Binding and immunogenicity of MHC class-I specific peptides and glycopeptides

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).