Hosn, R. R.; Marrone Mantovani, O.; Margaronis, A.; Wang, J. S.; Kriss, E.; Wu, C.; Kissner, M.; McFaline-Figueroa, J. L.; Correa, S.

Tertiary lymphoid structures (TLSs) are organized three-dimensional immune niches associated with improved antitumor immunity, which has galvanized efforts to induce them artificially using biomaterials. However, these strategies have largely focused on soluble cue delivery, leaving the role of scaffold physical properties poorly understood. Here, we developed injectable, liposome-crosslinked supramolecular hydrogels spanning soft and stiff formulations to determine how scaffold mechanical properties regulate in situ tertiary lymphoid neogenesis. The formulations differed in their viscoelastic properties while maintaining broadly comparable release of ovalbumin and LIGHT. Soft hydrogels underwent distributed cellular infiltration and material replacement, transitioning from an early myeloid-rich response to vascularized, lymphoid-dominant tissues containing B-cell-rich aggregates adjacent to T-cell regions, with B-cell organization peaking at day 14. Single-cell RNA sequencing revealed that transient interferon-associated neutrophil and macrophage states in the early niche preceded the emergence of TLS-associated transcriptional programs across lymphoid and myeloid populations. In contrast, stiff hydrogels resisted infiltration and perpetuated a niche dominated by activated myeloid cells with limited lymphoid organization. Prophylactically implanted soft hydrogels improved early melanoma control relative to stiff hydrogels under checkpoint blockade, and a single soft-hydrogel implantation restrained tumor growth even without checkpoint blockade. Together, these findings establish scaffold mechanics and remodeling as active regulators of engineered immune-tissue organization and provide design principles for directing the development of local TLS-like niches.