| Summary: | Gastric cancer (GC) therapies include gastrectomy and chemoradiotherapy. The tumor immune microenvironment (TME) has implications for potential immunotherapy. We analyzed the expression of PD-L1, CD8, CTLA-4 and IFN-γ in the tumor and regional lymph node (LN) of patients with GC and compared it with clinical and pathological data. Paraffin blocks were collected from 97 patients undergoing gastrectomy/lymphadenectomy for GC. Double immunohistochemistry was performed for CD8 and PD-L1 and double immunofluorescence for CTLA-4 and IFN-γ. Statistical significance was set at <i>p</i> < 0.05. PD-L1 expression in tumor cells was associated with intestinal GC type (<i>p</i> = 0.046), the density of macrophages and CD8 + T cells (<i>p</i> < 0.001, both). The median number of CD8+ T cells was higher in PD-L1-positive than in -negative tumors. A cut-off of 28.5 CD8 + T cells in one high-magnification field predicted PD-L1-positive tumors (AUROC 0.797, sensitivity 74.2%, specificity 77.3%). IFN-γ expression in tumor cells was found in 37 GCs and was positively associated with CTLA4<sup>+</sup> lymphocytes in the LN (<i>p</i> = 0.027) and CTLA4<sup>+</sup>/IFN-γ+ in tumors and the LN (all <i>p</i> < 0.001). The median overall survival (OS) was 17 months. In the group of deceased patients, IFN-γ expression in metastases correlated with lower OS (RHO = −0.314, <i>p</i> = 0.008). PD-L1 expression in tumor cells correlated with CD8 + T cells and macrophages in the TME and IFN-γ expression with suppressive CTLA4<sup>+</sup>/IFNγ+ immune cells in the TME and LN.
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