de Almeida, C. R. | bioRxiv Science

bioRxiv2025

Tissue-specific consequences of tag fusions on protein expression in transgenic mice

Genetic fusion of protein tags is widely used to study protein functions in vivo. It is well known that tag fusion can cause unwanted changes in protein stability, but whether this is an inherent property of the tagged protein, or can be influenced by the cell and tissue environment, is unclear. Using a series of genome edited mouse models, we show that tag-dependent changes in protein expression can vary across different primary cell and tissue contexts. In one case (Ncaph2), a C-terminal auxin-inducible degron fusion strongly increased protein stability in some tissues but decreased it in others. Destabilisation resulted from tissue-specific leakage of the auxin-inducible degron, which depended on TIR1 expression, and occurred selectively in the small intestine where basal concentrations of auxin / indole-3-acetic acid can reach levels that are sufficient to trigger protein degradation in cultured cells. Stabilisation occurred in post-mitotic cells via an endogenous degradation signal situated at the Ncaph2 C-terminus, which normally undergoes activation upon cell cycle exit, but is inactivated by C-terminal tag fusion. Our results highlight the underappreciated importance of cell and tissue environment in determining the consequences of tag fusions on protein expression, which may be particularly important in animal models that contain diverse cell types.

molecular biology↗

bioRxiv2024

Enhancing HLA-DR in Cytotoxic T Lymphocytes is crucial for the development of efficient adoptive T cell Therapies for Breast Cancer

BackgroundDespite advances in breast cancer (BC) therapies, more effective interventions are needed, especially for chemotherapy-resistant tumors. Immune checkpoint inhibitors show promise for triple-negative breast cancer, but their effectiveness across all BC subtypes remains challenging. Therefore, novel strategies, including adoptive cellular therapy, employing patients own T lymphocytes expanded ex vivo, are under investigation. Previously, we demonstrated that cytotoxic T lymphocytes (CTLs) expressing high HLA-DR levels in the tumor microenvironment are associated with a good response to neoadjuvant chemotherapy (NACT), due to their pronounced anti-tumor properties compared to CTLs with low or no HLA-DR expression. In this paper, we demonstrated that HLA-DR expression in CTLs is crucial for efficient T lymphocytes-based therapies. MethodsTo clarify the role of HLA-DR in CTLs anti-tumor abilities, we performed in vitro and in vivo experiments. We also improved a protocol to expand ex vivo HLA-DR-expressing CTLs and employed a 3D co-culture platform to test the potential of different immune agents, namely an anti-PD1, anti-OX40, anti-VEGF and anti-CD137, on CTLs cytotoxicity against BC cells. Additionally, we conducted a bioinformatic analysis of scRNA-seq data of BC patients to better understand the modulation of HLA-DR expression in CTLs. ResultsOur findings revealed that CTLs require HLA-DR expression to eliminate tumor cells. Additionally, we unveiled that blocking HLA-DR or depleting CD4+ T cells compromised CTLs activation and cytotoxicity, suggesting antigen presentation by CTLs through HLA-DR, and CD4+ T cells, as probable mechanisms for CTLs increased anti-tumor immune response and treatment efficacy. We refined an ex vivo stimulation and cytokine supplementation protocol, observing that short-term stimulation increases HLA-DR expression while boosting CTLs functionality, unlike prolonged expansion. This result highlights the importance of prioritizing cell quality, over quantity, for therapy efficiency. Additionally, we verified that anti-PD-1 further increases HLA-DR levels in CTLs, enhancing their anti-tumor efficiency. Notably, an in silico analysis revealed that PD-1 in CTLs shares 34 co-expressed genes with HLA-DR, including several non-coding RNAs, suggesting a PD-1-mediated regulation of HLA-DR expression. ConclusionsGlobally, our findings underscore that heightening HLA-DR expression in CTLs, by combining anti-PD-1 with short-term stimulation, offers promise for improving T lymphocyte-based therapies for BC. Key MessageO_LIWhat is already known on this topic: While immunotherapy holds promise for breast cancer (BC), its success is still limited. Novel strategies are under investigation to improve outcomes across all BC subtypes. Previously we established a correlation between HLA-DR expression on CTLs and a positive response to neoadjuvant chemotherapy, likely due to enhanced anti-tumor properties of HLA-DR-expressing CTLs. However, the specific role of HLA-DR on CTLs and its implications for T lymphocyte-based therapies requires further investigation. C_LIO_LIWhat this study adds: This study revealed that HLA-DR expression is essential for CTLs to effectively eliminate tumor cells. Blocking HLA-DR or depleting CD4+ T cells impairs CTLs activation and cytotoxicity, indicating that HLA-DR-mediated antigen presentation and interaction with CD4+ T cell are crucial for CTLs function. The study also shows that combining ex vivo short-term stimulation and anti-PD-1 treatment increases HLA-DR levels in CTLs, enhancing their anti-tumor activity. C_LIO_LIHow this study might affect research, practice or policy: By emphasizing the importance of optimizing CTLs quality over quantity, this approach has the potential to improve the design of more efficient T lymphocyte-based therapies for BC. This could influence future research directions, clinical practices, and treatment policies, leading to improved therapeutic outcomes. C_LI

immunology↗

bioRxiv2024

Macrophages drive the earliest anti-tumoral response to BCG therapy by directly killing bladder cancer through TNF signaling

The Bacillus Calmette-Guerin (BCG) vaccine is the cancer immunotherapy longest in use. Despite its effectiveness in bladder cancer (BC), its initial mechanisms of action remain largely unknown. Therefore, proper diagnostic assessments to identify patients who will not respond to treatment or develop resistance are lacking. Here, we set-out to unravel the earliest innate cellular mechanisms involved in BCG-induced clearance of tumors. We show that BCG induces a massive recruitment of macrophages to the tumor microenvironment and modulates their morphology and behavior towards a proinflammatory phenotype, while also promoting macrophage fusion-like events. We demonstrate that macrophages directly induce apoptosis and clearance of cancer cells through TNF-signaling and that they are indispensable for this antitumoral response since their depletion completely abrogates the BCG-anti tumor effect. Contrary to the general concept that macrophage antitumoral activities uniquely rely on stimulating an effective adaptive response, we demonstrate that macrophages alone can directly induce tumor killing and clearance; revealing an additional step to the BCG-induced tumor immunity model, that was not previously considered. In addition, we also provide proof-of-concept experiments demonstrating the potential of this unique in vivo preclinical model to test new innate immunomodulators.

cancer biology↗