAP1 genes were genotyped in 127 cervical carcinoma patients and 124 controls. Individual genotype and allele distributions were assessed by single-marker analysis. Effects of various SNP combinations were estimated by haplotype construction and subsequent haplotype interaction analysis. Significant haplotypes were modeled on disease risk. Allele distributions at the LMP7-145, TAP2-651, ERAP1-127, and ERAP1-730 loci differed significantly between cases and controls with the major allele at the LMP7 and TAP2 loci and the minor allele at both ERAP1 loci associated with increased cervical carcinoma risk. A combination of the two haplotypes spanning these loci was associated with a three-fold increased risk (OR = 3.024; P < 0.001); approximately 12% of all cervical carcinoma occurrences were attributable to this combination. Our data indicate that combined genetic variation in the TAP2, LMP7, and ERAP1 genes is associated with increased cervical carcinoma risk.     

MHC microsatellite diversity and linkage disequilibrium among common HLA-A, HLA-B, DRB1 haplotypes: implications for unrelated donor hematopoietic transplantation and disease association studies

Twenty-two human major histocompatibility complex (MHC) region microsatellite (Msat) markers were studied for diversity and linkage disequilibrium (LD) with HLA loci in hematopoietic cell transplant recipients and their HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 allele-matched unrelated donors. These Msats showed highly significant LD over much of the MHC region. The Msat diversity of five common Caucasian haplotypes (HLA-A1-B8-DR3, A3-B7-DR15, A2-B44-DR4, A29-B44-DR7, and A2-B7-DR15) was examined using a new measure called 'haplotype specific heterozygosity' (HSH). Each of the five haplotypes had at least one Msat marker with an HSH value of zero indicating that only one Msat allele was observed for the particular HLA haplotype. In addition, the ability of Msats to predict HLA-A-B-DRB1 haplotypes was studied. Over 90% prediction probability of two common haplotypes (HLA-A1-B8-DR3 and HLA-A3-B7-DR15) was achieved with information from three Msats (D6S265/D6S2787/D6S2894 and D6S510/D6S2810/D6S2876, respectively). We demonstrate how the HSH index can be used in the selection of informative Msats for transplantation and disease association studies. Markers with low HSH values can be used to predict specific HLA haplotypes or multilocus genotypes to supplement the screening of HLA-matched donors for transplantation. Markers with high HSH values will be most informative in studies investigating MHC region disease-susceptibility genes where HLA haplotypic effects are known to exist.     

Angiogenic factor VEGF is decreased in human colorectal neoplasms showing DNA microsatellite instability.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two important determinants of angiogenesis in human cancers. Expression of VEGF and bFGF was examined by immunohistochemistry in 120 colorectal cancers. Neoplasms were classified according to the presence or absence of microsatellite instability determined at six microsatellite loci and labelled as a high microsatellite instability (MSI-H), low microsatellite instability (MSI-L) or microsatellite stable (MSS). Only 4/30 MSI-H cancers expressed VEGF (13 per cent), compared with 24/64 MSS cancers (38 per cent; p< 0.01). Fewer MSI-H cancers showed bFGF expression (38 per cent) than MSS cancers (53 per cent; p< 0.09). MSI-L cancers showed the same pattern as MSS cancers. Western blotting and immunohistochemistry showed that the tumour suppressor gene p53 was mutated infrequently in MSI-H cancers (8 per cent; p< 0. 001). Microvessel density counts using CD31 and UEA-1 demonstrated no difference in the number of blood vessels in MSI-H and MSS cancers. Although these results are consistent with the known role of wild-type p53 in down-regulating VEGF, no association was found between a mutation in p53 and VEGF or bFGF levels in all colonic neoplasms. This is the first evidence that MSI-H cancers may follow a different pathway to angiogenesis. The low frequency of VEGF expression amongst MSI-H cancers may partially explain why these cancers are less aggressive, with a better overall prognosis.     

