Background: Immune checkpoint inhibitors (ICI) are currently included in the peri-operative standard of care for NSCLC with the objective of a curative strategy. In early stages of NSCLC, biomarkers predicting ICI efficacy should be more stringent than PD-L1 tumoral expression in order to improve the benefit-toxicity ratio. We deeply analyzed the tumor microenvironment of patients included in the IONESCO multicenter phase 2 trial (stage IB > 4cm–IIIA, non N2 resectable NSCLC). Diagnostic biopsies and surgical resection specimen after 3 cycles of Durvalumab were available. We previously showed that the % of residual viable tumor cells (RVT) was associated with disease free survival (DFS) and overall survival (OS). PD-L1 tumor positive score was not correlated to RVT nor survival. Methods: 46 patients were included in the IONESCO trial. Among them, diagnostic biopsy (n = 32), surgical resection specimen post durvalumab (n = 39) and paired tumor samples (n = 31) were analyzed. Immune environment was assessed using 7 quantitative 7-plex immunofluorescence panels generating 349 different cellular phenotypes in 3 different compartments (whole tumor, intra cytokeratin and stroma), focusing on T and B lymphocytes, macrophages, immune checkpoint, NK cells, apoptosis, innate and adaptive immunity, dendritic cells. Densities of cells were quantified using Fluorescent Multiplex immunohistochemistry performed on Leica Bond RX, using OpalTM technology. A fisher's exact test or chi2 test was used for demographics variables and RVT. HRs and 95% CIs were estimated using a Cox model. NCT number: NCT03030131. Results: With median follow-up of 4.5 years, 16/31 patients had a disease recurrence. Histology, sex, stage and survival were significantly associated with intra-tumor densities of CD3, CD8, PD1, TIM3, and CD163 cells in the diagnostic biopsy, and with the delta of CD3, FoxP3, CD4, CD163PD1, TIM3, PDL1 on immune cells and TIM3PDL1 cells, between biopsy and surgical specimen. DFS was significantly associated with high density of CD8+TIM3+ in biopsies (HR = 0.25 [0.09-0.71], p = 0.0092) and with the high density of CD20+ cells in surgical specimen (HR = 0.36 [0.13-0.97], p = 0.04). The RVT was significantly associated with CK+Caspase3- cells and CK+PDL1+ cells in the surgical specimen. No biomarker was associated with OS. Conclusions: Multiparameter analysis of the immune NSCLC environment of patients treated with neoadjuvant anti-PDL1 allows identification of markers associated with clinical, pathological parameters and DFS. Our findings highlight the substantial impact of high CD8+TIM3+ cell density pre-durvalumab and of high CD20+ cell density post-durvalumab on DFS. These findings deserve to be assessed in patients treated with neoadjuvant combined immunotherapy chemotherapy. Clinical trial information: NCT03030131.