CD49d Treg cells with high suppressive capacity are remarkably less efficient on activated CD45RA- than on naive CD45RA+ Teff cells.

BACKGROUND: Impairment of regulatory T cells (Tregs) in common autoimmune diseases seems likely. However, the extent of Treg deficiency (number, function) or differential susceptibility of T effector cells (Teffs) to suppression is not completely understood. We hypothesize that even in healthy individuals both cell populations are heterogeneous and differ in their suppressive capability and their susceptibility to suppression. MATERIAL AND METHODS: Lymphocytes were enriched by MACS for CD4(+)CD25(+) Tregs or CD4(+)CD25(-) Teffs. After multicolour staining (anti-CD25, anti-CD127, anti-CD49d or anti-CD45RA) highly purified Treg and Teff subpopulations were collected by FACS. Functional capacity of Tregs or suppressive susceptibility of Teffs was analyzed in an in vitro assay. RESULTS: When CD4(+)CD25(high)CD127(-/low) CD49d(-) Tregs were tested on naive CD4(+)CD127(+)CD25(-)CD45RA(+) Teffs (93.8 %) suppression was almost complete, while the suppressive capacity of CD4(+)CD25(high)CD127(-/low) CD49d(+) Tregs was significantly less (71.8 %). Suppressive activity was low when CD4(+)CD25(high)CD127(-/low) CD49d(+) Tregs were analyzed on CD4(+)CD127(+)CD25(-)CD45RA(-) Teffs (48.7%). CONCLUSION: Although CD49d(+) Tregs are functional, the suppressive capacity is significantly lower compared to CD49d(-) Tregs. CD45RA(+) Teffs can be completely suppressed, while CD45RA(-) Teffs display relative resistance. Phenotypic and functional heterogeneity of Tregs as well as Teffs has to be considered when analyzing deficiencies in immune regulation.

CD4+CD25+FoxP3+ T lymphocytes fail to suppress myelin basic protein-induced proliferation in patients with multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disorder directed against self antigens of the central nervous system. CD4(+)CD25(+)FoxP3(+) regulatory T cell (T(reg)) mediated suppression is an essential mechanism of self-tolerance. We studied whether changes in the suppressive function of a mixture of CD25(high) and CD25(intemediate) expressing T(reg) cells in myelin basic protein (MBP)-induced proliferation occurred in untreated MS patients. Suppression of MBP-induced proliferation was observed in 13 out of 29 (45%) MS patients; this was significantly (p<0.05) less compared with 17 out of 19 (89%) healthy individuals. Relative T(reg) counts was significantly increased in MS patients (mean+/-S.D.; 20+/-8%) compared with healthy individuals (15+/-5%). These findings suggest that impaired T(reg) function may be involved in pathogenesis of MS.

DNA methylation in promoter regions of red cell membrane protein genes in healthy individuals and patients with hereditary membrane disorders.

The methylation state of 5'-CG-3' sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, we examined the methylation profiles of several genes for human erythroid membrane proteins: ELB42 (protein 4.2), EPB3 (band 3), SPTB gene (beta-spectrin), and ANK1 (ankyrin). The bisulfite protocol of the genomic sequencing method was applied. The number of 5'-CG-3' dinucleotides was the most abundant in SPTB and ANK1, much less in EPB3, and the least in ELB42. In the DNA of peripheral blood mononuclear cells from healthy individuals, the promoter regions of EPB3 and ELB42 were extensively methylated, but the SPTB and ANK1 promoters were totally unmethylated. We also investigated methylation profiles in peripheral blood mononuclear cells from patients with red cell membrane diseases, such as complete protein 4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The DNA methylation states in these genes of erythroid cells, which we obtained at the second phase of the 2-phase liquid culture of erythroid precursor cells in the peripheral blood, were essentially identical or very similar to those of peripheral blood mononuclear cells. In disease states, the DNA methylation profiles of these red cell membrane protein genes were essentially not different from those in healthy individuals (statistically not significant).

Relationships between DNA methylation and expression in erythrocyte membrane protein (band 3, protein 4.2, and beta-spectrin) genes during human erythroid development and differentiation.

