Given the role of follicular immune dynamics, especially the germinal centre, for the development of pathogen-specific antibodies, their in situ characterisation is of great importance. We have developed a multiplex immunofluorescence imaging pipeline that allows the analysis of human follicular adaptive and innate immune cell subsets. Our data revealed the in situ phenotypic heterogeneity and differential localisation of follicular helper CD4 T (TFH) cell subsets across follicular areas in tonsils and reactive lymph nodes (LNs). Cell clustering analysis identified specific TFH subsets with differential prevalence between tonsils and LNs. Further, a multiplex RNAscope/protein imaging assay revealed the functional heterogeneity of TFH cells. No significant differences in follicular innate immune cell densities were found between tonsils and LNs. In conclusion, we present a combinatory experimental approach that provides a comprehensive analysis of human follicular and/or germinal centre immune dynamics and could be used to further understand the pathogenesis of diseases such as HIV and lymphomas.
Keywords: TFH cells; follicles; immune landscaping; multiplex imaging.
© 2025 The Author(s). Immunology published by John Wiley & Sons Ltd.