The clinical significance of programmed cell death protein 1 (PD-1) expression in the tumor microenvironment (TME) of lung cancer, particularly in the context of immunotherapy, remains poorly understood. This study aimed to evaluate PD-1 expression in tumor-infiltrating immune cells and its association with clinical outcomes in lung cancer patients. In a study of 20 patients (17 men and three women, average age 56 ± 6.9 years) with lung cancer, four key immune cell populations involved in the immunotherapy response were analyzed using multiplexed in situ immunofluorescence. The focus was on PD-1 expression patterns and their correlation with progression-free survival (PFS). Our findings revealed that PD-1 expression was predominantly observed on CD8⁺ lymphocytes, albeit at low levels (~5%), suggesting a state of T-cell exhaustion. Notably, PD-1-expressing immune cells were rare in both non-small-cell and small-cell lung cancer microenvironments, indicating that most immune cells remain functionally active. This deficit of PD-1⁺ cells may explain the limited therapeutic efficacy of antiPD-1 antibodies. Furthermore, we identified CD20⁺ B-cell infiltration as an independent predictor of poorer PFS (HR = 0.17, 95% CI: 0.02–0.65, p = 0.0454), highlighting a previously underappreciated role of B cells in lung cancer progression. Additionally, the presence of distant metastases (stage M1), a high proportion of PD-1⁺CD163⁺ macrophages, and a low proportion of PD-1⁺FoxP3⁺ lymphocytes were associated with shorter PFS, underscoring the complex interplay between immunosuppressive and immunostimulatory cell populations in the TME. These findings suggest that PD-1-expressing immune subsets, particularly cytotoxic lymphocytes and regulatory T cells, may serve as prognostic markers and potential therapeutic targets.