Identification of novel Tapasin polymorphisms and linkage disequilibrium to MHC class I alleles

Tapasin is a Mr 48,000 glycoprotein and has a specialized role in MHC class I-restricted antigen presentation. It is encoded by a gene which maps centromeric to the MHC class II region of human Chromosome 6 within 200 kb of HLA-DP. There is variable dependence upon tapasin for MHC class I expression among different MHC class I alleles. HLA-B*4402 and to a lesser extent HLA-A1 and B8 are tapasin dependent, whereas HLA-B27, A2 and to a lesser extent B7 and A3 are tapasin independent. We investigated whether tapasin is polymorphic and whether these Tapasin alleles are in linkage with any MHC class I alleles. We identified three new mutations within intron 4, which are in a particular linkage with the previously described exon 4 (G16003C) dimorphism. The intronic mutations are G16146T, G16232A, and T16317A (numbering according to cosmid clone F0811; GenBank accession number Z97184). The allele frequency of Tapasin*01 (G16003) was 0.47 and Tapasin*02 (C16003) was 0.53 in this UK population. Four of the eight possible intronic haplotypes were identified and their cis linkage with the tapasin dimorphism ascertained. Tapasin*01 was associated with all the identified haplotypes, while Tapasin*02 was only associated with the wild-type intronic sequence (GGT). There was no significant linkage (P>0.01) of the Tapasin dimorphism or new Tapasin alleles to any of the MHC class I A, B, or C alleles studied or to the extended A1 B8 DR3 haplotype.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Dendritic cells loaded with exogenous antigen by electroporation can enhance MHC class I–mediated antitumor immunity

To develop an efficient antitumor immunotherapy, we have examined if dendritic cells (DCs) loaded with soluble antigens by electroporation present more antigens via the MHC (major histocompatibility complex) class I pathway, which mediate a cytotoxic T-cell response. DCs loaded with ovalbumin (OVA) by electroporation presented more MHC class I–restricted determinants compared with DCs pulsed with OVA. When electroporated DCs were pulsed with OVA for additional times, both MHC class I– and II–restricted presentation of OVA were increased compared with each single procedure, including electroporation or simple pulse. Immunization with DCs loaded with OVA by electroporation induced higher cytotoxicity of splenocytes to E.G7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with DCs pulsed with OVA. In the animal study, immunization with DCs loaded with OVA or tumor cell lysates by electroporation induced an effective antitumor immunity against tumor of E.G7 cells or Lewis lung carcinoma cells, respectively. In addition, immunization with DCs loaded with antigen by combination of electroporation and pulse, completely protected mice from tumor formation, and prolonged survival, in both tumor models. These results demonstrated that electroporation would be a useful way to enhance MHC class I–mediated antitumor immunity without functional deterioration, and that the combination of electroporation and pulse could be a simple and efficient antigen-loading method and consequently lead to induction of strong antitumor immunity.Abbreviations DCs dendritic cells - MHC major histocompatibility complex - OVA ovalbumin - TAA tumor-associated antigen - CTL cytotoxic T lymphocyte - LDH lactate dehydrogenase     

Effective and new potent drug combination: Histone deacetylase and Wnt/β-catenin pathway inhibitors in lung carcinoma cells

Lung cancer is the most commonly diagnosed cancer worldwide with a high mortality rate. In this study, the therapeutic effect of combination valproic acid and niclosamide was investigated on human lung cancer cell line. The effects of the compounds alone and combination therapy on cell viability were determined by sulforhodamine B and adenosine 5′-triphosphate viability assays. Flow cytometry was used to determine the cell death mechanism and DNA damage levels responsible for the cytotoxic effects of combination therapy. The presence of apoptosis in cells was supported by fluorescence microscopy and also by using inhibitors of the apoptotic signaling pathway. The increase in cellular reactive oxygen species (ROS) level in combination therapy was determined by H2DCFDA staining. The effect of N-acetyl-l -cysteine combination on ROS increase was investigated on cell viability. In addition, the expression levels of the proteins associated with epigenetic regulation and cell death were analyzed by Western blotting and gene expression levels were determined using real-time quantitative polymerase chain reaction.It was observed that the combination therapy showed a cytotoxic effect on the A549 lung cancer cells compared to the individual use of the inhibitors. The absence of this effect on normal lung cells revealed the presence of a selective toxic effect. When the mechanism of cytotoxicity is examined, it has been observed that combination therapy initiates the activation of tumor necrosis receptors and causes apoptosis by activated caspase. It was also observed that this extrinsic apoptotic pathway was activated on the mitochondrial pathway. In addition, ER stress and mitochondrial membrane potential loss associated with increased ROS levels induce cell death. When the data in this study were evaluated, combination therapy caused a dramatic decrease in cell viability by inducing the extrinsic apoptotic pathway in lung cancer cell line. Therefore, it was concluded that it can be used as an effective and new treatment option for lung cancer.     