Coordinate downregulation of multiple MHC class I antigen processing genes in chemical-induced murine tumor cell lines of distinct origin

In murine tumor cell lines, downregulation of MHC class I surface expression has been frequently detected, but the underlying molecular mechanisms of such deficiencies have not been defined. In this study, murine tumor cell lines of different histology derived from spontaneous or from chemical-induced tumors were analyzed for the expression of multiple components of the major histocompatibility complex (MHC) class I antigen-processing machinery (APM), including the peptide transporter TAP, the interferon (IFN)-gamma inducible proteasome subunits and several chaperones. The tumor cell lines analyzed demonstrated a heterogeneous expression pattern of various APM components. In comparison to control cells an impaired coordinated expression of at least three APM components was detected. In particular, extensive APM deficiencies were found in cell lines derived from chemical-induced tumors. A strong coordinated downregulation of expression and/or function of TAP, the low molecular weight proteins (LMP) subunits, the proteasome activator PA28 and/or tapasin was found in 5 of 10 tumor cells, which was associated with impaired MHC class I surface expression. In contrast, the expression of beta2-microglobulin (beta2-m), PA28beta, the constitutive proteasome subunits X, Y, Z and of the chaperones calnexin, calreticulin, ER60 and phospho disulfide isomerase (PDI) was unaltered or only weakly decreased. The deficient expression of APM components could be corrected by IFN-gamma treatment, which also reconstituted MHC class I surface expression. However, impaired expression of APM molecules appears not to be the only cause of abnormal MHC class I expression, since it could neither be corrected by the addition of exogeneous MHC class I binding peptides nor by incubation at low temperature. These results suggest that one major mechanism of murine tumor cells, in particular chemical-induced tumors, to evade the immune system is the combined dysregulation of various APM components and other factors, which still have to be identified.     

Clinical Implications of Microsatellite Instability in T1 Colorectal Cancer

Purpose

The estimation of regional lymph node metastasis (LNM) risk in T1 colorectal cancer is based on histologic examination and imaging of the primary tumor. High-frequency microsatellite instability (MSI-H) is likely to decrease the possibility of metastasis to either regional lymph nodes or distant organs in colorectal cancers. This study evaluated the clinical implications of MSI in T1 colorectal cancer with emphasis on the usefulness of MSI as a predictive factor for regional LNM.

Materials and Methods

A total of 133 patients who underwent radical resection for T1 colorectal cancer were included. Genomic DNA was extracted from normal and tumor tissues and amplified by polymerase chain reaction (PCR). Five microsatellite markers, BAT-25, BAT-26, D2S123, D5S346, and D17S250, were used. MSI and clinicopathological parameters were evaluated as potential predictors of LNM using univariate and multivariate analyses.

Results

Among 133 T1 colorectal cancer patients, MSI-H, low-frequency microsatellite instability (MSI-L), and microsatellite stable (MSS) colorectal cancers accounted for 7.5%, 6%, and 86.5%, respectively. MSI-H tumors showed a female predominance, a proximal location and more retrieved lymph nodes. Twenty-two patients (16.5%) had regional LNM. Lymphovascular invasion and depth of invasion were significantly associated with LNM. There was no LNM in 10 MSI-H patients; however, MSI status was not significantly correlated with LNM. Disease-free survival did not differ between patients with MSI-H and those with MSI-L/MSS.

Conclusion

MSI status could serve as a negative predictive factor in estimating LNM in T1 colorectal cancer, given that LNM was not detected in MSI-H patients. However, validation of our result in a different cohort is necessary.     

APC mutation and tumour budding in colorectal cancer

AIM: To determine the frequency of tumour budding and somatic APC mutation in a series of colorectal cancers stratified according to DNA microsatellite instability (MSI) status. Material/Methods: Ninety five colorectal cancers were genotyped for APC mutation in the mutation cluster region (exon 15) and scored for the presence of tumour budding at the invasive margin in haematoxylin and eosin stained sections. A subset was immunostained for beta catenin and p16. RESULTS: The frequency of both somatic APC mutation and tumour budding increased pari passu in cancers stratified as sporadic MSI high (MSI-H), hereditary non-polyposis colorectal cancer (HNPCC), MSI low (MSI-L), and microsatellite stable (MSS). Both budding and APC mutation were significantly less frequent in sporadic MSI-H cancers than in MSI-L or MSS cancers. Tumour buds were characterised by increased immunostaining for both beta catenin and p16. CONCLUSION: Tumour budding is associated with an adverse prognosis. The lack of budding in MSI-H colorectal cancer may account for the improved prognosis of this subset and may be explained by an intact WNT signalling pathway and/or inactivated p16(INK4a).     

