HLA-DR expression in Cytotoxic T Lymphocytes: a key to boost the therapeutic potential of T cell-based strategies for Breast Cancer

Rute Salvador¹Bruna Filipa Correia¹Daniela Grosa¹Telma Martins^1,2Suelen Cristina Soares Baal³Diana Pereira Saraiva¹Sofia Cristovão-Ferreira⁴Isabel Lopes Pereira⁵Cátia Rebelo de Almeida⁶Rita Fior⁶Antonio Jacinto¹Carolina Mathias³Sofia Braga^1,2,5M Guadalupe Cabral^1*

• ¹Universidade Nova de Lisboa NOVA Medical School, Lisbon, Portugal
• ²Hospital Professor Doutor Fernando Fonseca EPE, Amadora, Portugal
• ³Universidade Federal do Parana, Curitiba, Brazil
• ⁴Instituto Portugues de Oncologia de Lisboa Francisco Gentil EPE, Lisbon, Portugal
• ⁵Instituto CUF de Oncologia, Lisbon, Portugal
• ⁶Champalimaud Centre for the Unknown, Champalimaud Foundation, Lisbon, Portugal

T cell-based therapies, involving ex vivo expansion of patients' T lymphocytes, hold significant promise for chemotherapy resistant cases of breast cancer (BC), although their effectiveness remains challenging. Building on our previous findings that the expression of the antigen presenting molecule, HLA-DR, is crucial on tumor-infiltrating cytotoxic T lymphocytes (CTLs) for a favorable response to neoadjuvant chemotherapy, we further investigated the role of HLA-DR-expressing CTLs in anti-tumor responses and evaluated strategies to amplify these cells. Through in vitro and in vivo experiments, we demonstrated that HLA-DR expression on CTLs is important for effective tumor cell elimination. Notably, blocking HLA-DR or depleting CD4+ T cells impaired CTLs activation, suggesting a critical role for antigen presentation by CTLs to CD4+ T cells through HLA-DR in promoting robust anti-tumor responses. Based on these findings we optimized an ex vivo stimulation protocol that increases the proportion of HLA-DR+CTLs with improved cytotoxicity, prioritizing cell quality over yield. Moreover, we showed that adding anti-PD-1 to the stimulation, further upregulated HLA-DR expression, and intensified CTLs' cytotoxic ability. This aligns with our in silico analysis suggesting a potential regulatory link between PD-1 and HLA-DR via non-coding RNAs. Overall, our findings open new avenues for advancing T cell-based therapies and improve the outcomes of chemotherapy-resistant-BC.