Final Report Summary - NKG2D IN T-CELLS (Memory Control; The role of NKG2D and the T-cell receptor in memory T cell biology.) The immune system provides us with the first and only line of defense against infections with bacteria, viruses and parasites. Over the last century, medical science has made enormous progress in the prevention of infections by ‘teaching’ the immune system to recognize specific pathogens via vaccination. Vaccination generates immunity against pathogens before they are encountered and its application has prevented loss of countless lives due to infectious diseases. Despite these developments, immunity is a process that is incompletely understood and further insight in its biology may help us fight present day medical problems. An important cell subset that mediates immunological memory is the cytotoxic memory CD8 T cell. Upon infection, naïve CD8 T cells are activated and form effector and memory cells. Effector CD8 T cells recognize infected cells and neutralize these threats by eliminating their targets. When an infection is cleared, effector cells die but a small fraction remains as memory cells. These cells are able to rapidly convert to effector cells upon re-infection and provide long-term protection. Previously, people had observed that both effector and memory, but not naïve CD8 T cells express the protein NKG2D. This receptor had been recognized as an important mediator of the target-killing (cytotoxic) potential of effector CD8 T cells. ‘Stressed’ cells, such as virally infected cells, up regulate ligands of NKG2D. Up-regulation of this molecule triggers their killing by CD8 T cells. However, it was unclear what the role of this receptor was on memory CD8 T cellsIn this project we aimed to elucidate the role of NKG2D in the formation, maintenance and function of memory CD8 T cells. Moreover, we wanted to elucidate the molecular signaling pathways that NKG2D uses to mediate its effects. To answer our research question, we made use of wild type mice and animals deficient for NKG2D (NKG2D-/-). CD8 T cells of these mice were directly compared after infection with murine cytomegalovirus (mCMV) or lymphocytic choriomeningitis virus (LCMV). Our initial observation was that NKG2D-/- cells generate a quantitatively normal effector response upon infection with LCMV or mCMV. In contrast, at later time points after infection, when no replicating virus was left and effector cells had died, NKG2D deficient cells had formed markedly reduced numbers of memory cells. This reduction of effector cell numbers had a clear clinical impact: immunological memory formed by NKG2D-deficient CD8 T cells had a significantly reduced capacity to fight viral re-infection. Next, we investigated whether NKG2D-deficient CD8 T cells were functionally impaired on a per cell basis. NKG2D-/- memory CD8 T cells expanded equally compared to wild type cells upon reinfection. Moreover, equal numbers of NKG2D-/- memory CD8 T cells cleared virus equally well as wild type cells. In addition, homeostatic proliferation was equal between wild type and NKG2D-/- memory CD8 T cells. This suggested that NKG2D-deficiency impaired memory formation, rather than memory cell function. Indeed, when memory precursor cell subsets were investigated early after infection, we noted that NKG2D-/- cells had an impaired ability to form central memory (TCM) precursor cells. Since TCM cells give rise to long-term memory, this observation is potentially of clinical implication. To elucidate how NKG2D mediates its effect on memory cell formation, we investigated various signaling pathways. We observed that NKG2D-mediated memory cell formation depends on the adaptor molecule DAP10. In addition, we found that NKG2D potentiates responsiveness to IL-15 an important cytokine that promotes memory cell formation. However, proliferation of memory precursors was not affected by NKG2D-deficiency, which indicated that this molecule enhances precursor cell survival. We generated a new in vitro memory differentiation model to further interrogate NKG2D-mediated signaling. We found that NKG2D enhances survival of memory precursors, after effector cell differentiation, but before terminal memory cell differentiation. We could confirm this model using an in vivo transfer model. On a molecular level, we could show that NKG2D-ligation potentiates IL-15 mediated signaling towards the important signaling molecule PI3K. We could also show that PI3K signaling was essential for the formation of memory cells during the same phase during which NKG2D mediates its effects. Finally, we demonstrated that NKG2D-signaling increases protein levels of the important pro-survival protein Mcl-1 by enhancing PI3K signaling. In summary, our findings show a new role for PI3K and the NKG2D/IL-15 axis in an underappreciated stage of effector to memory cell transition that is essential for the generation of antiviral immunity. Moreover, we provide novel insights how these receptors control both effector and memory T cell differentiation. Our findings have an impact on several levels. First, we show an important new function of an ‘old’ receptor. Many therapies are currently under development to enhance cytotoxic activity of CD8 T cells to virus-infected or tumor cells by targeting NKG2D. Our findings indicate that these therapies have the additional advantage that they promote memory cell formation against these targets. Second, we show that PI3K plays a detrimental role in the formation of memory cell formation, whereas current dogma states that PI3K activity promotes effector differentiation and impairs memory cell formation. This finding may have important implications, since inhibition of the PI3K/mTOR axis is currently under investigation as a strategy to promote memory formation upon vaccination. Our observations suggest that such therapies might be counterproductive when applied in the wrong timeframe. Finally, we provide better insight in the stages of memory cell formation during which various important signaling cascades mediate non-redundant functions. We demonstrate that NKG2D/IL-15 mediated survival signaling is crucial for the survival of memory precursors, during a phase during which IL-2 and IL-7 signaling are low. Therapies that aim to use either IL-2 or IL7 in order to enhance vaccine-induced immunity may therefore benefit greatly from the application of IL-15 during a crucial stage of memory cell formation. In addition to our scientific achievements, we have more than accomplished the knowledge-transfer goals described in the proposal. The fellow has gained much appreciated technological knowledge on the generation and use of genetically modified animals. He has been actively involved in the day-to-day supervision of two PhD. Students and has led the work-discussion sessions of the department. As such, he has obtained much professional maturity. Finally, the fellow has written several new research proposals and, together with Prof. Polić, has been awarded two research grants. Thus, this MC project has added much to the scientific and management skills of the fellow. In return, the fellow has contributed much to the host institution. In addition to supervising Ph.D. students, he has given several lectures to undergraduate and graduate students. Also, he has set-up and taught a work-shop on small animal imaging, as a part of the FP7 TransMedri program. Finally, he has educated students in various experimental techniques, and has actively participated in the development of research projects of his colleagues. Taken together, we conclude that this Marie Curie project has reached most, if not all of its objectives, both on a scientific and a socio-economical level. We therefore believe that this MC-IEF grant was a profitable investment in the career of an excellent European scientist.