Reanalysis of sequence-based HLA-A, -B and -Cw typings: how ambiguous is today's SBT typing tomorrow

The permanently increasing number of human leukocyte antigen (HLA)-alleles and the growing list of ambiguities require continuous updating of high-resolution HLA typing results. Two different kinds of ambiguities exist: the first, when two or more allele combinations have identical heterozygous sequences, and the second, when differences are located outside the analyzed region. The number of HLA-A, B and C alleles recognized in 1999 was almost tripled in 2006. Two hundred individuals, sequence-based typing (SBT) typed in the period from 1999 to 2002, were reanalyzed using the 2006 database. A final allele typing result of at least four digits was obtained for HLA-A, -B and -C by heterozygous sequencing of exons 2 and 3 and, if necessary, additional exons and/or allele-specific sequencing. Storage of the individual sequences in a specially developed database enabled reanalysis with all present and future HLA releases. In the 5-year period HLA-A, -B and -C typing results became ambiguous in 37%, 46% and 41% of the cases. Most were because of differences outside the analyzed region; ambiguities because of different allele combinations with identical heterozygous sequences were present in 7%, 8% and 13% of the HLA-A, -B and -C typings. These results indicate that within 5 years, approximately half of the HLA SBT typings become ambiguous.     

Interactions formed by individually expressed TAP1 and TAP2 polypeptide subunits

The transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptide-loading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.     

Selective co-evolution of the D6STNFa microsatellite region with HLA class I and II loci

Abstract: We analyzed the HLA-A, -B, -DR and -DQ phenotypes and 12 microsatellite locus genotypes within and close to the major histocompatibility complex in a panel of 98 randomly selected, healthy, unrelated Dutch Caucasoid individuals. Allele frequencies and Hardy-Weinberg equilibrium (HWE) were calculated. Also, the linkage disequilibrium patterns between HLA and microsatellite loci were studied. The HLA-A, -B, -DR, -DQ and six microsatellite loci centromeric of the HLA-A showed HWE. In contrast, all microsatellites telomeric of the HLA-A showed deviation from HWE due to excess of homozygosity. Linkage disequilibrium analyses provided strong evidence that among the tested microsatellite loci only the alleles of the D6STNFa locus are in linkage disequilibrium with both HLA-B and -DR. Our results suggest that selection acting on the HLA genes includes the D6STNFa locus and linked genes.     

Establishment of a monoclonal anti-pan HLA class I antibody suitable for immunostaining of formalin-fixed tissue: unusually high frequency of down-regulation in breast cancer tissues

A novel monoclonal anti-pan human leukocyte antigen (HLA) class I heavy chain antibody, EMR8-5, was established. It could detect HLA-A, -B, and -C antigens in formalin-fixed paraffin embedded tissues. By immunohistochemical staining using the EMR8-5 antibody, various cancer tissues from 246 cases were examined for HLA class I expression. It was found that HLA class I expression was decreased in 20% to 42% of the cases of lung cancer, hepatocellular carcinoma, colon cancer, renal cell carcinoma, and urothelial carcinoma. In contrast, 85% of breast cancer cases had loss of or decreased HLA class I expression. Of the 35 breast cancer cases that had decreased HLA class I heavy chain expression, 33 (94%) also had decreased beta2-microglobulin expression detected by immunohistochemical staining. It was suggested that HLA class I down-regulation might be a common characteristic of breast cancer mostly caused by the down-regulation of beta2-microglobulin expression.     

Bipartite regulation of different components of the MHC class I antigen-processing machinery during dendritic cell maturation.

Dendritic cells (DC) are professional antigen-presenting cells (APC) which proceed from immature to a mature stage during their final differentiation. Immature DC are highly effective in terms of antigen uptake and processing, whereas mature DC become potent immunostimulatory cells. Until now, the expression profiles of the major components of the MHC class I antigen-processing machinery (APM) during DC development have not been well characterized. In this study, the mRNA and protein expression levels of the IFN-gamma inducible proteasome subunits, of the proteasome activators PA28, and of key components required for peptide transport and MHC class I-peptide complex assembly have been evaluated in immature and mature stages of human monocyte-derived DC using semiquantitative RT-PCR and Western blot analyses. The IFN-gamma-responsive immunoproteasome subunits LMP2, LMP7 and MECL1 are up-regulated in immature DC, whereas the other components of the MHC class I presentation machinery, such as PA28, TAP, tapasin, and HLA heavy and light chains, were found to be more abundant in mature DC. These findings support the hypothesis that immature DC produced by the differentiation of monocytes in response to IL-4 and granulocyte macrophage colony stimulating factor first increase their capacity to capture antigens and process them into peptides, thereby switching from housekeeping to immunoproteasomes, while mature DC rather up-regulate the components required for peptide translocation and MHC class I-peptide complex formation, and thus specialize in antigen presentation. Our results establish that MHC class I, like MHC class II surface expression, is markedly regulated during DC development and maturation.     

