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- Abdel-Motal, Ussama M
(författare)
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Binding and immunogenicity of MHC class-I specific peptides and glycopeptides
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Short synthetic MHC class I (MHC-I) Db and Kb binding peptides were found to be strongly immunogenic and capable of inducing virus-specific cytotoxic T lymphocyte (CTL) responses. Restimulation of primed splenocytes with a low peptide concentration generated optimal CTL responses. CTL responses were found to correlate with the capacity of peptides to bind to MHC class I measured as MHC-I upregulation (I-II). Earlier studies using 15-16 mer peptides have demonstrated that CD4+ helperT-cells were required to induce optimal CTL responses. We investigated if optimal-length peptides still required CD4+ T-cell help to generate CTL. The results clearly established that activation of CTL responses by short optimal peptides did not require CD4+ T-cell help (III). MHC-I heavy chains can appear on the cell surface in different configurations, including forms which are designated as functionally "empty". Such empty MHC-I molecules can directly bind synthetic peptides having the correct motifs. A method to measure the fraction of "empty " Db molecules on different cells was established by using monoclonal antibodies, staining either for Db-bound glycopeptides and for Db itself (IV). This method has been used to measure "empty" MHC-I molecules on cells from TAP and b2-M gene targeted mice as well as TAP1/b2-M double mutant mice. The influence of pH on glycopeptide binding to MHC-I molecules was also investigated (V). MHC-I chains are known to internalize and recycle, to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC-I bound peptides also can recycle was not known. We have investigated this by two different approaches. First, glycopeptide recycling was tested with Gal2 specific monoclonal antibodies. By flow cytometry, a return of Gal2 epitopes to the surface was found, after cellular internalization and cell surface clearance by pronase treatment (VI). Recycling may be important for MHC-I processing of exogenous antigens. Second, this was tested using two different forms ofthe H-2Db molecule expressed in transgenic mice, either transmembraneous (Db-tm) or with a glycophosphatidylinositol (GPI)-lipid anchor (Db-GPI). Only the tm form of Db was found to readily internalize and recycle glycopeptides to the cell surface. When transgenic mice were immunized with influenza A virus and tested for CTL responses against an immunodominant nucleoprotein epitope, only Db-tm mice were found to generate specific CTL responses (VII). We have found that some Kb binding glycopeptides generated CTL which could recognize the carbohydrate present in a glycolipid form, in an MHC-I unrestricted way. An interesting finding in the present investigation was that glycopeptide and glycolipid specific CTL were found to use different T cell receptors (TCR:s) to recognize and kill target cells (VIII).
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| 2. |
- Abdel-Motal, Ussama M., et al.
(författare)
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Major histocompatibility complex class I binding glycopeptides for the estimation of 'empty' class I molecules
- 1995
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Ingår i: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 188:1, s. 21-31
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Tidskriftsartikel (refereegranskat)abstract
- Different forms of major histocompatibility complex (MHC) class I heavy chains are known to be expressed on the cell surface, including molecules which are functionally 'empty'. Direct peptide binding to cells is obvious during sensitization of target cells in vitro for cytotoxic T lymphocyte killing and 'empty' MHC-I molecules are comparatively abundant on TAP- 1 2 peptide transporter mutant cells. In the present work we have estimated the fraction of 'empty' MHC class I molecules using glycosylated peptides and cellular staining with carbohydrate specific monoclonal antibodies. Synthetic Db and Kb binding peptides were coupled at different positions with different di- or trisaccharides, using different spacing between the carbohydrate and the peptide backbone. Binding of sugar specific mAbs was compared in ELISA and cellular assays. An optimal Db binding glycopeptide was used for comparative staining with anti-Db and anti-carbohydrate monoclonal antibodies to estimate fractions of 'empty' molecules on different T lymphoid cells. On activated normal T cells, a large fraction of Db molecules were found to be 'empty'. The functional cole of such 'empty' MHC class I molecules on T cells is presently unclear. However, on antigen presenting cells they might participate in the antigen presentation process.
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