Understanding Cytokine-Driven Differentiation and Proliferation of Immunosuppressive Myeloid Cells for Targeting with Innovative Therapeutic Approaches

Numerous solid tumors exhibit a highly immunosuppressive tumor microenvironment (TME), which results in a significant deficiency or absence of effector T cells within the tumor site. Such "cold" tumors significantly challenge the efficacy of immunotherapy, as they lack the ability to generate or maintain an adaptive immune response. In particular, many brain tumors – i.e. Glioblastomas Multiforme (GBM) – are highly infiltrated by myeloid cells, which exert immunosuppressive functions supporting tumor aggressiveness. Our research team has identified a significant presence of immunosuppressive bone marrow-derived macrophages (BMDMs) within the TME of GBM, alongside myeloid-derived suppressor cells (MDSCs), which play a crucial role among the immune cell population infiltrating this microenvironment. These cells contribute significantly to creating an immunosuppressive environment that allows cancer cells to escape immune surveillance and elimination. Moreover, recent findings from Merad et al. 2024 indicate that MDSCs are induced by cytokines produced in the bone marrow (BM) in response to tumor-derived soluble factors, with IL-4 playing a key role in their generation. Our laboratory has established a protocol to generate MDSCs in vitro from the BM of healthy donors (HDs) using a mixture of cytokines. The first aim of my thesis is the optimization of an efficient in vitro model of BM-MDSCs to modulate their immunosuppressive activity and identify potential therapeutic targets. To this aim, BM aspirates from HDs were processed to isolate immature myeloid cells, which were cultured with G-CSF, GM-CSF, and IL-4. Hence, cells were examined by multicolor Flow cytometry to profile their phenotype and differentiation state. Then, to assess their suppressive activity, a functional assay was performed by co-culturing such in vitro-derived BM-MDSCs with activated T-cells. Results demonstrated that IL-4 priming at day 0 enhances the immunosuppressive capacity of BM-derived MDSCs, significantly increasing their ability to suppress T cell proliferation. Also, IL-4 promotes BM-MDSC proliferation and generates a more immature MDSC phenotype, blocking their default differentiation pathway. These findings emphasize that the receptor alpha of IL-4 (IL-4Rα) has a critical role in driving MDSC immunosuppressive capacity while blocking their default differentiation pathways. Future perspectives of this work will involve testing different pharmaceutical-grade compounds as potential treatments to counteract IL-4-driven immunosuppression and restore immune balance. The second aim of my thesis explore an in vitro model of immunosuppressive macrophages resembling BMDMs, to evaluate the efficacy of a novel nano-based therapeutic approach. Recent findings published by our team showed that there is a correlation between the immunosuppressive activity and the iron metabolism of BMDMs, regulated by the rate-limiting enzyme in heme catabolism, Heme-oxygenase (HO-1) (Magri, et al. 2022). Treating macrophages with Zinc protoporphyrin (ZnPPIX), an inhibitor of HO-1 activity, was found to restore T cell proliferation in vitro and downregulate immunosuppressive markers. We further investigated the therapeutic potential of encapsulating ZnPPIX, into a nanoemulsion (NE-ZnPPIX), to enhance its delivery. Pre-treatment of macrophages with either free ZnPPIX or NE-ZnPPIX, followed by co-culture with activated T cells confirmed its efficacy in restoring T cell proliferation. Future studies will focus on the phenotypic characterization of immunosuppressive macrophages, through specific markers – i.e. CD163 and PD-L1-, following treatment with encapsulated ZnPPIX to gain deeper insights into macrophage reprogramming.

Numerous solid tumors exhibit a highly immunosuppressive tumor microenvironment (TME), which results in a significant deficiency or absence of effector T cells within the tumor site. Such "cold" tumors significantly challenge the efficacy of immunotherapy, as they lack the ability to generate or maintain an adaptive immune response. In particular, many brain tumors – i.e. Glioblastomas Multiforme (GBM) – are highly infiltrated by myeloid cells, which exert immunosuppressive functions supporting tumor aggressiveness. Our research team has identified a significant presence of immunosuppressive bone marrow-derived macrophages (BMDMs) within the TME of GBM, alongside myeloid-derived suppressor cells (MDSCs), which play a crucial role among the immune cell population infiltrating this microenvironment. These cells contribute significantly to creating an immunosuppressive environment that allows cancer cells to escape immune surveillance and elimination. Moreover, recent findings from Merad et al. 2024 indicate that MDSCs are induced by cytokines produced in the bone marrow (BM) in response to tumor-derived soluble factors, with IL-4 playing a key role in their generation. Our laboratory has established a protocol to generate MDSCs in vitro from the BM of healthy donors (HDs) using a mixture of cytokines. The first aim of my thesis is the optimization of an efficient in vitro model of BM-MDSCs to modulate their immunosuppressive activity and identify potential therapeutic targets. To this aim, BM aspirates from HDs were processed to isolate immature myeloid cells, which were cultured with G-CSF, GM-CSF, and IL-4. Hence, cells were examined by multicolor Flow cytometry to profile their phenotype and differentiation state. Then, to assess their suppressive activity, a functional assay was performed by co-culturing such in vitro-derived BM-MDSCs with activated T-cells. Results demonstrated that IL-4 priming at day 0 enhances the immunosuppressive capacity of BM-derived MDSCs, significantly increasing their ability to suppress T cell proliferation. Also, IL-4 promotes BM-MDSC proliferation and generates a more immature MDSC phenotype, blocking their default differentiation pathway. These findings emphasize that the receptor alpha of IL-4 (IL-4Rα) has a critical role in driving MDSC immunosuppressive capacity while blocking their default differentiation pathways. Future perspectives of this work will involve testing different pharmaceutical-grade compounds as potential treatments to counteract IL-4-driven immunosuppression and restore immune balance. The second aim of my thesis explores an in vitro model of immunosuppressive macrophages resembling BMDMs, to evaluate the efficacy of a novel nano-based therapeutic approach. Recent findings published by our team showed that there is a correlation between the immunosuppressive activity and the iron metabolism of BMDMs, regulated by the rate-limiting enzyme in heme catabolism, Heme-oxygenase (HO-1) (Magri, et al. 2022). Treating macrophages with Zinc protoporphyrin (ZnPPIX), an inhibitor of HO-1 activity, was found to restore T cell proliferation in vitro and downregulate immunosuppressive markers. We further investigated the therapeutic potential of encapsulating ZnPPIX, into a nanoemulsion (NE-ZnPPIX), to enhance its delivery. Pre-treatment of macrophages with either free ZnPPIX or NE-ZnPPIX, followed by co-culture with activated T cells confirmed its efficacy in restoring T cell proliferation. Future studies will focus on the phenotypic characterization of immunosuppressive macrophages, through specific markers – i.e. CD163 and PD-L1-, following treatment with encapsulated ZnPPIX to gain deeper insights into macrophage reprogramming.