| Summary: | 博士 === 國立清華大學 === 分子醫學研究所 === 98 === IgE produced by IgE-secreting plasma cells mediates type-I hypersensitivity reactions responsible for various allergic diseases. The differentiation of IgE-secreting plasma cells must go through the membrane-bound IgE+ (mIgE+) lymphoblast stage. On mIgE+ B cells, the membrane-bound ε-chain (mε) exists predominantly in the long isoform, mεL, containing an extra 52-a.a. CεmX domain between CH4 and the C-terminal membrane-anchoring segment; the short isoform of mε, mεS, exists in minor proportions. CεmX, which resulted from an alternative splicing of the ε RNA transcript at 156-bp upstream of the splicing acceptor site used by mεS, is unique in the existing DNA and protein databases. CεmX thus provides an attractive site for immunologic targeting of mIgE+ B cells.
In this thesis, nine newly prepared CεmX-specific monoclonal antibodies (mAbs), as well as the previously reported a20, bound to mIgE.FcL-expressing CHO cells, while only 4B12 and 26H2 bound to mIgE.FcL-expressing B cell line Ramos cells. The mAb 4B12 bound to the N-terminal part, 26H2 the middle part and all others the C-terminal part of CεmX. Expression of Igα and Igβ on the mIgE.FcL-CHO cells reduces the binding of a20 to CεmX as compared with that of 4B12 and 26H2. The chimeric mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, lysed mIgE.FcL-Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 demonstrated antibody-dependent cellular cytotoxicity (ADCC) toward mIgE.FcL-Ramos cells in a dose-dependent fashion. In cultures of PBMCs from atopic dermatitis patients, c4B12 and c26H2 inhibited the synthesis of IgE driven by anti-CD40 and IL-4. These results suggest that 4B12 and 26H2 are potentially useful for targeting mIgE+ B cells to control IgE production.
Based on an analysis of the CεmX genomic DNA sequences of 320 subjects residing in Taiwan, single-nucleotide polymorphisms (SNPs) have been found at two positions, namely, G/T at #46 and A/G at #93 (along the 156 bp of CεmX), with the former creating an amino acid change from Val to Leu at #16 (along the 52 a.a. of CεmX) and the latter resulting in no change (Gly). Among the 640 CεmX sequences identified, the previously known 46G93A allelic form appeared 293 times, the newly discovered 46T93A allelic form (GeneBank accession no. GU208817) 26 times, and the 46G93G allelic form (GU208818) 321 times. No 46T93G allelic form was found. Serum IgE measurements showed that the SNPs did not correlate with the levels of serum IgE. The anti-CεmX mAb, 4B12, could bind equally well to mIgE.FcL(16V) and mIgE.FcL(16L). While genetic variation of CεmX of broader populations should also be investigated, these newly discovered genetic variants of CεmX in the Taiwanese population do not seem to affect the feasibility of using an anti-CεmX mAb, such as 4B12, to target mIgE+ B cells.
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