Microsatellite instability and alteration of the expression of hMLH1 and hMSH2 in ovarian clear cell carcinoma

Microsatellite instability (MSI) is commonly seen in tumors associated with the hereditary nonpolyposis colorectal cancer syndrome and is caused by defects in the DNA mismatch repair genes. MSI has also been observed in various sporadic cancers, including colorectal, gastric, and endometrial. The role and incidence of MSI in ovarian clear cell carcinoma remain unknown. This study was conducted to evaluate the frequency of MSI in ovarian clear cell carcinomas and to evaluate the sensitivity and specificity of immunohistochemistry in predicting mismatch-repair gene deficiency. A total of 42 ovarian clear cell carcinomas were analyzed for MSI using a panel of 5 microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250). Alterations in the expression of hMLH1 and hMSH2 proteins in these tumors were examined. Of the 42 ovarian clear cell tumors analyzed, 6 demonstrated a high level of MSI (MSI-H), 3 demonstrated a low level of MSI (MSI-L), and the remaining 33 exhibited microsatellite stability (MSS). No correlation was found between MSI level and patient age or tumor stage or size (P >0.05). Loss of expression of either hMLH1 or hMSH2 was observed in 4 of the 6 (67.7%) MSI-H tumors, whereas 34 of the 36 (94.4%) MSI-L or MSS tumors expressed both the hMLH1 and hMSH2 gene products. Our results indicate that MSI-H is involved in the development of a subset of ovarian clear cell carcinomas. A strong correlation exists between alterations in the expression of hMLH1 and hMSH2 and the presence of MSI-H in these tumors. However, immunohistochemical testing alone may miss a small fraction of cases with MSI-H.     

Genes in the HLA class I region may contribute to the HLA class II-associated genetic susceptibility to multiple sclerosis

In order to analyze whether loci in the human leukocyte antigen (HLA) class I region may contribute to the HLA class II-associated genetic susceptibility to multiple sclerosis (MS), we examined selected microsatellite markers in 177 Nordic sib-pair families, 222 British sib-pair families, 323 sporadic Norwegian MS patients and 386 Norwegian controls. All samples were, in addition, genotyped for the HLA-DR DQ haplotype, and the Norwegian case-control samples were also typed for HLA-A and -B loci. In the Norwegian sporadic MS patients association was seen with HLA-A, HLA-B, and with the D6S265 marker, located 100 kb centromeric to HLA-A. Associations with HLA-A and D6S265 loci were also suggested when restricting the analysis to HLA-DR15 haplotypes. In the sib-pair data a similar trend was seen with marker D6S265. Higher genotypic relative risk (GRR) was found for individuals who carry both HLA-DR15 and -A3 (GRR = 15), compared to those who carry only HLA-DR15 (GRR = 7), only HLA-A3 (GRR = 3) or none of these alleles (GRR = 1). The highest risk was conferred by a combination of HLA-DR15 and -A3 (odds ratio (OR) = 5.2). These results suggest that HLA-A or a gene in linkage disequilibrium with it may contribute to the HLA class II-associated genetic susceptibility to MS.     

Microsatellite analysis of microdissected tumor cells and 6p high density microsatellite analysis in head and neck squamous cell carcinomas with down-regulated human leukocyte antigen class I expression

Down-regulated human leukocyte antigen (HLA) class I expression is frequently correlated with allelic loss at 6p21.3, which is the location of the HLA coding sequence, in head and neck squamous cell carcinomas (HNSCCs). Previously, we have demonstrated loss of heterozygosity (LOH) at 6p21.3 for at least one locus in 49% of the HNSCCs using 5 microsatellite markers spanning the 4 megabase HLA region. In the present study, the detection threshold (25%) to assign LOH was addressed by laser-assisted microdissection of tumor cells from tumors containing marginal loss. In addition, we describe high density microsatellite analysis of chromosome 6p21.3 in HNSCC with down-regulated HLA class I expression. The purpose of this study was to refine the identification of genetic alterations at 6p21.3 and to pinpoint allelic loss to individual HLA class I genes, using additional markers closely located to the HLA-A, -B, and -C loci and the transporter associated with antigen processing (TAP) genes. LOH analysis by amplification of microsatellite markers and subsequent fluorescent detection is a rapid and sensitive technique to predict HLA class I loss phenotypes in tumors. LOH can be identified at 25% relative signal reduction. Analysis of heterogeneous tumor samples and samples containing a small amount of tumor cells is facilitated by laser-assisted microdissection of tumor cells. In addition, we showed that accurate HLA LOH analysis requires application of microsatellite markers in close proximity to HLA class I and TAP genes.