Final Report Summary - IFIMAP (Investigating the Functional Implications of MICA Polymorphisms) Over the past few years, it has become incredibly clear that the epithelium senses and responds to a wide variety of sterile and infectious insults which is then relayed to various components of the immune system, which we termed the lymphoid stress surveillance response (LSSR).In both mice and humans, a major component of the LSSR is the NKG2D axis, comprising the activating NKG2D receptor expressed by several immune subsets such as NK cells and gamma delta T cells, and its ligands (NKG2DL) expressed by stressed cells.Interestingly, these ligands are multiple (MIC and ULBP families in humans, Rae, H60 and Mult-1 in mice) and some of them display a high degree of polymorphism in humans. Although this diversity has probably emerged because of a constant arms race with pathogens (many of which target this pathway to achieve immune evasion), the resulting functional impact of these characteristics on the response of NKG2D-expressing immune cells are poorly understood.We are thus trying to clarify this issue with a particular focus on MICA, a human NKD2DL for which over 70 alleles have been described thus far.Before the start of the fellow's appointment, four different alleles of MICA were cloned and transduced into a Chinese Hamster cell line (CHO) so as to prevent the potential influence of other molecules on human immune cell responses. A novel genetic system of transduction leading to a single integration in a single region of the target cell genome was used in order to ensure a homogeneous expression of the transgene. Surprisingly, the expression levels for the four alleles were very different. As this should be prevented by the system we used, we concluded that different alleles might intrinsically have different expression potentials.Next, we monitored the responses of various immune cell subsets to these transgenic cells. Interestingly, various healthy donors displayed very different responses to each allele. For instance, some donors would respond equally to all alleles while others would preferentially respond to only two of them. We could also observe a response from both NK and gammadelta T cells, but never from CD8 T cells, suggesting that NKG2D triggering alone is not sufficient to elicit a response from the latter subset.The molecular mechanism driving a specific response to different alleles is likely to be complex and influenced by several parameters. However, as we observed that different alleles had different expression levels, we next investigated whether this parameter could at least partially explain the preferential responses observed for different donors. Indeed, we could show that manipulating the expression level of the different alleles could modify the hierarchies of responses of several donors. We have thus concluded that the observed variety in the response of different donors is at least partially determined by different 'tuning' levels, that is, cells from different donors are set to respond to a particular range of MICA expression level - anything above or below this prefered threshold will not trigger a functional response. This concept has recently been explored by other laboratories for other receptors of the immune system and has strong implications for our understanding of immune evasion and the pathophysiology of immune responses.As the expression of NKG2DL potentially has a complex impact on the outcome of a functional response, we next decided to re-focus the rest of the allocated time and resources for this project on a better understanding of the mechanisms regulating this expression, which are still ill-defined. While many different types of stress, either sterile or pathogens, are known to upregulate NKG2DL, the molecular pathways involved are poorly characterised. UVB irradiation and other physical or chemical stresses have been suggested to act through the activation of the DNA damage repair (DDR) pathway, by inducing the transcription of NKG2DL. Of note, while healthy tissues generally express NKG2DL at the mRNA level, cell surface expression of the protein is rarely detected, suggesting a tight post-transcriptional regulation.Preliminary results obtained several years ago by two post-doctoral fellows in the laboratory suggested that UVB irradiation could lead to the transactivation of the epidermal growth factor receptor (EGFR), leading to an increase in NKG2DL expression via the stabilisation of their mRNA, independent of transcription and the DDR We have thus decided to further explore these results to improve our understanding of NKG2DL regulation. First, we reproduced these preliminary results and also established that the NKG2DL upregulation triggered by UVB or EGF was sufficient to increase the response of both NK and gammadelta T cells to target cells after such treatments. In addition, we have elucidated the precise molecular mechanism responsible for the stabilisation of NKG2DL mRNA downstream of the EGFR. We have identified one or more (depending on the NKG2DL considered) classical Au-rich element type sequences in the 3' untranslated region of NKG2DL mRNAs. These sequences are known to be recognised by a variety of RNA-binding proteins which regulate the stability of mRNAS bearing such sequences. Using several reporter systems, we could demonstrate that such sequences in the 3'UTR of MICA are responsible for the poor stability of the mRNA. We have identified two previously described RNA-binding factors responsible for the regulation of NKG2DL (namely AUF1 and HuR) and dissected the precise signalling pathways downstream of the EGFR responsible for the inactivation of these factors and the consequent upregulation of NKG2DL.Overall, both projects have contributed to our understanding of the NKG2D axis, by describing and partially explaining the variety of responses to NKG2DL polymorphism and by completing our current knowledge of the regulation of these stress-induced proteins. As NKG2D and its ligands play an important role in the immune responses to various stresses and infectious agents as well as in diverse pathophysiological scenarios such as auto-immunity and transplantation, our work could potentially have a strong impact on the development of new clinical strategies.