The study introduces a novel imaging technique called CO-Detection by indEXing (CODEX) to create high-parameter imaging datasets of normal and lupus (MRL/lpr) mouse spleens. CODEX enables multiplexed imaging of multiple antibody-tagged epitopes using DNA barcodes and fluorescent dNTP analogs, allowing for the accurate quantification of membrane antigen levels on dissociated cells and tissue sections. The method was validated through experiments with isolated mouse spleen cells and tissue sections, demonstrating its ability to visualize complex cellular interactions and structural changes in autoimmune disease. Key findings include: 1. **Multiplexed Antigen Staining**: CODEX successfully visualized multiple antibody targets by iteratively extending DNA tags using primer extension, achieving high multiplexing capabilities. 2. **Quantitative Analysis**: The technique was validated against mass cytometry (CyTOF) data, showing consistent similarity in lineage-positive populations. 3. **Tissue Architecture Characterization**: CODEX provided a detailed view of splenic architecture, including the white pulp, B-cell follicle, PALS, and marginal zone. Automated algorithms were developed to segment and quantify cellular neighborhoods, revealing significant non-random distributions of cells. 4. **Cell-to-Cell Interactions**: The study identified 734,101 single-cell protein marker expression profiles and used X-shift clustering to annotate 58 phenotypic clusters. These clusters were manually annotated and matched to known cell types, confirming the presence of rare cell types in normal spleen. 5. **Disease-Associated Changes**: Analysis of MRL/lpr spleens showed dramatic changes in cell counts and interactions, particularly in the early stages of disease progression. These changes were driven by modulation of cell type frequencies rather than changes in cell-to-cell affinity. 6. **Reorganization of Tissue Substructures**: The study observed significant rearrangements in splenic architecture, including the disintegration of the marginal zone and the emergence of novel cell niches, which were associated with disease progression. Overall, CODEX provides a powerful tool for deep phenotyping and systematic characterization of complex tissue architecture in both normal and clinically aberrant samples, offering insights into the dynamics of cellular neighborhoods and their impact on immune cell function.The study introduces a novel imaging technique called CO-Detection by indEXing (CODEX) to create high-parameter imaging datasets of normal and lupus (MRL/lpr) mouse spleens. CODEX enables multiplexed imaging of multiple antibody-tagged epitopes using DNA barcodes and fluorescent dNTP analogs, allowing for the accurate quantification of membrane antigen levels on dissociated cells and tissue sections. The method was validated through experiments with isolated mouse spleen cells and tissue sections, demonstrating its ability to visualize complex cellular interactions and structural changes in autoimmune disease. Key findings include: 1. **Multiplexed Antigen Staining**: CODEX successfully visualized multiple antibody targets by iteratively extending DNA tags using primer extension, achieving high multiplexing capabilities. 2. **Quantitative Analysis**: The technique was validated against mass cytometry (CyTOF) data, showing consistent similarity in lineage-positive populations. 3. **Tissue Architecture Characterization**: CODEX provided a detailed view of splenic architecture, including the white pulp, B-cell follicle, PALS, and marginal zone. Automated algorithms were developed to segment and quantify cellular neighborhoods, revealing significant non-random distributions of cells. 4. **Cell-to-Cell Interactions**: The study identified 734,101 single-cell protein marker expression profiles and used X-shift clustering to annotate 58 phenotypic clusters. These clusters were manually annotated and matched to known cell types, confirming the presence of rare cell types in normal spleen. 5. **Disease-Associated Changes**: Analysis of MRL/lpr spleens showed dramatic changes in cell counts and interactions, particularly in the early stages of disease progression. These changes were driven by modulation of cell type frequencies rather than changes in cell-to-cell affinity. 6. **Reorganization of Tissue Substructures**: The study observed significant rearrangements in splenic architecture, including the disintegration of the marginal zone and the emergence of novel cell niches, which were associated with disease progression. Overall, CODEX provides a powerful tool for deep phenotyping and systematic characterization of complex tissue architecture in both normal and clinically aberrant samples, offering insights into the dynamics of cellular neighborhoods and their impact on immune cell function.