A novel regulator of T-cell motility: Fhl2 controls CD8 + T-cell migration and infiltration

For tumor immunotherapy to succeed, CD8 + T cells must efficiently migrate into tumor sites, yet the molecular pathways controlling this critical step of T-cell trafficking remain only partially understood. Using a novel reverse-engineered strategy-starting from antigen-experienced CD8 + T cells that either robustly rejected tumors in vivo or completely failed to do so despite comparable functional profiles-we identified Fhl2 (Four-and-a-half LIM domains 2) as a novel positive regulator of T-cell migration and infiltration, consistent with its role as a LIM-domain adaptor protein that integrates cytoskeletal and signaling inputs. Fhl2-deficient CD8 + T cells exhibited reduced migratory capacity, accompanied by impaired cytoskeletal remodeling and CXCR3-mediated chemotaxis. High-resolution confocal microscopy revealed that Fhl2-wild-type CD8 + T cells formed significantly more dendrite protrusions than knockout counterparts, consistent with enhanced motility. These findings were further supported by RNA-seq and Functional Proteomics Reverse Phase Protein Array (RPPA) analyses: RNA-seq revealed higher expression of several chemokine receptors in Fhl2-wild-type cells, including CXCR3 and CXCR6, while RPPA showed positive enrichment of multiple cell-migration-associated pathways in wild-type cells compared to the knockout. Importantly, transwell chemotaxis assays demonstrated that Fhl2-wild-type CD8⁺ T cells exhibit superior chemokine-driven migration compared with Fhl2-deficient cells-both in polyclonally activated populations and in SIINFEKL-stimulated OT-I cells. This enhanced migratory capacity of Fhl2-wild-type CD8⁺ T cells was also reflected in vivo. Collectively, these results establish Fhl2 as a key orchestrator of CD8 + T-cell motility and reveal a previously unappreciated molecular pathway that could be harnessed to improve tumor immune surveillance and T-cell-based therapies.