Investigating determinants of T cell exclusion exploiting germinal center dark zone/light zone interface as a model

Background: Germinal centers (GCs) are specialized microenvironments within secondary lymphoid organs (SLOs) that support antibody affinity maturation and the establishment of immune memory. These structures orchestrate the coordinated activity of B cells, stromal networks, and T-cell subsets, especially T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. However, the spatial and molecular cues that regulate T-cell entry, positioning, and function within GCs remain incompletely understood, and disruptions in these processes contribute to immune dysregulation and GC-derived lymphomas. To assess this, the current study employed spatial transcriptomics, multiplex imaging, and computational tissue modeling to characterize the T-cell compartment within the GC. The objectives of this study were to define the spatial organization and niche architecture of GC zones, and we have studied in detail the Tfh and plausible associated markers by integrating molecular and spatial data. This study aimed to provide a framework for understanding GC immune regulation and its contribution to lymphoma pathogenesis.Methods: T-cell localization within the GC was analyzed using Digital Spatial Profiling (DSP) on human tonsil, where CD20+ and CD271+ expression delineated the dark zone (DZ) and light zone (LZ) regions. DSP analysis revealed a mini signature of T cell–related in the LZ. To map T cells in situ, multiplex cyclic immunofluorescence was performed on formalin-fixed, paraffin-embedded (FFPE) tonsil sections using the MACSima platform, and T-cell subsets were quantified in DZ and LZ regions using HALO image analysis software. An independent DSP dataset comparing intrafollicular and interfollicular CD3⁺ T-cell populations was then used to define transcriptional programs associated with GC-resident T cells. Integrating spatial and transcriptional data enabled the identification of candidate molecular regulators linked to Tfh-cell localization and function within the GC microenvironment to enhance the efficacy of immunotherapy.Results: Spatial transcriptomic profiling revealed a marked reduction in T cell–associated and immune checkpoint gene expression within the GC DZ, consistent with limited T-cell infiltration in this compartment. Quantitative spatial mapping confirmed that CD3⁺ T cells are excluded from the DZ and instead accumulate in the LZ and perifollicular areas. Transcriptomic comparison between GC-resident and extrafollicular CD3⁺ T cells revealed enrichment of gene programs related to Tfh differentiation and co-stimulatory signaling. Integrated spatial and transcriptional analyses identified HAL, P2RY8, and ZFPM1 as candidate markers of Tfh-enriched regions within the GC.Conclusion: The identification of a Tfh-specific transcriptional signature, together with novel candidate markers, provides new molecular insights into the characterization of GC-associated T cells. These findings may hold diagnostic relevance for Tfh-derived T-cell lymphomas, for which specific molecular markers remain undefined. Further validation of independent patient cohorts is required to confirm the diagnostic and biological significance of these observations.