Representative gating protocols for Tfr cells based on CXCR5 and ICOS or on CXCR5 and PD-1 expression. Both strategies pregate on CD4⁺CD19⁻ FoxP3⁺cells. “Full minus CXCR5 biotin” indicates staining control in which the CXCR5 biotin is omitted.

Exposure to invading pathogens results in DC activation and antigen presentation in the T cell zone. A population of naïve T cells (Tnaive) that interact with DCs differentiate into T follicular helper (Tfh) cells. Tfh cells express CXCR5, which senses gradients of CXCL13 within the GC and directs Tfh cells into the GC. Simultaneously, a population of natural Tregs (nTreg) interacts with DCs and differentiates into T follicular regulatory (Tfr) cells. Box A shows the cues that modulate Tfr cell differentiation with positive regulators of differentiation denoted in green and negative regulators denoted in red font (OPN= osteopontin). Tfr cells in the T cell zone then diverge. A subset of Tfr cells with lower CXCR5 (and lower CD69) expression follows S1P gradients and exits the LN via the efferent lymph, and are destined to become memory-like cells. A second subset of Tfr cells with high CXCR5 (and high CD69) expression follows CXCL13 gradients to the GC. In the GC, Tfh cells interact with GC B cells, stimulating them to undergo activation, class switch recombination, affinity maturation, and differentiation into Memory B cells (Mem B) and plasma cells (PC). In the GC, Tfr cells interact with Tfh and GC B cells, leading to suppression of both Tfh and GC B cells. Box B shows a schematic of Tfr suppression of Tfh cells. Tfr cells suppress Tfh cell production of cytokines such as IL-21, IL-4 and IFNγ that stimulate B cells. Box C shows a schematic of Tfr suppression of GC B cells where positive mediators of Tfr suppression are in green and negative regulators of Tfr suppression are in red.

A) Model in which Tfr cells limit B cell stimulation by causing downregulation of MHC and B7-1 or B7-2 on the GC B cell. Tfr expressed CTLA-4, for example, may downregulate B7-1/B7-2. However, downregulation also may result from soluble factors (e.g., cytokine) or other receptor based mechanisms. B) Model in which Tfr cells produce suppressive cytokines, such as IL-10 and TGF-β which may inhibit activation of GC B and/or Tfh cells. C) Model in which Tfr cells suppress B cell responses by directly killing activated GC B and/or Tfh cells. Cytolysis could be mediated by granzymes produced by the Tfr cell and directed towards the GC B and/or Tfh cell. D) Model in which Tfr cells mechanically disrupt interactions between Tfh and B cells. Tfr cells bind to GC B cells and/or Tfh cells and push the cells apart, resulting in the separation of Tfh and GC B contacts. This separation reduces both Tfh and GC B activation since costimulation and antigen presentation are disrupted. E) Model in which a ligand on Tfr cells engages a receptor on GC B cells and/or Tfh cells, resulting in negative signaling into the GC B and/or Tfh cell and dampening of BCR and TCR signals.