<jats:title>Abstract</jats:title><jats:sec> <jats:title>Objectives</jats:title> <jats:p>A minority of NSCLC patients benefit from anti-PD1 immune checkpoint inhibitors. A rational combination of biomarkers is needed. The objective was to determine the predictive value of tumor mutational load (TML), CD8<jats:sup>+</jats:sup> T cell infiltration, HLA class-I and PD-L1 expression in the tumor.</jats:p> </jats:sec><jats:sec> <jats:title>Materials and methods</jats:title> <jats:p>Metastatic NSCLC patients were prospectively included in an immune-monitoring trial (NTR7015) between April 2016-August 2017, retrospectively analyzed in FFPE tissue for TML (NGS: 409 cancer-related-genes) and by IHC staining to score PD-L1, CD8<jats:sup>+</jats:sup> T cell infiltration, HLA class-I. PFS (RECISTv1.1) and OS were analyzed by Kaplan–Meier methodology.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>30 patients with adenocarcinoma (67%) or squamous cell carcinoma (33%) were included. High TML was associated with better PFS (<jats:italic>p</jats:italic> = 0.004) and OS (<jats:italic>p</jats:italic> = 0.025). Interaction analyses revealed that patients with both high TML and high total CD8<jats:sup>+</jats:sup> T cell infiltrate (<jats:italic>p</jats:italic> = 0.023) or no loss of HLA class-I (<jats:italic>p</jats:italic> = 0.026), patients with high total CD8<jats:sup>+</jats:sup> T cell infiltrate and no loss of HLA class-I (<jats:italic>p</jats:italic> = 0.041) or patients with both high PD-L1 and high TML (<jats:italic>p</jats:italic> = 0.003) or no loss of HLA class-I (<jats:italic>p</jats:italic> = 0.032) were significantly associated with better PFS. Unsupervised cluster analysis based on these markers revealed three sub-clusters, of which cluster-1A was overrepresented by patients with progressive disease (15 out of 16), with significant effect on PFS (<jats:italic>p</jats:italic> = 0.007).</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>This proof-of-concept study suggests that a combination of PD-L1 expression, TML, CD8<jats:sup>+</jats:sup> T cell infiltration and HLA class-I functions as a better predictive biomarker for response to anti-PD-1 immunotherapy. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted.</jats:p> </jats:sec>