Immunotherapy has revolutionized cancer treatment, but most patients are refractory to immunotherapy or acquire resistance. To explore immunotherapy resistance mechanisms, we characterized the transcriptomes of ~92000 single cells from 15 patients with non-small cell lung cancer (NSCLC) during neoadjuvant PD-1 blockade combined with chemotherapy. CD8+ T, natural killer, B, and dendritic cells were activated by therapy. Therapy also promoted differentiation of memory T cells into effector T cells. Macrophages were remodeled into an M0-like phenotype and neutrophils into an aged phenotype. Distinct therapy-induced cancer-cell transcriptomes were associated with clinical response. Major pathologic responders (MPRs) activated antigen presentation via major histocompatibility complex class II (MHC-II). Cancer cells of non-MPRs exhibited overexpression of estrogen metabolism enzymes and elevated serum estradiol. Elevated estradiol activated EGFR signaling and upregulated the expression of VEGFA, which promoted an immunosuppressive microenvironment. FCRL4+FCRL5+ memory B cells and CD16+CX3CR1+ monocytes were identified as biomarkers for positive immunotherapy response.