Background: Small-cell lung cancer (SCLC) accounts for 10-15% of all lung malignancies and is characterized by its neuroendocrine differentiation, prominent association with tobacco, elevated somatic mutational load, aggressive clinical course and limited therapeutic options. Early studies have shown promising results of PD-1 axis blockers and diverse trials using PD-1 monotherapy or combinations are ongoing. However, the expression of PD-L1, additional potentially actionable immune targets and immune infiltration remain poorly understood. Methods: Using multiplexed quantitative immunofluorescence (QIF), we measured the levels of PD-L1, B7-H3, B7-H4 and different tumor infiltrating lymphocyte (TIL) subsets in 90 SCLC samples represented in tissue microarray format from 2 retrospective collections (YTMA57 and YTMA259). Tumor PD-L1 (clone E1L3N), B7-H3 (D9M2L) and B7-H4 (D1M8I) were detected in serial sections. The major TIL subsets were determined using multiplex staining of the markers DAPI, cytokeratin (AE1/AE3), CD3 (E272), CD8 (C8/144B) and CD20 (L26). Associations between the marker levels, major clinico-pathological variables and survival were established. Results: PD-L1 protein was detected in 7.3%, B7-H3 in 64.9% and B7-H4 in 2.6% of SCLC cases. PD-L1 and B7-H3 showed limited co-expression and a mutually exclusive expression pattern. In preliminary analysis using the median score as cutpoint, expression of the markers was not significantly associated with age, stage, TILs or 5-year overall survival. In SCLC, the levels of CD3, CD8 and CD20 were significantly lower than in lung adenocarcinomas and squamous cell carcinomas. The ratio of CD8/CD3 signal was also prominently lower in SCLC. Conclusions: PD-L1 and B7-H4 are infrequently expressed in SCLC. B7-H3 is present in nearly 65% of cases and shows limited co-expression with PD-L1. Despite having a high mutational rate, the level of TILs in SCLC is low, suggesting prominent immune regulation/suppression in this malignancy. Our findings support the blockade of B7-H3 and immune regulatory pathways as candidate immunotherapeutic alternatives in SCLC. Validation of the findings in an independent SCLC population is ongoing.