TLR8 agonists remodel the tumor immune microenvironment through PF4-dependent T cell recruitment and ancillary mechanisms

Pattern Recognition Receptors (PRRs) modulate tumor immune microenvironments (TIME), but their comparative efficacy remains unclear. Platelet factor 4 (PF4/CXCL4) exhibits paradoxical roles in cancer, and its regulation by PRRs remains unexplored. We systematically evaluated PRR agonists to identify optimal TIME modulators and elucidate PF4's role. TLR8 agonists optimally remodel TIME by activating PF4-dependent T cell recruitment and engaging PF4-independent ancillary mechanisms. Context-dependent PF4 induction resolves its dual roles in cancer. These findings position TLR8 agonists as promising partners for immunotherapy. Overall design: Mouse tumor tissues (5 biological replicates per group) were rinsed with ice-cold PBS, mechanically dissociated into small fragments, and enzymatically digested using collagenase D/DNase I (Roche) with gentleMACS Tissue Dissociator (Miltenyi Biotec). The resulting single-cell suspensions were filtered through 70-µm strainers and PBS-washed before immune cell enrichment through CD45+ selection using biotin-conjugated anti-mouse CD45 antibody (BioLegend) and anti-biotin MicroBeads (Miltenyi Biotec), yielding >95% pure and >85% viable populations.