Pendrin and NIS antibodies are absent in healthy individuals and are rare in autoimmune thyroid disease: evidence from a Danish twin study.

OBJECTIVE: Antibodies against thyroglobulin, thyroid peroxidase and the TSH receptor are accepted as pathophysiological and diagnostic biomarkers in autoimmune thyroid disease (AITD). In contrast, the prevalence, aetiology and clinical relevance of autoantibodies against the human sodium-iodine symporter (NISAb) and pendrin (PenAb) remain unclear. The objectives of the study were to investigate the presence of NISAb and PenAb in Danish twins, with and without AITD, to study whether the published variations in NISAb and PenAb frequencies were related to differences in methodology or study populations, and to evaluate whether the presence of NISAb or PenAb most likely results from genetic or nongenetic factors. METHODS: Sera from 93 patients with AITD and 230 healthy controls were evaluated for NISAb and PenAb using radioligand binding assays (RBA). RESULTS: Patients with AITD had a higher prevalence than the controls: NISAb: 17% vs 0% (P < 0·001) and PenAb: 11% vs 0% (P < 0·001). Subdividing according to cause of AITD yielded similar results: 20% (11/56) of patients with Graves' disease (GD) and 14% (5/37) of patients with Hashimoto's thyroiditis (HT) had NISAb, (P < 0·05, vs control population). Seven of 56 (13%) patients with GD and three of 37 (8%) patients with HT had PenAb (P < 0·05 vs control population). No twin pairs were concordant for NISAb or PenAb, not even among twin pairs concordant for AITD. CONCLUSIONS: In accord with studies using the same RBAs, the frequency of NISAb and PenAb was low in Danish patients with AITD and absent in healthy individuals, suggesting that differences between studies rely on assay differences. The skewed distribution of NISAb and PenAb within AITD concordant twin pairs suggests that NISAb and PenAb are likely attributable to the effects of environmental factors acting in genetic susceptible individuals.

Distribution of NBCn2 (SLC4A10) splice variants in mouse brain.

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.

Slc26a9--anion exchanger, channel and Na+ transporter.

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl(-)-HCO(3)(-) exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO(3)(-) secretion; Na(+) electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl(-)-HCO(3)(-) exchanger, Cl(-) channel, and Na(+)-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.

T-Cell activation by t(9;22) acute lymphoblastic leukemia-derived dendritic-like cells is associated with increased tapasin expression.

Dendritic-like cells from t(9;22) acute lymphoblastic leukemia (ALL) blasts can activate T cells, while the original unmodified leukemic blasts cannot. To determine whether these functional differences were associated with differences in antigen-processing machinery (APM) component expression, we have measured the level of APM component expression in unmodified blasts and ALL-derived dendritic-like cells. Seven t(9;22) ALL patient samples and one cell line were studied for APM component expression utilizing a unique panel of recently developed monoclonal antibodies and a recently developed intracellular staining technique. In addition, the HLA class I antigen cell surface expression was measured. HLA class I antigens were similarly expressed on the unmodified blasts and on the autologous dendritic-like cells. Intracellular HLA class I antigen and tapasin expression (P=0.03 for both) were upregulated in all t(9;22) ALL-derived dendritic-like cells, in comparison to the unmodified blasts. These results provide a potential mechanism for the ability of t(9;22) ALL-derived dendritic-like cells to induce T-cell activation and, suggest that tapasin upregulation may serve as a marker to standardize and monitor the quality of the dendritic-like cells used in immunotherapy.

Accessory molecules in the assembly of major histocompatibility complex class I/peptide complexes: how essential are they for CD8(+) T-cell immune responses?

Assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum is a highly coordinated process that results in abundant class I/peptide complexes at the cell surface for recognition by CD8(+) T cells and natural killer cells. During the assembly process, a number of chaperones and accessory molecules, such as transporter associated with antigen processing, tapasin, ER60, and calreticulin, assist newly synthesized class I molecules to facilitate loading of antigenic peptides and to optimize the repertoire of surface class I/peptide complexes. This review focuses on the relative importance of these accessory molecules for CD8(+) T-cell responses in vivo and discusses reasons that may help explain why some CD8(+) T-cell responses develop normally in mice deficient in components of class I assembly, despite impaired antigen presentation.

The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex.

Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic beta(2) microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization.

Recognition of open conformers of classical MHC by chaperones and monoclonal antibodies.

