Abstract

Immunotherapy has shifted the paradigm of cancer treatment from directly targeting tumor cells to empowering the immune system to reject them. Most of these treatments, like immune checkpoint blockades, have focused on Tumor-Infiltrating T cells (TILs). However, considering the limited - yet significant-  response rates, these approaches need to be improved. One way could be to focus on the significant pool of Tumor-Associated Macrophages (TAMs) contained in the Tumor Microenvironment (TME). TIL and TAM have usually been considered to have antagonistic functions: TIL perform antigen-mediated cytotoxicity while TAMs suppress their activity and facilitate progression. Over the past few years, this view has been challenged by the capacity of immunotherapy to reprogram TAM functions. Using lung and breast cancer models in mice, our team previously reported that TAM were necessary alongside CD8+ TIL for tumor regression induced by type-I-interferon (IFN)-dependent immunotherapy.

Our objective was to further explore the parameters controlling this synergistic dialogue between TAM and CD8+ TIL. To do so we developed a flexible in vitro coculture system, to measure multiple parameters that could be associated with cell/cell cooperation. We monitored the capacity of anti-Kb/OVA-specific CD8+ T cells (OT-I) to kill B16OVA tumor cells, in the presence of bone-marrow-derived macrophages (BMDM), activated with a STING agonist to induce type I interferon (IFN-I). To get a grasp of the highly time-regulated interactions at stake, our system was multiplexed using a robotic cell culture platform, coupled to a custom-designed Python pipeline, to monitor at serial time points the killing, phenotype and cytokine secretion.

We first showed that a cooperation for tumor cell killing was taking place between LT CD8+ and macrophages in vitro. Enhanced killing was associated with a reciprocal activation of both partners with a rise in IFNγ release by CD8+ T cells and the upregulation the secretion of IL-12p70 by macrophages. Complete activation of BMDM in that context required contact with T cells. This effect was dependent on the reception of an IFN-I signal in macrophages.

We also reported that macrophages could help T cells functions in suboptimal conditions, by improving the speed of IFNγ production when tumors have downregulated MHCI-I expression, a common mechanism of resistance that we observed in vivo in our MMTV-PyMT breast cancer model, even during tumor regression.

Additionally, we focused on the possible therapeutic application of such cooperation by challenging the capacity of macrophages to enhance CAR-T cell antitumor activity. Our findings suggest that leveraging macrophages and T cell cooperation may offer promising CAR-T cell enhancement strategies, using autologous macrophages to boost anti-tumor efficacy.

Overall, this PhD project has provided insights into the parameters promoting synergistic activity between T cells and TAM after immunotherapy.

Keywords: Cancer Immunotherapy, Macrophage, T lymphocyte, CAR-T cells, TIL, TAM, Cellular cooperation