Memory control; Molecular mechanisms underlying the scope of immunological memory

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

Research objectives:
Immunological memory, i.e. immunity, provides us with protection against a second infection with a given pathogen. During the formation of memory, specificity must be balanced with efficiency. When memory is highly specific, it is also highly efficient, but only able to recognize a limited number of variants of the original pathogen. Vice versa, decreased specificity provides broader protection, but the efficiency per variant of the pathogen is reduced. 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. Research line one aimed to generate a new mouse model (CHT) with which we could specifically eliminate genes in cells that form immunological memory. Research line two aimed to identify regulators of diversity on B cells, which are the cells that generate antibodies that protect us against extracellular pathogens. Research line three aimed to identify regulators of diversity of memory CD8 T cells, which are the cells that provide protection against viral infection and tumors.

Description of Work:
The CHT model depends on the combination of two transgenes which are specific for memory cells. Only when both transgenes are expressed subsequently, a third gene is targeted. To achieve this goal, two transgenic alleles were generated in two separate mouse lines. These alleles can be combined in one animal through simple crossbreeding. Classical gene targeting techniques were used to achieve our goals.
To identify master regulators of diversity in activated B cells, we first generated new in vitro and in vivo models to allow us to study the impact of pathogen (antigen) specificity on B cell responses in an isolated way, in collaboration with Prof. Robert Brink at the Garvan institute in Sydney. Using these in vitro models, we successfully identified a key factor involved in elimination of low-affinity B cells in the first days after antigen encounter. Subsequently, we generated the appropriate in vivo models to confirm our findings in research animals.
To identify master regulators of diversity in 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 was performed to eliminate gene X specifically in T cells, or in an inducible fashion. In collaboration with Prof. Lemmermann of the Gutenberg University in Mainz, new mutated virus strains were generated to study the impact of antigen-affinity on memory cell formation in vivo. Our new mouse and viral models were combined to confirm the role of gene X in memory cell formation. Finally, we used high-throughput screening to identify the molecular mechanism via which gene X mediates memory cell diversity and could therapeutically target this mechanism to increase memory cell specificity.

Description of results:
For research line one, the transgenes were successfully produced in collaboration with Prof. Polić of the University of Rijeka and transgenic mice were generated close to the end of the CIG period. Animals are viable and are currently being tested for functionality of the transgenes.
Research line two has identified the master regulator of early activated B cell survival. We found that, in the first two days after activation, B cells depend on the cytokine BAFF for survival. Expression of BAFF receptors was induced in a B cell receptor-affinity dependent fashion, resulting in a survival advantage for high-affinity cells. Affinity-dependent expression of BAFF receptors mediated differential PI3K activation in response to BAFF stimulation, which controls levels of the pro-survival protein Mcl-1. Inhibition of PI3K lowered Mcl-1 protein levels and negated survival differences between B cells of high and low affinity. In the presence of excess BAFF protein, or in absence of the Mcl-1 antagonist Noxa, 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. In addition, our work on B cells has contributed to a project, which addressed the role of NKG2D in B1a cell formation. This work has been published in the Journal of Immunology by our collaborators.
Research line three has identified the master regulator of memory CD8 T cell diversity. In vitro characterization of memory precursors of high and low affinity revealed that the latter cells have reduced proliferative capacity, but 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. Gene X is induced upon T cell activation, but its expression is suppressed by gene Z in cells activated with high intensity. Mice conditionally lacking gene X in T cells therefore have a strong reduction in memory cell diversity, as a result of apoptosis of low-affinity memory precursors. We confirmed this finding using our new viral models. Finally, using our high-throughput screening model, we identified the key effector protein (protein Y) via which gene X mediates its effects. Using drugs that specifically target gene Y, we could show its importance for the control of memory cell diversity. Using animals lacking gene X, we could show that this drug had no additional impact on memory cell diversity, demonstrating that gene X functions via gene Y. Research line three gave rise to an additional line of inquiry in which we identified NKG2D as an important co-stimulatory molecule for effector T cell formation.

Overall conclusions and socio-economic impact:
The CHT model is a revolutionary in vivo system that opens up new ways to investigate the role of genes in biological processes. For us it allows a research platform which we can use to answer many new research questions in the future. We envision that the CHT template reaches beyond immunology and will help answer future research questions in many different fields of study.
We envision that our work on B cells will contribute to the development of vaccines with a broader scope of the immunological memory that it generates. This will provide protection against more strains of a given pathogen against which is vaccinated. Our findings are published in Scientific Reports.
Our work on T cells 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. Our proof-of-principle experiments using EMA-approved drugs shows that this approach is feasible. 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. A minor part of our work is accepted for publication in the European Journal of Immunology. The major part is approaching completion and a manuscript is planned to be submitted within the next two months.

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

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