Abstract T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody producing B-cells in human and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4 + T cell in vitro cultures. Further, adoptive transfer experiments using WT and Rinl-KO naïve CD4 + T cells unraveled T cell-intrinsic functions of Rinl. Mechanistically, Rinl regulates CD28 internalization and signaling, thereby shaping CD4 + T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore connect Rinl with CD28 internalization and signaling pathways in CD4 + T cells, demonstrating for the first-time the importance of endocytic processes for Tfh differentiation. Highlights Rinl-KO CD4 + T cells show increased Tfh differentiation in a context independent manner The regulation of Tfh differentiation is T cell-intrinsic Rinl controls CD28 endocytosis and shapes Tfh-specific CD28 signal transduction Human Tfh differentiation is regulated by Rinl

The guanine nucleotide exchange factor Rin-like acts as a gatekeeper for T follicular helper cell differentiation via regulating CD28 signaling