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barcoding approach for dendritic cells differentiation

Final Report Summary - BARDIF (Barcoding approach for dendritic cells differentiation)

Through technological advancements made over the last decade, immune responses can now be studied at the single cell resolution, thereby revealing single cell output and differentiation patterns. The objective of the project was to make use of cellular barcoding, a single cell resolution technology developed by one of the two host labs, to study two aspects of the immune system: The production of different types of immune cells from progenitor cells, and the formation of T cell responses upon infection.

With respect to the first issue, a variety of different blood cells are formed through a process called hematopoiesis. The current model of hematopoiesis, which results from years of collective research, has led to a hierarchical tree, in which multipotent progenitors such as LMPP have the capacity to each yield most of the known blood cell types. However, this model is still contentious and is largely based on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single cell complexity / variability.

To address this issue, multipotent progenitors (LMPPs) were labelled such that each progenitor contained a unique and heritable genetic barcode. When these progenitors divide and differentiate after in vivo transfer, these barcodes are inherited by their daughter cells, allowing us to identify the progeny of many different progenitors within the same animal and thereby reconstitute their lineage relationships with single cell resolution. As noted above, the most commonly held model of hematopoiesis proposes that all LMPPs have the potential to generate all cell types. Our data show that this is not the case; instead, LMPPs produce heterogeneous patterns of limited types of blood cell types. Furthermore, in contrast to the presumed myeloid and lymphoid origin of dendritic cells, we found that many LMPPs produce several types of dendritic cells without producing any lymphoid and myeloid cells, thereby redefining dendritic cells as a third lineage of immune cells. These results challenge the current model of hematopoiesis and show the value of the combination of lineage tracing technology and mathematical modelling to address this type of issue.

With respect to the second issue, T cells form an essential component of our immune system that continuously scout the body for signs of infection. The combined pool of T cells needs to be able to detect a very large variety of possible infections. As a consequence, only very few T cells (a few hundred in mice) that can recognize a specific pathogen are initially present. Thus, it is essential that these few cells collectively create a large number of offspring that together can fight the infection at the moment a pathogen is first encountered. In our experiments, we used cellular barcoding to follow individual T cells and their offspring during infection. By counting the number of barcodes that is present at different times after infection, we exactly calculated how many daughters each activated T cell produces. Following infection with a bacterium or virus, we discovered that not all activated T cells contribute to the same extent at the same point in time. Whereas some T cell families grow out to enormous numbers, most other T cells that are activated following infection only create very few offspring. Small consolation for the latter, following renewed infection, they often do most of the work, by providing the capacity to respond vigorously at this point in time. These results, together with those from the group of Dirk Busch, reveal that the reproducibility of T cell responses that has for long been known is actually the consequence of the averaging of highly divergent behaviour of individual cells.

Both of these studies were the result of an interdisciplinary collaboration between the experimental team of Ton Schumacher (NKI, Amsterdam) and the theoretical team of Rob de Boer (Utrecht University, Utrecht). My joint appointment in these groups, supported by the Marie Curie Seventh Framework Programme (FP7) programme, provided a solid basis for this interdisciplinary collaboration. The data obtained also illustrate well how such interdisciplinary research can lead to high impact output, with publications in Science and Nature, and can contribute to our understanding of basic aspects of the immune system that could lead to therapeutic applications in the future.