Background: One of the tumour escape mechanisms is the expression of programmed cell death-1 (PD-1) receptor on immune cells. Clinical trials have demonstrated that antibodies against PD-L1 or PD-1 molecules induce objective clinical responses in pre-treated patients with NSCLC. In this study, we evaluated the levels of PD-1⁺ and PD-L1⁺expressing- immune cells and their association with the clinical outcome in NSCLC patients. Methods: Peripheral blood was collected from 32 advanced/metastatic pre-treated NSCLC patients before anti-PD-1 therapy (Nivolumab). Flow cytometry was performed to identify and quantify PD-1 and PD-L1 expression on circulating immune cells with anti-tumor function (CD4⁺ and CD8⁺ T cells, B cells, Dendritic cells) , as well as on immunosuppressivecells [(CD4⁺ and CD8⁺ Treg cells, Bregs, Myeloid-derived Suppressor cells (MDSC)]. Correlation between the levels of PD-1⁺ and PD-L1⁺-expressing immune cells, as well as their association with overall (OS) and progression-free survival (PFS) was evaluated; high expression of immune cells was defined as the percentage of the cells above the mean value. Results: PD-1 and PD-L1 expression was detected on all tested immune cells with anti-tumor activity. PD-1 expression was found on all immunosuppressive cells, whereas only CD4⁺ and CD8⁺ Treg cells and Granulocytic -MDSCs expressed PD-L1. No PD-L1 expression was observed on Bregs and monocytic-MDSCs. There was no correlation of PD-1 or PD-L1-expressing cells with response to anti-PD1 treatment. A significantly decreased PFS was observed in the low CD4⁺ PD-L1⁺ T cells group compared with the high CD4⁺ PD-L1⁺ group (3.4 months vs. 5.2 months, p=0.03). Univariate analysis for PFS revealed that the low percentages of CD4⁺ PDL-1⁺ T cells were prognostic for shorter PFS (HR= 3.1, p=0.04). On the multivariate analysis, low PD-L1⁺ CD4⁺T cells were independently associated with decreased PFS (HR: 3.3, p=0.03). Conclusions: These data provide evidence that PD-L1⁺ expression on peripheral CD4⁺ T cells was a significant predictor of better PFS in NSCLC patients. Therefore, PD-L1 expression could be used for the selection of patients who might benefit from PD-1/PD-L1 inhibitors.