Spatial proximity sequencing maps developmental dynamics in the Germinal Center 2332687

Abstract

Abstract Introduction Spatial profiling of proteins and protein interactions is essential for understanding immune responses, yet existing methods cannot simultaneously measure protein complexes in situ. We developed Spatial Proximity-Sequencing (Sprox-seq), a multi-omic technique that simultaneously profiles proteins, protein complexes, and mRNAs with spatial resolution in intact tissues. Methods Sprox-seq integrates proximity ligation assay with spatial transcriptomics on the 10x Visium platform. Antibody-oligonucleotide conjugate pairs targeting 32 proteins were applied to human tonsil sections. Upon spatial proximity, probes ligate to generate PLA products captured alongside mRNAs via spatial barcodes. Fisher’s Exact Test and fractional overlap analysis were used to identify and quantify protein complexes. Results We profiled 32 proteins, 528 pairwise interactions, and thousands of mRNAs across human tonsil germinal centers. Protein interaction networks revealed higher complexity in the Light zone compared to Dark zone, consistent with B cell selection dynamics. Trajectories inferred from protein interactions uncovered a B cell state transition distinct from that inferred by RNA. We identified spatially enriched complexes including CD19-CD21, CD21-CD35, and directly captured VLA-4—VCAM1 mediated B cell-FDC interactions in the Light zone. Conclusion Sprox-seq enables simultaneous spatial profiling of transcriptomes, proteomes, and interactomes, revealing protein interaction dynamics during B cell state transition that cannot be inferred from RNA or protein expression alone. This technology provides new insights into cell-cell communication in immune tissues. Funding Source NIH grants AI175371, R35GM148231, and GM127527 (S.T.), P. Allen Distinguished Investigator Award (S.T), and Chan-Zuckerbeg Chicago Biohub Investigator Award (S.T. and M. R. C.). Topic Categories Technological Innovations in Immunology (TECH)