| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 94 === HLA class I molecules plays an important role in the host immune response. These molecules present antigenic peptides to cytotoxic lymphocytes and thereby are important in the immune surveillance of cells infected with virus, other intracellular pathogens, or altered by malignant transformation. It is well recognized that a lack or deficiency of expression of such antigens in a variety of human malignancies. In this thesis, we examined the HLA class I heavy chain (HC), β2-microglobulin (β2m), LMP (LMP2 and LMP7), and TAP (TAP1 and TAP2) genes in six human renal cell carcinoma cell lines (RCC52, RCC98, HH050, HH244, HH332 and HOKN-9). These cell lines exhibited varying degrees of deficiencies in expression of the heavy chain allospecificities and/or β2m gene products, as determined by immunofluorescence/ flow cytometric analysis. RCC52 was only cell line, in which no surface HLA class I antigen expression was detected. The RCC98 expressed the most abundant surface HLA class I antigens among the six cell lines, while in the HOKN-9, HH050 and HH244cell lines, only 80, 66 and 24 % of cells respectively were positive with surface HLA class I antigens, and the percent positive cells did not increase even after cells were treated with IFN-γ, signifying that the proportion of HLA-class I negative cells would be difficult to be eradicated even if potent and corresponding cytotoxic T lymphocytes were generated. While the basal levels of cytoplasmic components LMP2 and TAP2 proteins were expressed in all 6 RCC cell lines, which were either weakly enhanced or not enhanced at all by IFN-γ, In contrast, none of LMP7 and TAP1 proteins were expressed at either the baseline level or the induction level upon IFN-γ exposure. However, baseline levels and IFN-γ-mediated enhancement of mRNAs of β2m, LMP2, LMP7, TAP1 and TAP2 were all demonstrated in each of the 6 RCC cell lines including RCC52. The discrepancy between mRNAs and proteins for LMP7 and TAP1 is yet to be determined. To elucidate the possible mechanism involved in the totally lack of surface HLA class I expression in RCC52 cells, genomic DNAs from RCC52 and RCC98 were extracted and sequencing analysis of the β2m gene was performed, which revealed two important and interesting finding: (i) a single base guanine (G) deletion (delG) was found in the β2m gene exon 1. It changed codon 6 from GCC to CCT resulting in a subsequent introduction of a premature stop at codon 7; and (ii) in normal wild type β2m gene, there are four cytosine and thymine (CT) dinucleotide repeats at codon13 to 15. However, we found a CT dinucleotide deletion (delCT) in RCC52 cell line with three dinucleotide repeats, leading to a premature stop on codon 55. These results may account for the instability of β2m to associate with HLA class I heavy chain and for lack of reactivity with the anti-β2m monoclonal antibody tested. We believe that these unique lesions in the β2m gene were not caused by mutations acquired during cell growth in vitro, since surface HLA class I antigens of RCC52 cells in the early passages of this cell line could not be detected either. The site of delG mutation identified is distinct from those identified by other reported tumor types such as melanoma, and may represent the first such case reported with RCC, specifically in the sarcomatoid subtype. Since we have examined only a small penal of RCC cell lines representing different subtypes including sarcomatoid, chromophobe, papillary and clear cell, it is not possible at this point to determine whether the defect lesions in HLA class I gene(s) identified could be the general representation of the given subtype of RCC. Therefore, in-depth studies with a larger number of RCC tumor tissues and cell lines are warranted. Finally, the development of efficient methodology to assess HLA-class I expression may be useful to select suitable patients with RCC to be enrolled in T cell-based immunotherapy.
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