Delu Song

Supplementary Figure 5: Granzyme B and Ki-67 expression in CD8+ T cells in the MPDOs.

Supplementary Figure 3. Mouse body weight during treatment.

The Toll-like receptor (TLR) 7/8 agonist resiquimod shows promise for treating cutaneous T-cell lymphoma and actinic keratosis, yet its mechanism of action remains unclear. We demonstrated that topical resiquimod significantly inhibited melanoma growth across various genetic and syngeneic mouse models, prolonged survival, and reduced lymph node metastasis in vivo. Resiquimod suppressed B16 melanoma growth, with an effect superior to that of imiquimod. The therapeutic effect was CD8+ T cell–dependent, as evidenced by the loss of efficacy upon CD8+ T-cell depletion or in Rag2−/− mice. Resiquimod increased intratumoral CD45+ inflammatory cells, particularly antigen-experienced PD-1+CD62L−CD8+ effector T cells, and enhanced their Ki-67 and granzyme B expression. Resiquimod significantly expanded Pmel- and Trp2-specific CD8+ T cells in the presence of dendritic cells (DC). Topical treatment of melanoma-bearing mice induced systemic protection in rechallenge experiments. In addition, combining topical resiquimod with anti–PD-1 antibodies led to superior inhibition of tumor growth and metastasis across multiple melanoma models. Proteomic analysis revealed increased granzyme B and CD26 and decreased phosphorylated FOXO3a after treatment. In patient-derived organoids and melanoma slice cultures, resiquimod induced significant tumor killing and CD8+ T-cell activation, further augmented by PD-1 antibodies. Our findings support the conclusion that resiquimod promotes CD8+ T-cell priming via DCs and enhances the therapeutic efficacy of anti–PD-1 checkpoint blockade in melanoma.

Supplementary Figure 4: Effects of resiquimod on CD45-and CD45+ cell fractions in MPDOs.

Supplementary Figure 1. Representative mouse genotypes by PCR. Different mouse genotypes were illustrated.

Supplementary Figure 2. Cytokine expression in Tyr::creER, Brafca, Ptenlox/lox mouse tumors after resiquimod treatment.

The Toll-like receptor (TLR) 7/8 agonist resiquimod shows promise for treating cutaneous T cell lymphoma and actinic keratosis, yet its mechanism of action remains unclear. We demonstrated that topical resiquimod significantly inhibited melanoma growth across various genetic and syngeneic mouse models, prolonged survival, and reduced lymph node metastasis in vivo. Resiquimod suppressed B16 melanoma growth, with an effect superior to that of imiquimod. The therapeutic effect was CD8+ T cell-dependent, as evidenced by the loss of efficacy upon CD8+ T cell depletion or in Rag2-/- mice. Resiquimod increased intratumoral CD45+ inflammatory cells, particularly antigen-experienced PD1+CD62L-CD8+ effector T cells, and enhanced their Ki-67 and Granzyme B expression. Resiquimod significantly expanded Pmel- and Trp2-specific CD8+ T cells in the presence of dendritic cells. Topical treatment of melanoma-bearing mice induced systemic protection in rechallenge experiments. In addition, combining topical resiquimod with anti-PD-1 antibodies led to superior inhibition of tumor growth and metastasis across multiple melanoma models. Proteomic analysis revealed increased Granzyme B and CD26, and decreased phosphorylated FOXO3a, post-treatment. In patient-derived organoids and melanoma slice cultures, resiquimod induced significant tumor killing and CD8+ T cell activation, further augmented by PD-1 antibodies. Our findings support the conclusion that resiquimod promotes CD8+ T-cell priming via dendritic cells and enhances the therapeutic efficacy of anti-PD-1 checkpoint blockade in melanoma.

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.