1．The Preparation And Application For HLA-A2/HPV6E7 Tetramer 2．Linkage Of A Locus Determining FPH To Chromosome 19p13.1-19pter

Objectives: MHC tetramer is a powerful tool for detecting the antigen specific T cells with exquisitely antigen-specific and highly sensitive. Since HLA-A*0201 is the most common allele of human MHC I molecules; Human Papillomavirus 6 (HPV6) is the major cause of Condyloma Acuminata (CA), we choose HLA-A*0201 and its restricted peptide HPV6E7 (22-30) to construct sHLA-A2/HPV6E7 tetramer. With the tetramer, we detected the antigen specific T cells in the different courses of CA.Methods: The two subunits of the human leucocyte antigen (HLA-A2) have been expressed at high levels as insoluble aggregates in bacterial cells. They were induced by IPTG to overexpress the target proteins. In the presence of an HLA-A2 restricted antigenic peptide (HPV6E7), refolding was initiated by dilution of the denaturant in the solution of heavy chain andβ2m. After concentrate and buffer exchange of refolding products, the BirA enzyme was used to biotinylate the refolded complex. Biotinylation was performing at the COOH-terminus of the heavy chain named BirA substrate peptide (BSP). The refolded and biotinylated products were detected by ELISA and Western blot with mAbW6/32 and HRP-streptavidin. sHLA-A2/HPV6E7 tetramer was prepared by mixing the biotinylated peptide-HLA complex with phycoerythrin (PE) labeled avidin at a molar ratio of 4:1.Peripheral blood was obtained from HLA-A2 positive healthy individual and different course of CA. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Paque density gradient centrifugation and then placed in sterile tissue culture dishes for separation of adherent from nonadherent cells by 2h incubation at 37℃. The nonadherent cells (PBLs) were plated into tube. With the PE-tetramer and FITC-CD8 mAb, a group of double positive cells can be observed by FACS.Here we use the T2 (HLA-A2+) cell pulsed with HPV6E7 as stimulating cells to stimulate the HLA-A2+ CA with HPV6 infected PBLs to generate the peptide-specific CTLs with the long term mixed lymphocytes culture. After culture, the specific CTLs will proliferate, and are used to monitor our sHLA-A2/ HPV6E7 tetramer. To detect the function of these specific CTLs, T2 cells loading peptide act as target cells, we detect their killing activation by LDH assay.In situ tetramer (IST) staining has opened the door to exploration of antigen specific T cells in tissues. The development of MHC tetramer staining has revolutionized our ability to study antigen specific T cells. This technique permits visualization of antigen-specific T cells in tissue sections along with their spatial relationship to their microenvironment. The quantities and qualities of CTLs infiltrating in tissues reflect the status of cell immunity. All have an effect on prognosis of CA. Condyloma tissues sections were incubated with the tetramers, washed with PBS and frozen in Tissue-Tek OCT compound. To monitor tetramer binding to CD8-positive antigen specific CTL in the cryopreserved tissue, 40μm cryosections were prepared, fixed in acetone for 10 min at RT and pre-incubated for 30 min in 5% (v /v) normal cow serum. Then they were incubated with the relevant mAb CD8+ and analyzed by fluorescence microscope.Results: After initial purification, the aggregates were dissolved in 8M urea. The yields are 450mg and 350mg for theβ2m and heavy chain from 6 liter saturant baterial cells, respectively. The purity of theβ2m and heavy chain is 65.3% and 69.8%, respectively. The western blot result shows monoclonal antibody (mAb) W6/32, which recognizes a determinant present on the heavy chain only when the heavy chain is associated withβ2m, binds to the refolded component. Sandwich ELISA with mAb W6/32 and rabbit anti-β2m antibody demonstrates the success of refolding. The sHLA-A2/ HPV6E7 tetramer were constructed successfully.The frequency of double positive cells in PBL of healthy dividual is 0.02%; but it is 0.11% in paramedic CA and is 0.10% in recurrence CA. We apply our prepared sHLA-A2/ HPV6E7 tetramer to stain the cells after long term mixed lymphocytes culture. The results shows our tetramer is able to discriminate the frequencies of specific CTL induced by HLA-A2/HPV6E7. The frequency of specific CTLs in paramedic CA is very high (0.23% vs 1.19%,P<0.05). The frequencies of specific CTLs in recurrence CA is obvious high too (0.23% vs 1.16%,P<0.05). The CTLs killing frequency in paramedic CA is (64.5±2.5) %. But the CTLs killing frequency in recurrence CA is (51.5±1.5) %. The