CD4⁺ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8⁺ T cells, whereas combination therapy expanded both CD4⁺ and CD8⁺ T cells. Such CD4⁺ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4⁺ and CD8⁺ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4⁺ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4⁺ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.