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In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1), indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

A major group of murine inhibitory receptors on Natural Killer (NK) cells belong to the Ly49 receptor family and recognize MHC class I molecules. Infected or transformed target cells frequently downmodulate MHC class I molecules and can thus avoid CD8(+) T cell attack, but may at the same time develop NK cell sensitivity, due to failure to express inhibitory ligands for Ly49 receptors. The extent of MHC class I downregulation needed on normal cells to trigger NK cell effector functions is not known. In this study, we show that cells expressing MHC class I to levels well below half of the host level are tolerated in an in vivo assay in mice. Hemizygous expression (expression from only one allele) of MHC class I was sufficient to induce Ly49 receptor downmodulation on NK cells to a similar degree as homozygous expression, despite a strongly reduced cell surface level of MHC class I. Co-expression of weaker MHC class I ligands in the host did not have any further effect on the degree of Ly49 downmodulation. Furthermore, a single MHC class I allele could downmodulate up to three Ly49 receptors on individual NK cells. Only when NK cells simultaneously expressed several Ly49 receptors and hemizygous MHC class I levels, a putative threshold for Ly49 downmodulation was reached. Collectively, our findings suggest that in interactions between NK cells and normal untransformed cells, MHC class I molecules are in most cases expressed in excess compared to what is functionally needed to ensure self tolerance and to induce maximal Ly49 downmodulation. We speculate that the reason for this is to maintain a safety margin for otherwise normal, autologous cells over a range of MHC class I expression levels, in order to ensure robustness in NK cell tolerance.

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for "missing self" recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an "educating impact" on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.