Stability and Progressive Differentiation of Tfr cells are intrinsically and extrinsically controlled by Tfh programs

Follicular regulatory T (Tfr) cells restrain follicular helper T (Tfh) cell-mediated B cell responses in the germinal center (GC) reaction to optimize humoral immunity while limiting autoimmunity. The immune system partially regulates GC responses by controlling the stepwise differentiation of Tfh cells. Whether Tfr cell development requires sequential developmental stages and how the immune system regulates these processes to control humoral immunity is poorly understood. Here, we used longitudinal sampling of lymphoid organs along with fate mapping and matched single-cell RNASeq/TCRseq to assess developmental dynamics of Tfr cells. We found that Tfr cells undergo dynamic clonal expansion. During this process, Tfr cells also undergo progressive differentiation through progenitor-like, early effector, and late effector stages. Late effector Tfr cells possess inherent instability, with a propensity to lose expression of the transcription factor FoxP3 to become ExTfr cells. ExTfr have unique features and can be redeemed to become suppressive Tfr cells. Acquisition of a Tfh-like transcriptional program in Tfr cells was an intrinsic predictor of progeny instability. Extrinsically, Tfh cells enhanced late effector Tfr cell differentiation by diverting early effector Tfr cells away from a default Blimp1-expressing pathway to a Bcl6-expressing one. Moreover, Tfh induce Tcf7 in Tfr cells which promotes late effector Tfr differentiation. Together, these data indicate that Tfr cells are a dynamic and plastic cell subset, the progressive differentiation of which is controlled at later effector stages by intrinsic and extrinsic programs that work together to provide a negative feedback loop to control humoral immunity.