A distinct subset of CD25 negative T-follicular regulatory cells localizes in the germinal centers

T-follicular helper cells (Tfh) differentiate through a multistep process culminating in germinal center (GC) resident GC-Tfh that provide support to GC B-cells. T-follicular regulatory cells (Tfr) have been shown to have critical roles in the control of Tfh and germinal center formation. While Tfh cells are inhibited by IL-2, Treg cells depend on it. Here we describe a novel CD25 negative subset within both murine and human PD1+CXCR5+Foxp3+ Tfr that is preferentially located in the GC and can be clearly differentiated from non-GC Tfr, Tfh and effector Tregs by expression of a wide range of molecules. In comparison to Tfr and effector Tregs, GC-Tfr cells partially downregulate IL-2 dependent canonical Treg features, but retain suppressive function, while simultaneously upregulating genes associated with Tfh and GC-Tfh. We suggest that, similar to Tfh, Tfr follow a differentiation pathway culminating in a distinct GC resident subset, GC-Tfr.