There is considerable evidence that the conformation and stability of class I and class II major histocompatibility complex (MHC) proteins is dependent upon high-affinity peptide ligation, but structural data for an empty MHC protein unfortunately is lacking. However, several monoclonal antibodies (mAbs) that specifically detect open MHC conformers have been characterized, and they provide insights into the changes associated with peptide loading and unloading. Here, the structural changes make the argument that certain of these open conformer-specific mAbs recognize analogous MHC segments as the molecular chaperones tapasin and DM. MHC residues located in regions flanking the peptide-terminal anchoring pockets have been implicated in both chaperone and monoclonal antibody binding. Indeed, we propose these regions serve as peptide-binding hinges that are uniquely accessible in open MHC.

Restoration of the expression of transporters associated with antigen processing in lung carcinoma increases tumor-specific immune responses and survival.

A wide variety of human carcinomas have low expression of tumor-associated antigen presentation in the context of MHC class I antigens due to defects in the antigen presentation pathway. This immune evasion mechanism renders many tumors unrecognizable by host immune surveillance mechanisms. The present study examines the expression of HLA, tapasin, transporter associated with antigen processing 1 (TAP1), and beta2 microglobulin in human small cell lung carcinoma and non-small cell lung carcinoma. Immunohistochemical staining showed severe impairment of the antigen presentation pathway in all patients. In order to recover tumor immunogenicity, a nonreplicating adenovirus expressing human TAP1 (AdhTAP1) was used to restore the expression of TAP1 in the antigen presentation pathway-deficient mouse lung carcinoma cell line, CMT.64. Infection of CMT.64 cells with AdhTAP1 increased MHC class I antigen surface expression, antigen presentation, and susceptibility to antigen-specific CTLs. Fluorescence-activated cell sorting and ELISPOT analysis showed that AdhTAP1 treatment significantly increased dendritic cell cross-presentation and cross-priming of tumor antigens. Furthermore, ex vivo and in vivo AdhTAP1 treatment significantly retarded tumor growth and increased survival of mice bearing CMT.64 tumors. Fluorescence-activated cell sorting analysis and immunohistochemical staining showed a significant increase in CD8+ and CD4+ T cells and CD11c+ dendritic cells infiltrating the tumors. The results show that TAP should be considered as a part of the immunotherapies for various cancers because it is likely to provide a general method for increasing immune responses against tumors regardless of the antigenic composition of the tumor or the MHC haplotypes of the host.

Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene.

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

Stoichiometric tapasin interactions in the catalysis of major histocompatibility complex class I molecule assembly.

The assembly of major histocompatibility complex (MHC) class I molecules with their peptide ligands in the endoplasmic reticulum (ER) requires the assistance of many proteins that form a multimolecular assemblage termed the 'peptide-loading complex'. Tapasin is the central stabilizer of this complex, which also includes the transporter associated with antigen processing (TAP), MHC class I molecules, the ER chaperone, calreticulin, and the thiol-oxidoreductase ERp57. In the present report, we investigated the requirements of these interactions for tapasin protein stability and MHC class I dissociation from the peptide-loading complex. We established that tapasin is stable in the absence of either TAP or MHC class I interaction. In the absence of TAP, tapasin interaction with MHC class I molecules is long-lived and results in the sequestration of existing tapasin molecules. In contrast, in TAP-sufficient cells, tapasin is re-utilized to interact with and facilitate the assembly of many MHC class I molecules sequentially. Furthermore, chemical cross-linking has been utilized to characterize the interactions within this complex. We demonstrate that tapasin and MHC class I molecules exist in a 1 : 1 complex without evidence of higher-order tapasin multimers. Together these studies shed light on the tapasin protein life cycle and how it functions in MHC class I assembly with peptide for presentation to CD8(+) T cells.

HLA-E surface expression is independent of the availability of HLA class I signal sequence-derived peptides in human tumor cell lines.

