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MEMORY CONTROL Résumé de rapport

Project ID: 630827
Financé au titre de: FP7-PEOPLE
Pays: Netherlands

Periodic Report Summary 1 - MEMORY CONTROL (Memory control; Molecular mechanisms underlying the scope of immunological memory)

Research objectives:

The overall goal of this research project was to identify master regulators that control the diversity of antigen-specific cells of the adaptive immune system in response to infection or immunization. We focused (but not restricted) our research efforts to memory CD8 T cells. Our research plan contained three primary objectives: (1) generation of a new mouse model (CHT) to study memory CD8 T cell formation, (2) identification and validation of master regulators of diversity in mouse cells and (3) identification and validation of master regulators of diversity in human cells.

Description of Work:

The budget made available by the CIG was mostly applied to outsource the work required to achieve research objective 1, to our collaborators of the University of Rijeka. At the time of writing, the complex cloning required for the generation targeting constructs has been completed. The generated DNA is currently being used to generate targeted ES cells. We expect to have CHT mice available for experiments within six months. Budget to cover their costs will be allocated to them in the near future.
Research objective 2 has been segregated in two separate lines of research. The first research line focuses on the identification of master regulators of diversity in activated B cells during the first days after antigen encounter. To identify these factors, we generated new in vitro and in vivo models and combined up to five genetically modified alleles in a single mouse through breeding, to answer our research questions. We successfully identified a key factor involved in elimination of low-affinity B cells in the first days after antigen encounter. This research line has been completed successfully and a manuscript is currently under revision for Nature Communications. The second line of research tries to identify master regulators of diversity of memory CD8 T cells. We have invested a significant amount of time to establish mouse models and in vitro culture systems that allowed us to characterize the impact of antigen-affinity on memory CD8 T cell formation. Moreover, it provided us with a model system in which we could screen for potential targets involved in memory cell diversification. High-throughput analysis of differential gene expression in our culture system identified several potential candidates. Validation prompted us to continue with one particular gene of interest (gene X). Mice with floxed alleles for Gene X were obtained from the Jackson Laboratories and complex breeding schedules were set-up to specifically eliminate this gene in memory precursor cells. In collaboration with the Johannes Gutenberg University in Mainz, new mutated virus strains were generated in order to study the impact of antigen-affinity on memory cell formation in vivo. Currently, we are combining our mouse models with viral tools to confirm the role of gene X in memory cell formation. Following validation of the role of gene X, we will confirm whether the murine situation applies to human cells, as indicated in research objective 3.

Description of main results:

The work that addresses research objective 1 is in the process of generating our new mouse models. We expect to have mice available for experiments within the next six months.
Research line 1 of research objective 2 has indeed identified a master regulator of early activated B cell survival. We found that, in the first two days after activation, B cells depend on cytokine Y for survival. Expression of receptors for cytokine Y was induced in a B cell receptor-affinity dependent fashion, resulting in a survival advantage for high-affinity cells. Affinity-dependent expression of Y receptors mediated differential PI3K activation in response to cytokine Y, which controls levels of the pro-survival protein Z. Inhibition of PI3K lowered Z protein levels and negated survival differences between B cells of high and low affinity. In the presence of excess protein Y, or in absence of the protein Z antagonist X, more low-affinity B cells survived the first two days after antigen encounter due to relaxed negative selection. This resulted in increased cell numbers, but reduced overall affinity of the B cells that contributed to the germinal center reaction. Combined, our findings elucidate a crucial molecular pathway of antigen-affinity dependent selection in the earliest phase of B cell activation.
Research line 2 has identified a potential master regulator of memory CD8 T cell diversity, but requires additional experiments to validate its role and identify the molecular mechanism via which it mediates its effects. In vitro characterization of memory precursors of high and low affinity revealed that the latter cells have reduced proliferative capacity, but demonstrate increased survival compared to high-affinity cells. In contrast, low-affinity effector cells have both reduced proliferative and survival capacity. When in direct competition, the effector cell response is therefore dominated by high-affinity cells, whereas the memory cell pool is much more diverse. Comparison of transcriptional profiles using high-throughput screening methods between high-and low affinity cells during various stages of memory T cell formation identified a specific transcription factor (gene X) of particular interest. Validation of our screening confirmed the importance of this molecule and mice were obtained in which its gene was floxed. Subsequently, these animals were backcrossed on various Cre-expressing lines to eliminate its expression at various stages of T cell development. In parallel, we generated new model viruses that can be used to present antigen of high- and low affinity to these cells. Currently, we are in the process of confirming the role of gene X in the survival of low-affinity memory cells and measuring its impact on fighting re-infection with antigens of high- and low affinity. Research objective 3 will commence as soon as we have confirmed the role of gene X in memory cell diversification in vivo.

Description of expected final results:

The CHT mouse represents a revolutionary mouse model that opens up new ways to investigate the role of genes in biological processes. This particular CHT model allows study of memory CD8 T cells. For us it allows a research platform which we can use to answer many new research questions in the future. However, we envision that the CHT template reaches beyond immunology and will help answer future research questions in many different fields of study.
Our work on B cells is mostly finished and we do not expect our main conclusions to change dramatically. Our findings are primarily of scientific interest, since it identifies a new phase of B cell selection and future research is required in order for it to be used in therapeutic settings. Nevertheless, we envision that our findings may contribute to vaccination strategies that aim to broaden the scope of vaccines, thereby providing protection against more strains of a given pathogen against which is vaccinated.
Our work on T cells is still ongoing, but has promising leads towards reaching its final goals regarding the identification of master regulators of memory T cell formation. Again, our work is mostly fundamental by nature and has no direct practical applications. Nevertheless, similar to our B cell work, we expect that our T cell project will lead to the ability of broadening or narrowing memory CD8 T cell responses. The application of these therapies will differ from our B cell work, because CD8 T cells target intracellular pathogens and cancer cells. The field of personalized therapies that use autologous CD8 T cells to treat cancer is currently rapidly developing. We therefore envision that our work will provide a great contribution to enhancing its potency.


Edwin Groenewegen van Wijk, (Project controller EU subsidies)
Tél.: +31205660075
Fax: +31205669698


Life Sciences
Numéro d'enregistrement: 183853 / Dernière mise à jour le: 2016-06-13
Source d'information: SESAM