Background: Therapies targeting immune checkpoints are changing our understanding of the biology and treatment of cancer. Analysing the immune landscape in esophageal adenocarcinoma (EA) may help future prognostication and therapeutic decision-making. Methods: We assembled 310 EA cases in a tissue microarray format with associated clinicopathological information, including a discovery cohort of 156 EA from Northern Ireland and a 154 EA validation cohort from Aberdeen. We carried out validated immunohistochemistry (IHC), stained for range of adaptive immune (CD3, CD4, CD8 and CD45RO) and immune checkpoint biomarkers (ICOS and IDO-1). Slides were digitised and assessed using QuPath image analysis software program to quantify their expression and correlate them with outcome. Results: In the discovery cohort we identified a group of patients highly expressing several immune biomarkers, conferring a significant positive survival advantage (p = 0.022). CD3, CD4, CD8, CD45RO, and ICOS were individually prognostic for better overall survival (Log rank p = 0.0003; p = 0.0292; p = 0.0015; p = 0.0008; p = 0.0051 and p = 0.0264 respectively). Multivariate and correlation analysis identified a subgroup of CD45RO+/ICOS+ patients with significantly improved overall survival (p = 0.0002). The co-expression of CD45RO+/ICOS+ immunophenotype was investigated in the validation cohort and a confirmed survival advantage was seen (p = 0.042). Additionally, the Opal Multiplex IHC technology revealed the much higher frequency of single-cell, dual labelling of CD45RO+/ICOS+ in immune hot cases. Conclusions: These data demonstrate the advantage of immune markers other than the traditional CD3/CD4/CD8 in EA prognostication. The fact that one of these biomarkers is an immune checkpoint inhibitor may have therapeutic implications.