Single-cell transcriptomic analysis reveals the synergistic effect of gut microbiota and immunotherapy through modulating tumor microenvironment

The gut microbiota crucially regulates the efficacy of immune checkpoint inhibitor (ICI) based immunotherapy, but the underlying mechanisms remains unclear at single cell resolution. In this study, we employed single-cell RNA sequencing and subsequent validations to investigate the synergistic effects of ICIs and gut microbiota through profiling the tumor microenvironment (TME) and elucidating critical cellular interactions in mouse models. Our results demonstrate that combination of intact gut microbiota and ICI treatment would synergistically increase the proportions of CD8+, CD4+, and γδ T cells, reduce their glycolysis metabolism, and reverse the exhausted CD8+ T cells to memory/effector CD8+ T cells, suggesting enhanced antitumor response. Moreover, the synergistic effect induces macrophage reprogramming from M2 polarization-related protumor Spp1+ tumor-associated macrophages (TAMs) to Cd74+ TAMs, which act as antigen-presenting cells (APCs). Depleting Spp1+ TAMs in Spp1 conditional knockout mice enhances ICI efficacy and promotes T cell infiltration regardless of gut microbiota clearance, underscoring the crucial negative role of Spp1+ TAMs and importance of macrophage reprogramming in immunotherapy outcomes. Mechanistically, we propose a γδ T cell-APC-CD8+ T cell axis, where gut microbiota and ICI enhance Cd40lg expression on γδ T cells, activating Cd40 overexpressed APCs (e.g., Cd74+ TAMs) through CD40-CD40L related NF-κB signaling, and boosting CD8+ T cell responses through CD86-CD28 interactions. These findings highlight the vital role of γδ T cells and SPP1¬-related macrophage reprogramming in activating CD8+ T cells and the synergistic effect of gut microbiota and ICIs, offering new insights into the cellular and molecular mechanisms that enhance immunotherapy efficacy.