M2 macrophages modulate the differentiation of CD8 + CD101-TIM3 + T cells via the SPP1‒CD44 pathway, influencing the immunotherapeutic response in NSCLC

Non-small cell lung cancer (NSCLC) patients present greatly different responses to immunotherapy. The main clinical challenge lies in the poor response to immune checkpoint inhibitors (ICIs), and the underlying mechanisms remain unclear. Here, single-cell RNA sequencing and bulk RNA sequencing data were comprehensively analyzed. Tumour samples from NSCLC patients treated with ICIs were subjected to multiplexed immunofluorescence staining, and PBMCs were extracted from peripheral blood for flow cytometry. Primary cells were isolated from the PBMCs of NSCLC patients for in vitro coculture experiments, and a murine subcutaneous Lewis lung carcinoma (LLC) model was used for in vivo experiments. This study proposed the hypothesis that CD8 + CD101 + TIM3 + T cells (CCT T cells) are differentiated from CD8 + CD101-TIM3 + T cells (Pre-CCT T cells). This differentiation process is mediated by M2 macrophages via the SPP1‒CD44 pathway and influences the immunotherapeutic response of NSCLC. Analysis of NSCLC clinical samples revealed that lower proportions of tumour-infiltrating CCT T cells and M2-TAMs were associated with a better immunotherapeutic response. According to in vitro experiments, coculture with M2 macrophages significantly increased the CD101 expression in Pre-CCT T cells, whereas in vivo experiments revealed that the removal of bone marrow-derived M2-TAMs significantly lowered the proportion of CCT T cells, inhibited tumour growth, and improved the response to anti-PD-1 therapy. Besides, blockade of the SPP1‒CD44 pathway remarkably weakened the CD101 expression in Pre-CCT T cells in coculture experiments. Moreover, blockade of the SPP1‒CD44 pathway in vivo significantly inhibited tumour growth and enhanced the response to anti-PD-1 therapy in C57BL/6 mice. Our study provides insights into the promotion of Pre-CCT T-cell differentiation by M2 macrophages through the SPP1‒CD44 pathway and elucidates its important role in anti-PD-1 therapy, thus providing potential strategies for increasing immunotherapy efficacy in NSCLC patients.