Human leukocyte antigen (HLA)-E is a nonclassic HLA class I molecule whose expression at the cell surface of tumor cells might allow them to escape T- and natural killer (NK)-cell immune surveillance. In this study, we analyzed HLA-E expression in a panel of human HLA-typed tumor cell lines of different histotypes by flow cytometry with anti-HLA-E monoclonal antibodies and by reverse transcriptase-polymerase chain reaction. Although specific HLA-E transcripts were detected in all cell lines, except in HELA, surface expression was detected at different intensities on seven (23%) of 30 cell lines with higher frequency and intensity among osteosarcoma cell lines. HLA-E-positive tumor cell lines mainly expressed the HLA-A*02 class I allele. Some tumor cell lines demonstrating HLA class I A* or Cw* alleles, which we expected to allow HLA-E surface expression on the basis of reported data on lymphoid cells, instead were HLA-E negative. All tumor cell lines were either tapasin and TAP-1 positive by flow cytometry, except two osteosarcoma cell lines, a finding that suggests an intact assembly machinery for peptide loading. We conclude that the concomitant presence of the appropriate HLA class I alleles with leader sequence-derived peptides and HLA-E heavy chain may not be sufficient to allow HLA-E surface expression in tumor cell lines as opposed to lymphoid cells.

TAP genes and immunity.

The transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette transporter family that specializes in delivering cytosolic peptides to class I molecules in the endoplasmic reticulum. The TAP is a major target of genetic alteration in tumours and disruption by viral inhibitors. In some species, TAP genes have co-evolved with MHC class I molecules to deliver peptides that are customised for particular alleles. In humans, MHC class I polymorphism determines the level of tapasin-mediated association with TAP and subsequent peptide optimisation within the peptide-loading complex (PLC). MHC class I molecules that still load peptides without complexing to the TAP might be more resistant to viral interference of the PLC and less sensitive to competition for TAP by other class I allotypes.

Species-specific differences in proteasomal processing and tapasin-mediated loading influence peptide presentation by HLA-B27 in murine cells.

Expression of HLA-B27 in murine cells has been used to establish animal models for human spondyloarthritis and for antigen presentation studies, but the effects of xenogeneic HLA-B27 expression on peptide presentation are little known. The issue was addressed in this study. HLA-B27-bound peptide repertoires from human and murine cells overlapped by 75-85%, indicating that many endogenous HLA-B27 ligands are generated and presented in both species. Of 20 differentially presented peptides that were sequenced, only 40% arose from obvious inter-species protein polymorphism, suggesting that differences in antigen processing-loading accounted for many species-specific ligands. Digestion of synthetic substrates with human and murine 20 S proteasomes revealed cleavage differences that accounted for or correlated with differential expression of particular peptides. One HLA-B27 ligand found only in human cells was similarly generated in vitro by human and murine proteasomes. Differential presentation correlated with significantly decreased amounts of this ligand in human tapasin-deficient cells reconstituted with murine tapasin, indicating that species-specific interactions between HLA-B27, tapasin, and/or other proteins in the peptide-loading complex influenced presentation of this peptide. Our results indicate that differences in proteasomal specificity and in interactions involving tapasin determine differential processing and presentation of a significant number of HLA-B27 ligands in human and murine cells.

Cytologic findings of epithelioid angiosarcoma of the thyroid. A case report.

BACKGROUND: Angiosarcoma of the thyroid is a rare and aggressive tumor and occurs mainly in patients from central Europe, especially the alpine region. The fine needle aspiration findings of a keratin-positive epithelioid angiosarcoma of the thyroid occurring in a nonmountainous area in South America is described. CASE: A 65-year-old male from São Paulo, Brazil, presented with a mass in the anterior part of the neck with progressive enlargement for three months. The cytologic findings on the fine needle aspirate were a cellular smear composed of single cells and small clusters of neoplastic cells, oval and round. Cell borders were indistinct, and the cytoplasm was vacuolated. The nuclei were eccentrically located, with irregular nuclear membranes; single, prominent nucleoli; and a coarse chromatin pattern. Features suggestive of intracytoplasmic lumens were identified. Open surgical biopsy demonstrated a tumor infiltrating the thyroid gland and composed of large, round, atypical epithelioid cells lining vascular spaces. These neoplastic cells were immunoreactive for AE1:AE3, CK7, vimentin, CD31 and factor VIII. CONCLUSION: Epithelioid angiosarcoma should be considered in the differential diagnosis of epithelioid neoplasms of the thyroid. An immunohistochemical panel should include vascular markers even in the presence of immunoreactivity for epithelial markers.

Optimization of the MHC class I peptide cargo is dependent on tapasin.

