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Defining the immunological interplay between fibroblastic reticular cells and anti-tumour T cells

Periodic Reporting for period 1 - FIBROCAN (Defining the immunological interplay between fibroblastic reticular cells and anti-tumour T cells)

Reporting period: 2016-07-01 to 2018-06-30

Our project was designed to answer the question of why and how particular cells called fibroblastic reticular cells (FRCs) found in lymph nodes allow metastasis to happen. The FRCs are the most frequent stromal cell (non-white blood cell) in the lymph node and recent work shows they heavily regulate immune cell activity and survival. We predicted that this regulation is exactly why tumour cells reaching lymph nodes escape detection and deletion. An important part of this detection and deletion process are the T cells that are present in the lymph nodes. T cells are specialized Immune cells that patrol their environment constantly and delete cells that are harmful such as cancer cells.
The persistent dogma has been that lymph nodes are a hostile environment for tumour cells. However, a variety of cancers preferentially metastasise to the lymph nodes that are close in proximity to the origin of the tumours. Research is slowly overturning the initial dogma by showing that lymph nodes are a site where T cell responses are censored. The reason for this is probably that the lymph node site can be rich in stimulants that would otherwise continuously activate the T cells and lead to chronic inflammation.
At the commencement of the project, the state of the art was that mouse FRCs prevent T cell activation in the lymph node by secreting signals that dampen the T cell response. Our research sought to test this observation in human systems and pose the following question in relation to tumour metastasis: if the escape mechanism of tumour cells is related to these FRCs, how does it occur, and can we identify new targets for therapies that would reverse the inhibitory effect that they place on T cells, to allow early deletion of metastatic tumour cells. This would be important to the wider community as it would open up a new range of therapies that target metastasis specifically and even perhaps allow preventative treatment in high risk patients.
Our overall objectives were to SO1: To chart the fate of anti-tumour T cells following suppression by FRCs. SO2: To identify tumour immunotherapy targets presented by tumour cells but not FRCs. SO3: To test therapeutic fibroblast populations for censorship of anti-tumour T cells.
Overall the fellowship was a successful endeavour and lasted a total of 15 months from the intended 24 months. I withdrew early as a result of being offered a prestigious position at Monash University in Australia, leading the Stromal Immunology research group, largely due to the work outlined in this report.

We have progressed through our milestones with effectiveness and have achieved SO1 and SO2 within the lifespan of the fellowship.

In our application, some proposed experiments were mouse-based, as we (and others) believed mouse models needed to be used to address some of our hypothesis. However, our work on human samples revealed that mechanisms in human FRCs were very different that those in mice. Therefore, we amended our plans to answer the same questions using human cells and systems, which in turn made our results more immediately applicable and has resulted in more significant outcomes. Our results can be summarized as follows:

1) We have shown that human T cell proliferation is effectively halted by human FRCs, as is activation and differentiation. This is novel.
2) We have established that human T cells are still fully functional after their encounter with FRCs and are able to activate to normal levels.
3) We found that inhibition by FRCs was induced by secreted factors as conditioned media alone is able to halt proliferation and activation.
4) We have determined 4 mechanisms that are responsible for suppression of activation of T cells by FRCs and have applied specific inhibitors for each one that partially reverse this effect but if applied together do so completely.
5) We have run RNA-sequencing for FRCs and MSCs to highlight their similarities and differences and applied that expertise in collaborations that have been recently published (Tamblyn et al. 2017 BioScientifica).
6) We have developed new assays to confirm in situ our observations and show proliferation of T cells after treatment in patient samples.
7) In recent days we have submitted our manuscript for publication that summarizes this work in a high impact journal and we are still developing further work for two more publications that will be the result of data generated by this work.
8) As a result of this fellowship I have obtained $1.3M AUD in funding and a job co-leading a laboratory, where I plan to continue the work I started with this fellowship.

Some of the work that was carried out was presented at the International Congress of Immunology (Melbourne, 2016) and an abstract published at the European Journal of Immunology (Knoblich et al. 2016). The work will also be presented at the next international T cell meeting (ThymOz, Heron Island, 2018). We have also recently submitted the work for publication (decision pending). As a result of this work I obtained a position as a Lab Head that allows me to carry on the work that was made possible by the Marie Curie fellowship. In addition, data generated during the fellowship will allow me to apply for future grants.
We have shown for the first time that human FRCs are able to halt proliferation and activation of T cells and that we can reverse this effect with a series of inhibitors. We have identified 4 individually druggable targets and as a result have begun discussions with industry. We intend to pursue development of novel therapeutics to target one or more of these inhibitors with the intent of developing a new drug, relevant to stimulating immunity in hard-to-immunise groups (eg the elderly, and cancer patients).

Uncovering the mechanisms involved drives the field forward as it allows future research to focus on delivering drugs that target these pathways exclusively. With the era of bispecific antibodies imminent, we are looking into delivering new therapeutic antibodies. This would most likely require us to team up with industry for impact.

In recent months we have had a lot of interest in our research from commercial entities and entrepreneurs experienced in fostering and funding start-ups, and are hopeful now that our research has matured this interest will lead to a full collaboration. Thanks to this funding from MSC, an exciting new avenue of therapeutically-relevant research is starting to unfold for us and our field, and we are glad to be part of it.
Tonsil tissue treated with stimulants and inhibitors shows increase of active cells (blue) ex vivo