Multimodal analysis resolves GNG4 as a distinguishing feature of activated germinal center-resident T follicular helper cells in humans 2258382

Abstract Introduction CD4 T follicular helper (Tfh) cells coordinate humoral immune responses within germinal centers (GC) of lymphoid tissues. Although Tfh are known to play critical roles in vaccination and autoimmunity, gene expression programs that define distinct Tfh states in humans remain unclear. Further, the precise molecular programs spatially engaged by Tfh within the GC compartment are undefined. Such ambiguity has limited translational efforts to monitor or target specific GC Tfh states for clinical benefit. Methods Here, we delineated CD4 T cell heterogeneity in human tonsils and blood using paired spectral flow cytometry and a trimodal single-cell sequencing platform to define protein, transcriptional, and epigenetic features of distinct Tfh states. To identify features that are anatomically restricted to GC-resident Tfh, we performed spatial transcriptomics on human tonsils. Finally, we tested our findings by reanalyzing longitudinal human lymph node samples following vaccination. Results Tfh with an activated GC-like phenotype were highly enriched in chromatin accessibility, transcripts, and protein expression of GNG4 (encoding G protein subunit gamma 4). Integrated analyses of Tfh epigenomic and transcriptional states implicated NFATC1 in GNG4 induction during Tfh activation. In spatial transcriptomic analysis of tonsils, GNG4 expression distinguished activated Tfh states within spatially resolved GC rather than nonGC anatomic compartments, outperforming conventionally GC-associated features of Tfh such as BCL6, TOX2, and B3GAT1. Finally, we found that GNG4 was specifically induced in Tfh in vivo and increased over time in a reanalysis of previously published BNT162b2 mRNA and quadrivalent influenza vaccination data. Conclusion Together, these data identify GNG4 as a distinguishing feature of activated GC Tfh states in humans. In ongoing work, we will investigate the role of GNG4 in Tfh function, with implications for GC responses in human health and disease. Funding Source Sam Barnett Dubensky was supported by the NIH Medical Scientist Training Program T32 (GM007170). This research was supported by the Doris Duke Foundation (2021190) and NIH (K08AI136660) (to Laura A. Vella); the Parker Institute for Cancer Immunotherapy and V Foundation for Cancer Research, co-sponsoring a Parker Bridge Fellow Award (to Derek A. Oldridge); the NIAID (AI179680) and Jeffery Modell Foundation (to Neil Romberg); as well as the Burroughs Wellcome Fund and CHOP Research Institute (to Sarah E. Henrickson). Topic Categories Immune Mechanisms of Human Disease (HUM)