Immune checkpoint blockade (ICB) has revolutionized the treatment of advanced malignancies such as melanoma. However, a significant proportion of patients fail to respond, owing to mechanisms that remain poorly understood. Here, we developed murine melanoma cell lines (R1 and R2) derived from MO4 tumors and that are resistant to anti-PD-1 therapy. These resistant cells exhibited increased tumor initiation capacity in vivo correlating with enhanced spheroid formation in vitro, and greater invasiveness and metastatic potential in a lung macrometastasis model. Mechanistically, both R1- and R2- derived tumors showed enrichment of a cancer stem cell (CSC)-like population overexpressing Wnt3a, which correlated with reduced infiltration and impaired function of CD8⁺ tissue-resident memory (TRMs) T cells. This suggested a functional link between Wnt-driven cancer stemness and TRMs dysfunction. Pharmacological inhibition of Wnt/β-catenin signaling in vitro enhanced CD8⁺ T cell proliferation and expression of functional markers. Notably, Wnt/β-catenin inhibition in vivo reversed anti-PD-1 resistance in resistant tumors, coinciding with reduced OCT4⁺Wnt3a⁺ CSC-like cells and restoration of TNFα⁺CD8⁺ TRMs. Finally, transcriptomic analysis of publicly available melanoma cohorts showed that enrichment of the Wnt pathway correlates with poor prognosis and ICB resistance, highlighting this pathway as a potential druggable negative regulator of ICB efficacy in melanoma.

Background: Bacterial lysates are known to modulate the immune response against respiratory infections. However, the effects of the commercial bacterial lysate Pulmonarom^® on dendritic cells—particularly human monocyte-derived dendritic cells (moDCs)—have not been studied. Additionally, limited data are available on the expression of Toll-like receptors (TLRs) and cytokines following stimulation with bacterial lysates. Methods: Human monocytes were isolated from buffy coats and differentiated into moDCs. Pulmonarom^® was lyophilized, quantified, and used to stimulate moDCs. Ultrastructural changes were evaluated using transmission electron microscopy. The expression of TLRs and selected cytokines was analyzed by flow cytometry. Results: Pulmonarom^® stimulation induced morphological changes in moDCs, including an increased number of dendrites and lysosomes. It also led to the upregulation of MHC class II molecules and TLRs 2, 3, 6, and 7. Additionally, the production of IL-4, IL-6, IL-8, and MCP-1 was significantly increased. Conclusions: Pulmonarom^® promotes moDC maturation, characterized by enhanced antigen presentation capabilities and lysosomal activity, along with increased expression of specific TLRs and cytokines. These features suggest a trained immunity phenotype in moDCs, potentially improving their ability to initiate adaptive immune responses against respiratory pathogens. To our knowledge, this is the first study to investigate the immunomodulatory effects of Pulmonarom^® on human moDCs, providing novel insights into its potential as an immunotherapeutic adjuvant.

Background Evidence shows bacterial lysates may participate in the immune response against respiratory infections. However, few studies have been conducted on dendritic cells, and, to our knowledge, none on human dendritic cells, and few have analyzed certain TLRs and cytokines. Materials and Methods Monocytes were obtained from the buffy coat and subsequently differentiated into dendritic cells (moDCs). The protein of commercial bacterial lysate Pulmonarom® was lyophilized and quantified. Ultrastructural changes induced by Pulmonarom® were evaluated in moDC by electron microscopy, and the expression of TLRs and some cytokines in these cells was evaluated by flow cytometry. Results Pulmonarom®-stimulated moDCs showed increased numbers of dendrites and the presence of lysosomes; they also showed increased expression of MHC-II and TLRs 2, 3, 6, and 7, IL-4, IL-6, IL-8, and MCP-1. Major conclusions The effects of Pulmonarom® on moDC correlate with an increased capacity to mobilize to lymph nodes and present antigens, initiating an effective adaptive immune response. Lysosomal activity and maturation of moDC, increased expression of TLR2, TLR3, TLR6, and TLR7 receptors, and increased production of the cytokines IL-8, MCP-1, IL-6, and IL-4, indicate a training process in moDC after their activation with Pulmonarom® bacterial lysate. This suggests that Pulmonarom® could enhance responses against viruses and bacteria by activating moDC. To our knowledge, this is the first study to address the effect of Pulmonarom® on one of the most important cells orchestrating the innate and acquired immune responses.