Ly49A allelic variation and MHC class I specificity

The Ly49 family of natural killer (NK) cell receptors is encoded by a polygenic genetic locus. Allelic forms have been described and their expression appears to be regulated. The best-characterized Ly49 molecule, the C57BL/6 form of Ly49A, is an NK cell inhibitory receptor that binds H2Dd. To determine whether differences between Ly49a alleles may have functional consequences, allelic variants of Ly49a were cloned from several inbred mouse strains. Stable transfectants expressing each Ly49a allelic variant were generated and tested for reactivity with a panel of monoclonal antibodies (mAbs A1, JR9.318, YE1/32, and YE1/48) that recognize the C57BL/6 form of Ly49A. Binding to H2Dd was also assessed using fluorescently labeled H2Dd tetramers. Furthermore, cytotoxicity assays were performed using anti-Ly49A mAb-separated interleukin-2-activated NK cells. We show that despite binding to fluorescently labeled H2Dd tetramers, the Ly49A+ NK cells from representative mouse strains displayed significantly different degrees of inhibition with H2Dd targets. These results can be interpreted in the light of recent structural data on the Ly49A-H2Dd complex. Thus, the Ly49 family displays functionally significant allelic polymorphism which adds to the repertoire of NK cell receptors.     

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.     

MicroRNA-148/152 impair innate response and antigen presentation of TLR-triggered dendritic cells by targeting CaMKIIα

MicroRNAs (miRNAs) are involved in the regulation of immunity, including the lymphocyte development and differentiation, and inflammatory cytokine production. Dendritic cells (DCs) play important roles in linking innate and adaptive immune responses. However, few miRNAs have been found to regulate the innate response and APC function of DCs to date. Calcium/calmodulin-dependent protein kinase II (CaMKII), a major downstream effector of calcium (Ca(2+)), has been shown to be an important regulator of the maturation and function of DCs. Our previous study showed that CaMKIIα could promote TLR-triggered production of proinflammatory cytokines and type I IFN. Inspired by the observations that dicer mutant Drosophila display defect in endogenous miRNA generation and higher CaMKII expression, we wondered whether miRNAs can regulate the innate response and APC function of DCs by targeting CaMKIIα. By predicting with software and confirming with functional experiments, we demonstrate that three members of the miRNA (miR)-148 family, miR-148a, miR-148b, and miR-152, are negative regulators of the innate response and Ag-presenting capacity of DCs. miR-148/152 expression was upregulated, whereas CaMKIIα expression was downregulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. We showed that miR-148/152 in turn inhibited the production of cytokines including IL-12, IL-6, TNF-α, and IFN-β upregulation of MHC class II expression and DC-initiated Ag-specific T cell proliferation by targeting CaMKIIα. Therefore, miRNA-148/152 can act as fine-tuner in regulating the innate response and Ag-presenting capacity of DCs, which may contribute to the immune homeostasis and immune regulation.     

Autophagy facilitates major histocompatibility complex class I expression induced by IFN-γ in B16 melanoma cells

The reduction or loss of MHC-I antigen surface expression in human and murine tumor cells is partly attributable to the dysregulation of various components of the MHC-I antigen-processing machinery. Accumulating evidence suggests that autophagy, besides its vital role in maintaining the cellular homeostasis, plays an important role in MHC-II surface expression. Here, we report that autophagy is a negative regulator of MHC-I antigen expression in B16 melanoma cells; however, in the presence of IFN-γ, it is converted to a positive regulator. We show that autophagy not only participates in the degradation of MHC-I antigen but also plays a role in the generation of MHC-I-binding peptides. For these two processes, IFN-γ interferes with MHC-I antigen degradation, rather than affecting peptide generation. Using B16 melanoma mouse model, we further show that autophagy may enhance the cytolysis of CTL to melanoma cells at the early stage of melanoma, but impairs the cytolysis at the late stage. Such different consequences may be explained by the different levels of IFN-γ during tumor progression. Taken together, our findings demonstrate that autophagy is involved in the regulation of MHC-I antigen expression, through which autophagy can play different roles in tumor immunity.     