The loading of MHC class I molecules with their peptide cargo is undertaken by a multimolecular peptide loading complex within the endoplasmic reticulum. We show that MHC class I molecules can optimize their peptide repertoire over time and that this process is dependent on tapasin. Optimization of the peptide repertoire is both quantitatively and qualitatively improved by tapasin. The extent of optimization is maximal when MHC class I molecules are allowed to load within the fully assembled peptide loading complex. Finally, we identify a single natural polymorphism (116D>Y) in HLA-B*4402 that permits tapasin-independent loading of HLA-B*4405 (116Y). In the presence of tapasin, the tapasin-independent allele B*4405 (116Y) acquires a repertoire of peptides that is less optimal than the tapasin-dependent allele B*4402 (116D).

HLA-DM, HLA-DO and tapasin: functional similarities and differences.

In both the MHC class II and class I pathways of antigen presentation, accessory molecules influence formation of MHC-peptide complexes. In the MHC class II pathway, DM functions in the loading and editing of peptides; recent work demonstrated that it is acting not only in late endosomal compartments but also in recycling compartments and on the surface of B cells and immature dendritic cells. DM activity is modulated by another accessory molecule, DO, but this modulation is mainly operative in B cells, where it may lead to preferential activation of B cells producing high-affinity antibodies. In the MHC class I pathway of antigen presentation, recent in vivo experiments with knockout mice confirmed the role of tapasin in antigen presentation and indicate that it acts as a peptide editor and as a chaperone for TAP and the MHC class I heavy chain. In the class I loading complex, calreticulin and the thiol-dependent oxidoreductase ER60/ERp57 appear to support the function of tapasin in an as-yet-unknown fashion. The picture emerges that DM and tapasin have analogous functions in shaping the peptide repertoire presented by the respective MHC class II and class I molecules.

A region of tapasin that affects L(d) binding and assembly.

Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-L(d) cells. One mutant has a deletion of nine amino acid residues (tapasin Delta334-342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with L(d) and their effects on L(d) surface expression. We found that tapasin Delta334-342 was unable to bind to the L(d) H chain, and yet it facilitated L(d) assembly and expression. Tapasin Delta334-342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of L(d). Tapasin mutant H334F/H335Y, unlike tapasin Delta334-342, bound to L(d). Expression of tapasin H334F/H335Y in 721.220-L(d) reduced the proportion of cell surface open forms of L(d) and retarded the migration of L(d) from the endoplasmic reticulum. In total, our results indicate that the 334-342 region of tapasin influences L(d) assembly and transport.

Tapasin: an ER chaperone that controls MHC class I assembly with peptide.

The stable assembly of MHC class I molecules with peptides in the endoplasmic reticulum (ER) involves several accessory molecules. One of these accessory molecules is tapasin, a transmembrane protein that tethers empty class I molecules to the peptide transporter associated with antigen processing (TAP). Here, evidence is presented that tapasin retains class I molecules in the ER until they acquire high-affinity peptides.

Lack of HLA-class I antigens in human neuroblastoma cells: analysis of its relationship to TAP and tapasin expression.

We studied the constitutive and the interferon (IFN)-gamma-induced expression of HLA class I antigen heavy chain, beta2-microglobulin (beta2m), TAP-1, TAP-2 and tapasin in a panel of eleven neuroblastoma cell lines. Surface expression of HLA class I antigens was low in eight out of eight neuroblastoma cell lines bearing MYC-N amplification and/or 1p deletion, while two out of three neuroblastoma cell lines lacking these genetic alterations showed normal expression. IFN-gamma treatment restored HLA class I antigen surface expression in all neuroblastoma cell lines. Eight out of 11 neuroblastoma cell lines did not express TAP-1 mRNA and three of them also lacked TAP-2 mRNA. beta2 m mRNA was barely detectable or absent in five neuroblastoma cell lines, while tapasin mRNA was always expressed. IFN-gamma upregulated the expression of HLA class I heavy chain, beta2 m, TAP-1, TAP-2 and tapasin, as detected at mRNA or protein level. Post-transcriptional events were involved in altered TAP-1 and beta2 m expression in one peculiar neuroblastoma cell line. These data indicate that multiple mechanisms play a role in the HLA class I antigen-deficient phenotype of human neuroblastoma.

Thermal stability of MHC class I-beta 2-microglobulin peptide complexes in the endoplasmic reticulum is determined by the peptide occupancy of the transporter associated with antigen processing complex.

Once MHC class I heavy chain binds beta(2)-microglobulin (beta(2)m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated K(b)beta(2)m complexes within microsomes (TAP(+)/TAPasin(+)) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of K(b)-beta(2)m-peptide complexes depends on the timing of peptide binding to K(b)beta(2)m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in K(b)beta(2)m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of K(b)beta(2)m-peptide complexes.