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Development and immunological control of dendritic cell cancer

Periodic Reporting for period 1 - DC cancer (Development and immunological control of dendritic cell cancer)

Reporting period: 2015-05-01 to 2017-04-30

Dendritic cells (DCs) are powerful cells of the innate immune system that are central to immunity against infection and cancer. This prominent role of DCs in the initiation of immune responses has led to the hypothesis that the immunological potential of DCs hinders DC cancer development. Diagnosis of DC cancer in humans is complicated by the fact that histiocytosis – defined as a hyperplasia of APC like DC or macrophages - can be a consequence of immunological rather than neoplastic transformation. Recent reports suggest that DC cancer can indeed form in humans and account for some of the histiocytic malignancies observed in the clinic. However, the steps that occur during DC cancer development have not been studied so far, due to the lack of appropriate models. We thus generated a novel, genetically engineered cancer model resulting in spontaneous neoplastic transformation in cells of the DC lineage in mice. These Clec9a Kras-G12D mice allowed us to characterise DC cancer formation and study the immunological consequences of neoplastic transformation of DCs and DC precursors. Our work establishes that oncogenic transformation of DCs leads to rapid development of DC cancer in multiple organs with 100% penetrance. This development of DC cancer could be induced by a small number of cells and was not prevented in the presence of untransformed DCs. Furthermore, DC cancer developed despite a lack of overt immune function and transformed DCs could be sufficiently activated upon stimulation. Importantly, DC cancer cells were immunogenic upon transplantation into immune competent mice despite their ability to grow unabated in the original host, thus indicating that immunological control of DC cancer is in principle possible but does not occur during its spontaneous generation. In summary, our data provide a detailed characterisation of DC cancer development and immunological properties of transformed DCs. DC precursors, if transformed, can give rise to haematological malignancy in mice, raising the question of whether this might also occur in some circumstances in humans and might be the underlying cause of certain histiocytic pathologies observed in human patients.
Main results

We generated a new genetically-engineered cancer mouse model that makes use of the recent finding by the host lab that the Clec9a gene is expressed specifically within cells of the DC lineage. We used this model to study the aetiology of DC cancer development. DC cancer was observed with 100% penetrance in Clec9a Kras-G12D mice, which all succumbed to neoplastic transformation within the DC lineage. Flow cytometric analyses over time established that high accumulation of transformed DCs occurred in multiple lymphoid and non-lymphoid organs and was evident early in life, from week 4 after birth. DC cancer formation showed 100% penetrance in mice and we did only observe minor heterogeneity between individual mice. However, further analyses revealed that cancer formation is dominated by the CD8a+ subset of DCs, which accumulated over time, indicating that this subset has a stronger potential to form tumors in Clec9a Kras-G12D mice.
We additionally analysed whether oncogenic transformation of DCs affects their immunological phenotype and function. Cancer DCs did not show signs of activation but could be activated by stimulation, evident by the upregulation of costimulatory markers and cytokine production. Furthermore, in vitro culture assays with T cells established that cancer DCs were capable to induce T cell activation and stimulation, suggesting that DCs in Clec9a Kras-G12D mice retained a normal phenotype and functional ability despite their oncogenic transformation. Furthermore, we did not detect changes in the immune cell repertoire in Clec9a Kras-G12D mice, suggesting that cancer DCs are immunologically invisible and ignored by the immune system. This was supported by the finding that crossing Clec9a Kras-G12D mice to Rag1-/- mice did not show accelerated DC cancer development, despite T cell deficiency. However, adoptive transfer of cancer DCs into immune competent mice resulted in immune rejection, an important finding with implications for DC cancer therapy. We complemented our analyses of T cells by analysing further potential immunological barriers that could hinder DC tumor development. To this end, we generated bone marrow chimeric mice in which neoplastic transformation is restricted to a very low frequency of transformed DC. In these experiments, we discovered that a small number of transformed DC precursors is sufficient to evoke rapid DC cancer development and DC cancer development can occur in the presence of untransformed DC.
Taken together, our data provide an in-depth characterisation and fundamental insights into the biology of DC cancer development. Our experiments establish that DC cancer formation is not controlled by immunological barriers in mice, and DC cancer cells seem to be ignored by the immune system. These findings have important implications for the design of targeted immunotherapy against DC tumors.

Dissemination of results
Most of the results of this project have been published in a peer-reviewed manuscript in the Journal of Immunology (See uploaded publication).
Furthermore, results from this project have been disseminated to the scientific community by several oral presentations at national and international meetings.

Further exploitation
This project allowed us to generate several DC cancer cell lines, which are available as a biological tool to the scientific community and already used by the several labs at the Francis Crick Institute.
Furthermore, Clec9a Kras-G12D mice are available as a tool to study the formation of DC cancer. In addition to this and beyond the original proposal, these mice have been crossed to Batf3-/- mice, allowing to study neoplastic transformation of CD11b+ DCs; and Rag1-/-Clec9a Kras-G12D mice to study immune-editing of DC cancer in future projects.
Firstly, work from this project proposal allowed me to acquire skills and expertise in the field of cancer biology and immunology. The skills and expertise I gained during the last two years of work as a postdoc within the scientific environment at the Francis Crick Institute are reflected in several projects published in highly renowned journals: with Julie Helft, I investigated the development and biology of DC subsets in in vitro culture systems (Helft, Böttcher et al., Immunity 2015 and Helft, Böttcher et al., Immunity 2016). Furthermore, with Santiago Zelenay I investigated immune evasion mechanisms of cancer cells and the suppression of dendritic cells by PGE2 production (Zelenay, Van der Veen, Böttcher et al., Cell, 2015).

Secondly, my postdoctoral work on DCs and cancer allowed me to start a follow up project in which I am investigating the mechanisms that cancers use to impair DCs within tumours. This project was recently submitted for publication.

Thirdly and most importantly, the funding of my postdoctoral work in Caetano Reis e Sousa’s lab allowed me to develop my own area of research interest within the field of cancer immunology. The unique combination of expertise, tools and techniques I acquired during my research as a Marie Sklodowska-Curie fellow ideally positioned me to reach the next level of professional maturity and I successfully acquired grant money for a 5-year junior research group at the TU Munich, Germany, starting next year.