Characterization of MHC class I and β2-microglobulin sequences in Atlantic cod reveals an unusually high number of expressed class I genes

 Degenerate polymerase chain reaction (PCR) primers based on conserved residues from alignments of species with already characterized major histocompatibility complex (MHC)-encoded sequences were used in the search for class I and β₂-microglobulin (b ₂ m) genes in Atlantic cod (Gadus morhua L.). After PCR amplification and subsequent sequencing a putative class I sequence was identified, from which a probe was designed and used to screen a spleen cDNA library from one single individual. The full-length clone obtained was sequenced and shown to be a classical Mhc class I-encoded sequence. It revealed the characteristic α1-, α2-, and α3-domains and transmembrane and cytoplasmic region, with several conserved amino acids. A PCR amplification from the α2-domain to the CY-region was performed on the same library, using a proof-reading enzyme. At least 11 unique additional sequences were isolated. Moreover, sequencing of the additional cDNA clones resulted in a total of 17 different Mhc class I sequences in this individual. A Southern hybridization of DNA from four different individuals using an α3-specific probe confirmed this large number of genes. Interestingly, based on differences mainly in their transmembrane region, the sequences obtained could be divided into two distinct groups. Within the groups no support could be obtained for any further subdivision. Southern experiments using an α1-specific probe gave almost the same restriction fragment length polymorphism with a high number of hybridizing bands, suggesting a low divergence in this part of the gene. Sequencing of PCR clones obtained with a proof-reading enzyme confirmed this at the nucleotide level. PCR amplification to isolate and characterize the b ₂ m gene resulted in a sequence which was used to screen a thymus cDNA library. Two different alleles were obtained and these showed the characteristic features of known teleostean β₂m sequences. A Southern hybridization with genomic DNA from four different individuals suggested the presence of one b ₂ m locus in Atlantic cod. Received: 10 March 1999 / Revised: 1 June 1999     

Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae)

In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC.Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AY258333–AY258335, AY428561–AY428570, and AY530534–AY530535     

Physical mapping and evolution of the centromeric class I gene-containing region of the rat MHC

We physically mapped the centromeric part of the BN rat MHC (RT1n haplotype) in a contig of overlapping P1-derived artificial chromosome (PAC) clones encompassing about 300 kb. The following genes were identified and ordered as: (Syngap, Hset, Daxx, Bing1)-Tapbp-Rgl2-Ke2-Bing4-B3galt4- Rps18-Sacm2l-RT1-A1-RT1-A2-RT1-A3-Ring1-Ring2-++ +Ke4-Rxrb-Col11a2-RT1-Hb-Ring3-RT1-DMb. Thus, in contrast to other RT1 haplotypes, RT1n contains three class I genes, RT1-A1, RT1-A2, and RT1-A3, mapping between the Sacm2l and Ring1 genes. Comparisons of the sequences flanking the Sacm2L and Ring1 genes in rat, human, and mouse suggest that the class I gene-containing region was inserted between these genes in rat and mouse at a similar position. Thus, this insertion is likely to have occurred in a common ancestor of these rodents, although the presence of a site particularly permissive for insertions cannot be excluded.     

Functional redundancy between thymic CD8α+ and Sirpα+ conventional dendritic cells in presentation of blood-derived lysozyme by MHC class II proteins

Dimorphic fungi collectively account for 5-10 million new infections annually worldwide. Ongoing efforts seek to clarify mechanisms of cellular resistance to these agents and develop vaccines. A major limitation in studying the development of protective T cells in this group of organisms is the lack of tools to detect, enumerate, and characterize fungus-specific T cells during vaccination and infection. We generated a TCR transgenic mouse (Bd 1807) whose CD4(+) T cells respond to a native epitope in Blastomyces dermatitidis and also in Histoplasma capsulatum. In this study, we characterize the mouse, reveal its applications, and extend our analysis showing that 1807 cells also respond to the related dimorphic fungi Coccidioides posadasii and Paracoccidioides lutzii. On adoptive transfer into vaccinated wild-type mice, 1807 cells become activated, proliferate, and expand in the draining lymph nodes, and they differentiate into T1 effectors after trafficking to the lung upon lethal experimental challenge. Bd 1807 cells confer vaccine-induced resistance against B. dermatitidis, H. capsulatum, and C. posadasii. Transfer of naive 1807 cells at serial intervals postvaccination uncovered the prolonged duration of fungal Ag presentation. Using 1807 cells, we also found that the administration of vaccine only once induced a maximal pool of effector/memory CD4(+) cells and protective immunity by 4 wk after vaccination. The autologous adoptive transfer system described in this study reveals novel features of antifungal immunity and offers a powerful approach to study the differentiation of Ag-specific T cells responsive to multiple dimorphic fungi and the development of CD4(+) T cell memory needed to protect against fungal infection.     

