Determinants of Fate Progression Beyond the Human Tfh Cell Stage

T follicular helper (Tfh) cells have been reported to have multiple trajectories, including terminally differentiated Tfh, memory-like circulatory Tfh (cTfh), and, most recently, induced T follicular regulatory (iTfr) cells (1, 2, 3). While sufficient data have validated the presence of each of these paths, the molecular machinery determining Tfh cell fate remains unclear due to the complex temporal and spatial influences within tissues. To bridge this gap in knowledge, we have applied the human tonsillar organoid (TO) system to study Tfh cell development longitudinally in a tissue-like setting. TOs are an all-human model system containing a range of cell types and stimulatory molecules that recapitulate key features of human follicular biology (4, 2). Our preliminary data suggest that when cultured alone, human tonsillar Tfh cells appear terminally differentiated, but when incorporated into autologous TOs, the same cells spontaneously divide and differentiate. Using intravital labeling of Tfh cells cultured in TOs combined with single-cell analysis, we have mapped the transcriptome of differentiating cells for each cell fate on a per-division basis. We have found that Tfh cells can either: 1) remain undivided and maintain their identities, 2) divide and upregulate markers associated with tissue egress (CCR6, S1PR1, and KLF2) and memory (CCR7), or 3) divide and differentiate into FOXP3-expressing iTfr cells. Using spatial transcriptomics of healthy tonsils, we have verified that each Tfh developmental trajectory maps to a distinct anatomic niche, linking cellular fate decisions to specific tissue microenvironments. In common variable immunodeficiency (CVID) and likely other immunodeficiencies, Tfh cell fate decisions appear to be disrupted (5). Patients often display skewed Tfh subset ratios and impaired humoral responses, suggesting a breakdown in the spatial and molecular cues that normally guide Tfh development. A better understanding of these developmental pathways and their drivers could inform translational strategies to improve therapeutic targeting and enhance humoral immunity.