ICOS costimulation is indispensable for the differentiation of T follicular regulatory cells

ICOS is a T cell costimulatory receptor critical for Tfh cell generation and function. However, the role of ICOS in Tfr cell differentiation remains unclear. Using Foxp3-Cre-mediated ICOS knockout (ICOS FC) mice, we show that ICOS deficiency in Treg-lineage cells drastically reduces the number of Tfr cells during GC reactions but has a minimal impact on conventional Treg cells. Single-cell transcriptome analysis of Foxp3+ cells at an early stage of the GC reaction suggests that ICOS normally inhibits Klf2 expression to promote follicular features including Bcl6 upregulation. Further, ICOS costimulation promotes nuclear localization of NFAT2, a known driver of CXCR5 expression. Notably, ICOS FC mice had an unaltered overall GC B cell output but showed signs of expanded autoreactive B cells along with elevated autoantibody titers. Thus, our study demonstrates that ICOS costimulation is critical for Tfr cell differentiation and highlights the importance of Tfr cells in maintaining humoral immune tolerance during GC reactions. Splenocytes from Foxp3YFP-creIcos+/+(ICOS WT) and Foxp3YFP-creIcosfl/fl(ICOS FC) male mice were isolated 6 dpi with NP-OVA/alum and stained with anti-CD4 alexa fluor 647 (GK1.5, BioLegend), anti-TCRβ PeCy7 (H57-597, BioLegend) and propidium iodide (ThermoFisher). Live (PI-) conventional (YFP-) and regulatory (YFP+) CD4+TCRβ+ T cells were sorted with a BD FACSAria (BD Biosciences) and mixed in a 1:10 ratio. A total of 13500 cells from ICOS WT and ICOS FC mice were sent for library preparation. Libraries were generated using the following components from 10x Genomics: Chromium Next GEM Chip G Single Cell kit, Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead kit v3.1, Chromium i7 Multiplex kit. Sequencing was performed by Genome Québecusing a NovaSeq 6000 (Illumina) with a flow cell S1 PE28*91.