Differential modulation of exhausted Th1 cell migration by immune checkpoint blockade: insights from 3D microfluidic...

Effective anti-tumor immunity is critically dependent on the functional capacity and robust infiltration of type-1 helper T (Th1) cells into the tumor microenvironment (TME). However, persistent antigenic stimulation leads to T cell exhaustion, determining the efficacy of immunotherapy approaches such as immune checkpoint inhibitors (ICIs). A key barrier to successful immunotherapy therapy remains the impaired motility and infiltration of exhausted T cells. To address this, we investigated the dual impact of anti-PD-1 and anti-CTLA-4 blockade on the migratory efficacy of ex vivo generated exhausted Th1 (Th1-Ex) cells. Recognizing the limitations of 2D culture, we utilized a 3D microfluidic (lab-on-a-chip) platform to simulate the TME's complex physical and chemical constraints, alongside in vivo patient-derived xenograft (PDX) models. While ICI treatment did not alter static adhesion, it distinctly modulated focal adhesion dynamics. Critically, in the highly relevant 3D microfluidic environment, ICI-treated Th1-Ex cells exhibited significantly enhanced motility and directional persistence compared to untreated cells. Mechanistically, anti-PD-1/CTLA-4 treatment activated signalling pathways associated with both amoeboid and mesenchymal-like migration, but functional inhibition identified RAC1-dependent mesenchymal-like migration as the predominant contributor to the enhanced migratory response. These results were strongly corroborated in vivo, where the anti-PD-1/CTLA-4 combination conferred remarkable and selective tumor and lymph node infiltration capacity to the adoptively transferred Th1-Ex cells. Collectively, our findings highlight a dual role for ICIs on restoring Th1 cell functionality and actively promoting tumor-directed migration by altering adhesion-migration pathways, offering novel mechanistic insights for optimizing immunotherapies in solid tumors.