Mycobacterium bovis bacillus Calmette-Guérin secreting active cathepsin S stimulates expression of mature MHC class II molecules and antigen presentation in human macrophages

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-gamma, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4(+) T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4(+) Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.     

Genetic polymorphism of the swine major histocompatibility complex (SLA) class I genes, SLA-1, -2 and -3

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex (Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC (HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.The DNA sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank nucleotide databases and have been assigned the accession numbers, AB105379, AB105380, AB105381, AB105382, AB105383, AB105384, AB105385, AB105386, AB105388, AB105389, AB105390 and AB105391     

Design,synthesis, in vitro inhibition and toxicological evaluation of human carbonic anhydrases I,II and IX inhibitors in 5-nitroimidazole series

Abstract

With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhydrases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the toxicity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed efficient inhibition against hCA II (KI = 58.6?nM). Compound 10 showed moderate inhibition against hCA II (KI = 199.2?nM) and hCA IX (KI = 147.3?nM), whereas it inhibited hCA I less weakly at micromolar concentrations (KI = 6428.4?nM). All other inhibition constants for these compounds were in the submicromolar range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study suggests that these compounds are suitable for further preclinical characterisation as potential inhibitors of hCA I, II and IX.     

Carbonic anhydrase inhibitors: Two-prong versus mono-prong inhibitors of isoforms I,II, IX,and XII exemplified by photochromic cis-1,2-α-dithienylethene derivatives

We investigated the inhibition of five physiologically relevant CA isoforms with photochromic cis-1,2-α-dithienylethene-based compounds incorporating either a benzenesulfonamide and Cu(II)-iminodiacetic acid (IDA)-, bis-benzenesulfonamide-, bis-Cu(II)-IDA-, and bis-ethyleneglycol-methyl ether moieties, in both their open- and closed-ring forms. For hCA I the best inhibitors were the mono-prong bis-sulfonamide and the bis-Cu-IDA complexes (K_Is of 2–3 nM) in their open form. For hCA II, best inhibitors were the open and closed forms of the mono-prong bis-sulfonamide (K_Is of 13–18 nM). hCA IX was moderately inhibited by these compounds (K_Is of 9–376 nM) whereas hCA XII and XIV were less susceptible to inhibition (K_Is of 1.12–16.7 μM).     

MHC class I-like genes in cattle,<Emphasis Type="Italic"> MHCLA,</Emphasis> with similarity to genes encoding NK cell stimulatory ligands

A comparative genomics approach for mining databases of expressed sequence tags (ESTs) was used to identify two members of a novel MHC class I gene family in cattle. These paralogous genes, named MHC class I-like gene family A1 ( MHCLA1) and MHCLA2, were shown by phylogenetic analysis to be related to human and mouse genes encoding NK cell stimulatory ligands, ULBP, RAET, H60 and Raet-1. Radiation hybrid mapping placed cattle MHCLA1 on BTA9, which, on the basis of existing comparative mapping data, identified the ULBP, RAET1, H60 and Raet1 genes as homologues of the cattle MHCLA genes. However, the human and mouse orthologues of MHCLA1 and MHCLA2 could not be defined due to extensive sequence divergence from all known members of the ULBP1/ RAET1/H60/Raet1 gene family. The cattle MHCLA1 molecule is predicted to be missing an alpha(3) domain, similar to the human and mouse homologues. Like the human ULBP genes, MHCLA1 was found to be transcribed constitutively in a variety of fetal and adult tissues by RT-PCR. The patterns of hybridization obtained by Southern blotting using MHCLA1 as a probe and DNA from 14 species representing five mammalian orders suggests that the MHCLA genes evolved rapidly in the Cetartiodactyla. Previous findings demonstrating that ULBPs serve as ligands for the NK cell NKG2D stimulatory receptor, and that this interaction can be blocked by a human cytomegalovirus glycoprotein that binds to ULBPs, suggests that the extensive divergence found among the cattle, human and mouse MHCLA homologues is due to selection exerted by viral pathogens.