Meremikwu, I. C. | bioRxiv Science

bioRxiv2025

Multimodal analysis defines GNG4 as a distinguishing feature of germinal center-positioned CD4 T follicular helper cells in humans

CD4 T follicular helper (Tfh) cells coordinate humoral immune responses within germinal centers (GC) of lymphoid tissue. Despite their critical roles in vaccination and autoimmunity, the gene expression programs that define functionally distinct human Tfh states-- and the molecular pathways engaged by Tfh positioned within the GC niche--remain incompletely understood. This gap has limited translational efforts to monitor or therapeutically target specific Tfh states for clinical benefit. Here, we delineate human CD4 T cell heterogeneity in tonsils and peripheral blood using trimodal single-cell sequencing and spectral flow cytometry to define epigenomic, transcriptional, and proteomic features of distinct Tfh states. Tfh with a GC-like phenotype exhibited markedly increased chromatin accessibility and both mRNA and protein expression of G protein subunit gamma 4 (GNG4). In tonsil, single-cell spatial transcriptomics defined GNG4 expression as a distinguishing feature of activated Tfh states within spatially demarcated GC compartments, with greater specificity than conventionally GC-associated features such as BCL6, TOX2, and S1PR2. In contrast, GNG4- Tfh primarily localized to nonGC regions and exhibited a resting, Th17-polarized phenotype. Together, these data highlight GNG4 as a central feature of activated, GC-positioned Tfh cell identity in humans. One Sentence SummaryGNG4 expression defines activated CD4 T follicular helper cells localized to the germinal center of human lymphoid tissue.

immunology↗

bioRxiv2022

Large scale functional screen identifies genetic variants with splicing effects in modern and archaic humans

Humans co-existed and interbred with other hominins which later became extinct. These archaic hominins are known to us only through fossil records and for two cases, genome sequences. Here we engineer Neanderthal and Denisovan sequences into thousands of artificial genes to reconstruct the pre-mRNA processing patterns of these extinct populations. Of the 5,224 alleles tested in this massively parallel splicing reporter assay (MaPSy), we report 969 exonic splicing mutations (ESMs) that correspond to differences in exon recognition between extant and extinct hominins. Using MaPSy splicing variants, predicted splicing variants, and splicing quantitative trait loci, we show that splice-disrupting variants experienced greater purifying selection in anatomically modern humans than in Neanderthals. Adaptively introgressed variants were enriched for moderate effect splicing variants, consistent with positive selection for alternative spliced alleles following introgression. As particularly compelling examples, we characterized a novel tissue-specific alternative splicing variant at the adaptively introgressed innate immunity gene TLR1, as well as a novel Neanderthal introgressed alternative splicing variant in the gene HSPG2 that encodes perlecan. We further identified potentially pathogenic splicing variants found only in Neanderthals and Denisovans in genes related to sperm maturation and immunity. Finally, we found splicing variants that may contribute to variation among modern humans in total bilirubin, balding, hemoglobin levels, and lung capacity. Our findings provide novel insights into natural selection acting on splicing in human evolution and demonstrate how functional assays can be used to identify candidate causal variants underlying differences in gene regulation and phenotype.

genomics↗