Poster #25 - Sam Barnett Dubensky

Multimodal analysis resolves GNG4 as a distinguishing feature of activated germinal center-resident Tfh in humans

Barnett Dubensky S, BS 1,2, Gallagher M, MS 3†, Kumashie KG, PhD 3, Lu T, MS 4, Zhu Y, MS 5, Tedesco J, BA 3, De Luna N, BS 2, Premo K, BS 2, Qi Y, MSE 5, Rachimi S, BS 2, Cruz Cabrera E, MS 6, Fulmer F, BS 2, Meremikwu IC, BS 7, Carter A, MS 9,10, Henrickson SE, MD, PhD 4,6,8, Romberg N, MD 6,8, Baxter AE, PhD 8‡, Oldridge DA, MD, PhD 9,10* & Vella LA, MD, PhD 3,8* 1 Medical Scientist Training Program, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 2 Immunology Graduate Group, Biomedical Graduate Studies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 3 Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA. 4 Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 5 Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA. 6 Division of Allergy and Immunology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA. 7 Graduate Group in Genomics and Computational Biology, Biomedical Graduate Studies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 8 Institute for Immunology and Immune Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 9 Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA. 10 Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA. *Co-corresponding authors. † Present Address - Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA. ‡ Present Address - Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA and Institute for Immunology and Immune Health, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

CD4 T follicular helper (Tfh) cells are critical for robust humoral immunity. Though canonically active within lymphoid tissue germinal centers (GC), Tfh can also migrate into nonGC regions and peripheral circulation. Resolving the spectrum of Tfh cellular programs between these anatomic compartments in humans has been technically challenging, as T cell states defined by epigenomic versus transcriptomic or proteomic features diverge. Further, gene expression programs of anatomically defined GC-resident versus nonGC Tfh have not been delineated in humans. Such ambiguity has precluded translational efforts to monitor or target precise human Tfh states for clinical benefit. Here, we define human Tfh states in tonsils and blood using trimodal single-cell sequencing, spectral flow cytometry, and spatial transcriptomics to resolve compartment-dependent Tfh gene expression. Tfh with an activated GC-like phenotype were highly enriched in G Protein Subunit Gamma 4 (Gγ4, encoded by GNG4). By integrated analyses of Tfh transcription and chromatin accessibility, we inferred transcription factors driving GNG4 expression that are known to mediate T cell activation. Concordantly, Gγ4 protein was induced in T cells from tonsil and peripheral blood upon stimulation. In tonsils, we found that GNG4 expression distinguishes activated Tfh states within anatomically defined GC. Together, these data resolve GNG4 as a central feature of the GC Tfh activation program in humans.