Red cell membrane proteins are sequentially expressed during erythroid development and differentiation. Spectrins have already been synthesized in early erythroid precursors such as pronormoblasts, and band 3 (B3) appears at nearly the same stage. Protein 4.1 appears next, followed by protein 4.2 (P4.2) at the very late erythroblast stage. The methylation states of the promoter 5'-CG-3' sites are known to be linked to the regulation of promoter function by modulating DNA-protein interactions and the structure of chromatin. Hence, the genes for B3, P4.2, and beta-spectrin (beta-SP) appear to be suitable models to study the relationship between methylation of promoter 5'-CG-3' sites and the sequential expression of genes during human erythroid development and differentiation. We have examined methylation profiles in the promoter regions of the genes (ELB42, EPB3, and SPTB) for the human erythroid membrane proteins P4.2, B3, and beta-SP by applying the bisulfite genomic sequencing method. Our results demon strate the following: (1) The promoter regions of EPB3 and ELB42 are extensively methylated in DNA from human peripheral blood mononuclear cells, but the SPTB promoter is totally unmethylated. (2) During erythroid differentiation, DNA methylation patterns change as follows: (a) ELB42 is unmethylated in DNA from erythroid-committed blastic cells, such as the human cell lin UT-7/EPO, but is methylated in erythroblasts from peripheral blood burst-forming unit erythroid (BFU-E) in the second phase of the liquid-culture method. Messenger RNA (mRNA) from ELB42 is first detected in early erythroblasts, and P4.2 is expressed in late erythroblasts. (b) In contrast, EPB3 is consistently methylated in UT-7/EPO cells and in cultured erythroblasts from BFU-E from human peripheral blood. B3 mRNA and protein are already expressed in early erythroblasts. (c) SPTB remains unmethylated in human DNA from UT-7/EPO cells and from cultured erythroblasts. These results document the diversity of the reactions of human promoter sequences to the modulating influence of DNA methylation. Whereas the human SPTB promoter conforms to expectations in that it is unmethylated and fully active throughout erythroid development, high levels of promoter methylation correlate with promoter activity for the EPB3 and ELB42 genes during their sequential activation in erythrocyte differentiation.

Gene structure and expression of the 5'-(CGG)(n)-3'-binding protein (CGGBP1).

The human nuclear 5'-(CGG)(n)-3'-binding protein (CGGBP1) influences the expression of the fragile X mental retardation (FMR1) gene by specifically interacting with the 5'-(CGG)(n>5)-3' repeat in its 5' untranslated region. Here, we show that CGGBP1 binds to 5'-(CGG)(n)-3' repeats with n>or=5 and to interrupted repeats. The genomic and mRNA organization of the human and murine CGGBP1 genes was studied and the human gene was mapped to chromosome 3p. Due to alternative polyadenylation, mRNAs of 1.2 and 4.5 kb are transcribed at varying ratios in human and murine cells and in embryonic, fetal, and adult tissues. The human and the murine genes, including promoters and large parts of the untranslated regions, are highly conserved. A sequence of 235 nucleotides 5' upstream of CGGBP1 is essential for promoter activity in transfection experiments. Complete in vitro methylation inactivates the promoter, which is unmethylated in human cells as shown by bisulfite genomic sequencing.

Intraperitoneal dissemination of Ad12-induced undifferentiated neuroectodermal hamster tumors: de novo methylation and transcription patterns of integrated viral and of cellular genes.

The intramuscular (i.m.) injection of human adenovirus type 12 (Ad12) into newborn Syrian hamsters caused widespread dissemination of up to 15 tumors over the entire peritoneal cavity in 70-90% of the animals within 30-50 days. Subcutaneous (s.c.) injections led to local tumor formation only. Independent of location, tumor histology revealed Homer-Wright rosette-like structures typical for primitive neuroectodermal tumors (PNET). All tumor cells showed markers indicative of neuroectodermal and mesenchymal derivations. Each Ad12-induced tumor cell carried multiple copies of integrated Ad12 genomes at one chromosomal site which was different for each tumor. For Ad12 tumor induction in hamsters, the patterns of Ad12 viral and cellular gene expression were important and were affected by changes in DNA methylation, both in the integrated Ad12 DNA and the cellular genome. By applying the bisulfite protocol, the de novo DNA methylation in the integrated Ad12 genomes was determined. These patterns were complex, characterized by regional initiation and by excluding genome segments in the E1A and E1B promoters. In all tumors, the Ad12 segments E1A, E1B, E2A, parts of E3 and E4 were similarly transcribed, as shown by the RT-PCR and DNA microarray methods. Changes in the transcription of a large number of cellular genes was assessed by using mouse gene microarrays encompassing about 1980 different mouse genes with 87-96% homology to hamster genes. Similarities and differences existed in the transcription of cellular genes of different functional classes among the different Ad12-induced tumors. These alterations in cellular gene transcription may be an important parameter in the oncogenic transformation by Ad12.

Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family.

In an effort to identify psoriasis-associated genes, we compared gene expression in normal and psoriatic skin, using differential display RT-PCR technique. Sequence analysis of a 650-bp cDNA fragment (clone 110) that was highly up-regulated in lesional skin revealed homology to a noncoding cDNA (NICE-2). By subsequent cDNA cloning, using RNA from psoriatic skin, we have identified two alternatively spliced mRNA-isoforms (0.5 and 4.4 kb), which differ in composition of their untranslated regions. By sequence comparison, we have mapped the novel gene, named S100A15, to the S100 gene cluster within the epidermal differentiation complex (chromosome 1q21). Analysis of the deduced amino acid sequence revealed a protein of 101 amino acids containing two potential EF-hand motifs with high homology to the S100A7. Northern blot hybridization and semiquantitative RT-PCR analysis confirmed the S100A15 overexpression in psoriasis, showing different levels of expression of the S100A15 mRNA isoforms. In situ hybridization of the S100A15 revealed a markedly increased staining of basal and suprabasal epidermal layers of psoriatic skin compared with healthy tissue. Our data suggest an involvement of the novel S100A15 in epidermal differentiation and inflammation and might therefore be important for the pathogenesis of psoriasis and other diseases.