TCR clonotyping of paired blood and tonsillar T-follicular cells reveals the mixed origin of CD38high Tfrs.

Circulating T follicular regulatory (cTfr) cells are measured as blood biomarkers after vaccination, in autoimmune disease (e.g., lupus, rheumatoid arthritis) and cancer immunotherapy, yet whether they originate from natural Tregs (yielding nTfr) or germinal center T follicular helper (GC Tfh) cells (yielding iTfr) remains unknown. This distinction is critical for interpreting cTfr expansion as Treg-derived regulation versus Tfh-derived negative feedback. To characterize tonsillar follicular T cell heterogeneity and resolve the origins of cTfr cells we applied single-cell multiomics and bulk T cell receptor sequencing (TCRβ-seq) analysis of matched human tonsil and blood. Tonsillar nTfr and iTfr cells distribute across shared transcriptional states, with CD38 expression distinguishing iTfr from nTfr in most contexts. However, within a transitional cluster positioned between activated Tfh and Tfr cells, nTfr-like cells also upregulated CD38, indicating that its specificity for iTfr is not absolute. Peripheral blood CD38- cTfr cells shared clonotypes exclusively with non-follicular Tregs and lacked overlap with GC Tfh, indicating predominantly Treg origin. CD38high cTfr cells captured a mixed population: some shared clonotypes with tonsillar GC Tfh (iTfr), while others shared clonotypes with non-follicular Tregs (nTfr). These findings establish that accurate discrimination of iTfr versus nTfr within the CD38high compartment requires TCR clonal reference to matched tissue.