Dissecting the dynamic changes in the innate immune cells of tumours to enhance immune checkpoint blockade therapies

Aims
We will utilise novel models of in vivo cellular migration to reveal new understanding of tumour infiltrating immune cells. Focusing on the innate immune response, we will establish a detailed order of how the innate composition of the tumour microenvironment is established and how it changes both over time and in response to different therapeutic interventions targeting T cell checkpoint mechanisms.
Background
The generation of photoconvertible Kaede transgenic mice where violet light can label cells at a specific site has enabled direct assessment of cellular migration in vivo [1]. We recently established an approach to temporally label all the cells within a peripheral LN and then assess cellular migration into and out of this tissue (Figure 1) [2]. Through photoconversion of a tumour draining LN or the tumour itself, we reason that TILs and other immune cells can be specifically labelled in a temporal manner providing a novel and superior means of assessing this population. Specifically, cells that had only recently entered the tissue could be distinguished and dynamic phenotypic changes after tumour entry could be determined. Furthermore, this system can then be utilised to compare dynamic changes induced by current treatments and with understanding the surface expression changes in different molecules, devise new combination strategies to enhance anti-tumour responses. Innate immune cell populations in tumours contribute to an immunosuppressive
environment. Several studies have identified different innate lymphoid cell (ILC) populations in tumours [3-5], but whether these cells are beneficial or suppressive is far from clear [6, 7]. Furthermore, ILC expression of molecules such as PDL1 and PD1 indicates these cells may be affected by checkpoint inhibition. Beyond ILCs, other innate immune cell populations such as MDSCs are considered key cells in limiting anti-tumour responses.
Hypothesis
We hypothesise that assessing dynamic changes in innate cell populations and their function within the tumour microenvironment will enhance the understanding of the mechanisms driving the effectiveness of immune checkpoint blockade therapies.
Experimental Methods and Research Plan
Characterising innate cell dynamics:
Initial experiments will assess the migration and retention of innate immune cells in syngeneic tumours following the injection of cell lines subcutaneously at a single site on the flank of Kaede mice. Three tumour cell lines: B16/F10, MC38 and CT26 which show differing T and innate cell infiltrations and differing responsiveness to checkpoint inhibition have been selected [8]. The inguinal LN or the tumour itself will be photoconverted (Figure 1 shows how the entire cellular compartment of a peripheral lymph node can be labelled) to track migration into and out of the tumour [1]. Standardised cell lines and validated protocols will be provided by MedImmune. We will then extend these studies to primary tumours building on existing collaborations (Awen Gallimore, Cardiff; Jessica Strid, Imperial). We will develop the use of new Dendra photoconvertible mice which show a prolonged label half-life (collaboration with Oliver Pabst, Aachen). Analysis of immune cell populations will utilise flow cytometry and immunofluorescence approaches.
Checkpoint blockade therapies:
Having characterised the innate immune cell infiltrates, we will then dissect the effects of checkpoint blockade strategies. Current (anti-PDL1 and/or anti-CTLA4 blockade) and novel (T cell agonists e.g.
anti-OX40) approaches being developed by Medimmune will be employed providing critical new data on their effects in vivo.
Expected outcomes and Impact
The findings of the study will 1) build understanding of the dynamic changes in innate populations that contribute to an immunosuppressive tumour environment, and 2) further our understanding of how immunomodulatory therapies alter the suppressive tumour microenvironment.