Enhancing HLA-DR in Cytotoxic T Lymphocytes is crucial for the development of efficient adoptive T cell Therapies for Breast Cancer
Authors: Salvador R , Correia BF , Grosa D , Martins T , Baal SC Soares , Saraiva DP , Cristóvão-Ferreira S , Pereira IL , de Almeida C Rebelo , Fior R , Jacinto A , Mathias C , Braga S , Cabral MG 
Published: September 2024

AbstractBackgroundDespite 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 expandedex 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 performedin vitroandin vivoexperiments. We also improved a protocol to expandex vivoHLA-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 anex vivostimulation 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, anin silicoanalysis 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 MessageWhat 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.What 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.How 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.