difference is obvious in the CTLs killing frequency different course CA. Additionally, antigen specific T cells infiltrating in tissues in paramedic CA can be observed, but not observed in recurrence CA.Conclusions: In this study the HLA-A2 / HPV6E7tetramer were successfully constructed. The tetramer can be applied to directly visualize antigen-specific CTLs efficiently and become the critical approach in assessment of T cell immune responses in CA patients.Objectives: In this study, we undertook a linkage analysis with microsatellite polymorphic markers?short tandem repeat (STR) in a three-generation Familial Progressive Hyperpigmentation (FPH, MIM 145250) family with 17 members, including 6 affected individuals residing in Hubei Province. The immortal B Lymphocyte Cell Lines (B-LCLs) of the family were established by EBV transformation and addition of cyclosporin A (CysA) to inhibit the activity of T cells. Additional, the plasmid pcDNA3.0-STK11 was constructed from 3 affected individuals in this FPH family.Methods: DNA was isolated from peripheral blood cells from all 17 individuals. Genome screening was performed using 176 STR microsatellite markers from 22 autosomes, 8 for each chromosome, these STR markers were selected for their polymorphism in Chinese population and their physic locations which were able to represent a given chromosome segment. Additional 6 STR markers were chosen from the short arm of 19th chromosome to define the borders of the cosegregating region. These markers were amplified by polymerase chain reaction (PCR) based on the protocol reported previous. PCR products were analyzed by vertical electrophoresis polyacrylamide gels (PAGE) and silver staining. According to the guideline recommended by the DNA Commission of the International Society for Forensic Haemogenetics (ISFH), every allele of STR locus was named by the repeat number of its repetitive sequence. After purification, PCR products of 2 alleles from every STR locus were directly sequenced for their repetitive numbers on an Applied Biosystems 377 automatic sequencer. PCR products were synelectrophoresed with molecular weight standard and then every allele was named by its repeat number. As FPH was demonstrated to be an autosomal dominantly inherited disease, principles for judging linkage between its genotype and phenotype are as follows: (1) If an allele or haplotype is present in all affected individuals and not present in all unaffected individuals, it is suggested to be linked to the pathogenic gene; (2) If an allele or haplotype is present in some, but not all, affected individuals and not present in all unaffected individuals, it is possible that this allele or haplotype links to the pathogenic gene; (3) If an allele or haplotype is present randomly in affected and unaffected individuals, it is excluded to be linked to the pathogenic gene.The total cell RNA is extracted from the Peripheral blood mononuclear cells; and the cDNA encoding the STK11 was acquired by RT-PCR technique and then inserted into the plasmid pcDNA3.0.Results: the FPH locus is observed to link to the region covering D19S714 and D19S591 which are markers for 19p13.1-19pter. The genetic distance of region in Marshfield is 45.48 cM and physical distance in UCSC is 17.17Mb, harboring 628 defined genes. 15 immortal cell lines were established. The results showed that cellular volume of these immortal cells was enlarged and their morphology was diversity with glitch. There was increasing development of cell aggregates of proliferative lymphocyte cells. The recombinant plasmid pcDNA3.0-STK11 was identified to be correct by restriction endoniclease digestion and sequence analysis.Conclusions: the FPH locus is linked to chromosome 19p13.1-19pter. Interestingly, this region harbors STK11 gene, germline mutations in which were shown to be associated with Peutz-Jeghers Syndrome (PJS). PJS and FPH share the disorder of hyperpigmentation. But construcated STK11 sequences were completely coincidence with Genebank. So, the STK11 was not the virulence gene for FPH. There is a new gene not found for FPH in 19p13.1-19pter. For further fine mapping, it is obvious that more familial or/and sporadic FPH cases are required for the further fine mapping. The fine mapping of FPH gene is expected to lead to a better understanding of the etiology for both FPH and PJS.The immortal B-LCLs acquired successfully provide a long and plentiful DNA material for mapping and cloning causative gene for FPH and studying punctually the molecular mechanism of pigment metabolism in the skin.