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Background Diffuse Large B-Cell Lymphoma (DLBCL), the most common non-Hodgkin lymphoma (>18,000 US cases/yr), achieves durable remission in approximately 60% of patients, but outcomes remain poor for those with refractory disease. The immune landscape of DLBCL is highly complex and a deeper understanding of the interplay between T cell activation and immunosuppression within the tumor microenvironment (TME) is crucial for improving patient stratification and predicting therapeutic responses. Given the heterogeneity of these tumors and the importance of spatial relationships among cell types within the TME, there is a need for technologies that can simultaneously visualize multiple biomarkers and cell phenotypes. Multiplex immunohistochemistry (mIHC) enables this level of analysis.Methods In this study, we developed a 20-plex SignalStar mIHC panel designed to profile T cell activation and suppression states, as well as phenotype tumor, myeloid, and vascular cells within the TME. All 20 antibodies were applied in a single primary incubation, followed by the introduction of fluorescently-labeled oligonucleotide networks that amplified the signal from four antibody-oligo conjugates across five imaging rounds. After each round, the fluorescent signal was enzymatically removed.The resulting five images were processed through the Elucidate Bio Spatial Analysis pipeline. Images were co-registered and segmented based on DAPI; marker-specific classifiers labeled cells as positive or negative for each stain. Expression intensities were normalized and clustered via Leiden to assign major lineage phenotypes. Graph-based community detection identified higher-order cellular neighborhoods, facilitating downstream quantification of biomarker expression and the spatial relationships between immune and tumor cells.Results Our analysis revealed several distinct cell neighborhoods with unique spatial organization and cellular composition. As the architecture of the DLBCL TME and the nature of these cellular communities remain poorly characterized, our findings provide valuable insights into the complexity of this disease.Conclusions Overall, our results demonstrate that the SignalStar mIHC assay is a robust tool for dissecting the immunosuppressive TME, offering new opportunities to better understand responses to immunotherapy and combination chemotherapy strategies.