Studies of cytotoxic T lymphocytes and natural killer cells in relation to MHC class I presented peptides

Sammanfattning: In the defense against disease it is essential for the immune system to be able to discriminate self/normal from non-self/abnormal. This key feature of the immune system relies on the presentation of antigens by MHC class I and class II molecules. MHC class I molecules present short peptides derived from the degradiation of proteins passing or resident in the cytoplasm. MHC class I molecules are expressed by all nucleated cells in the body, and they are scrutinized by CD8+ cytotoxic T Iymphocytes (CTL) and Natural Killer (NK) cells. To study T-cell mediated immume responses in transplantation and cancer, I established a biochemical technique to isolate naturally processed peptide antigens, adopted from related work on MHC class I and class II presented peptides in the literature. Application of this technique to a murine tumor model identified one epitope. In two murine minor histocompatibility antigen (MiHa) systems, CTL generated against complex antigenic challenges recognized only three immunodominant epitopes respectively. However, CTL generated against single MiHa identified three novel thus subdominant/cryptic epitopes, and also allowed us to assign one immunodominant epitope to a known MiHa locus. The data suggest that dominance mechanisms in this case operate distal to the level of antigen processing/presentation. We could use minute amounts of naturally processed peptides loaded on TAP2 deficient murine RMA-S Iymphoma cells to prime for cytotoxic T cell responses in vivo, a strategy of possible use in the clinic. To develop the biochemical approach, we applied capillary zone electrophoresis (CZE) to resolve the peptides in identified rpHPLC fractions for further characterization. Application of these techniques in combination with mass spectrometry have so far generated two partial and one full peptide sequence, ESFSDYPPL (derived from elongation factor lalpha), used in our studies of MHC class I mediated resistance to NK cells. Summarizing our own experiences and the literature regarding the search for and characterization of antigenic peptides as well as the studies of immume responses to complex antigenic challenges, it can generally be recommended: 1) For known proteins - generate and screen peptides libraries. 2) For unknown proteins - generate and screen cDNA libraries from antigen expressing cells. 3) For combinations of unknown antigenic sequences - screen eluted naturally processed peptides, and also consider "4)". 4) Create a defined system by using synthetic peptides of known antigenic sequences. Natural killer cells detect "missing self', i.e. the failure of target cells to express adequate types and levels of MHC class I molecules at the cell surface. We could show for the first time that restoration of TAP (transporter associated with antigen presentation) function in RMA-S cells results in reduced NK sensitivity, and increased tumorigenicity. We also demonstrated that transduction or transfection of the murine wild type Iymphoma, RMA, with Interleukin-10 impaired antigen presentation, increased NK sensitivity, and decreased sensitivity to CTL. Thus, (patho-)physiological regulation can control both CTL and NK cell responses; mutations or other gross defects affecting MHC class I expression are not required. Further, we studied peptide induced resistance to NK cells. The naturally processed and H-2Kb presented "self" peptide, ESFSDYPPL (as described above) did not protect, whereas the strongly immumogenic chicken ovalbumin derived "non-self" peptide SIINFEKL conferred strong protection to Iysis mediated by NK cells expressing the murine inhibitory receptor Ly-49C. Protection was thus not limited to specific self peptides. The further analysis based on modified peptides suggest that inhibitory receptors interact with conformational epitopes on MHC class I molecules, possibly encompassing the bound peptide. While IL-2 activated NK cells incubated with antibodies specific for Ly-49C or MHC class I killed MHC class I expressing tumor cells more efficiently, we surprisingly found that they kill MHC class I deficient targets worse than untreated effector cells. These and other data suggest that MHC class I expression on the NK cell itself may affect MHC class I specific receptors/function. Finally, we could induce cytotoxic and cytokine (IFN-y) responses by local NK cells by intraperitoneal tumor cell challenges. This was observed specifically in situations where inoculated tumor cells were deficient in TAP- or ß2m-expression or mismatched vis-a-vis the host with respect to MHC class I expression, and suggest an adaptive component in the NK response. Generally, the main fimctions of natural killer cells may be to participate in the combat of infections and in certain cases malignancy, surveil the peptide surveillance machinery by eliminating antigen presentation escape phenotypes, and possibly regulate adaptive immune responses by eliminating activated APC.