Abstract 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. Citation Format: Achintya Perumal, Grant Brennan, Jiayao Ye, Hu Chen, Roya Solhi, Omar I. Avila, Kathleen B. Yates, Robert T. Manguso, Stanley C. Huang, Haoxin Zhao, Giovanni Nigita, Zihai Li, No-Joon Song, Hazem E. Ghoneim, Ava Lowin, Kelley Ptak, Qin Ma, Hakimeh Ebrahimi-Nik. A novel regulator of T-cell motility: Fhl2 controls CD8+ T-cell migration and infiltration abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6580.
Perumal et al. (Fri,) studied this question.