ICOS-ICOSL Interactions Orchestrate Follicular Helper T Cell Differentiation And Function

High affinity antibodies generated by germinal center (GC) B cells is one of the mostimportant arms to protect human health from long-term infection diseases. However, GCB cells cannot survival and proliferate normally without helper signaling from T cells.CD4+T follicular helper (Tfh) cells specifically support GC response in B cell zone orcalled follicle. Now we know that Tfh cells is absolutely necessary for GC reactions.However, the detail mechanisms under how T cells differentiate into Tfh cells in follicularregion and how Tfh cells support and regulate GC reaction are still unclear.Inducible co-stimulator (ICOS) molecule belongs to CD28co-stimulatory familyand it is highly expressed on Tfh cells. Firstly, based on the in vitro retrovirus overexpression system combined with antigen specific lymphocyte transfer animal model, wefound that ICOS deficient T cells failed to migrate into follicle even in the CXCR5overexpression condition. The location of activated T cells in μMT:Icosl-/-bone marrowchimeras showed that ICOSL on bystander B cells was necessary for ICOS dependentrecruitment process. In addition, we analyzed the T cell motility in vitro on lipid bi-layerwith TIRF microscopy and found that ICOS stimulation can promote the pseudopodgeneration and persistent motility of T cells. In addition, the function of ICOS infacilitating cell motility were verified in vivo with intravital2-photon microscopy. Thenthe in vivo imaging experiments further shown that ICOSL on bystander B cells werenecessary for T cells persistent motility.Tfh cells support GC reaction through co-localization and physical contact withcognate B cells. The mix chimera model constructed with Icosl+/+: Icosl-/-bone marrowshown that ICOSL had intrinsic function in GC B cells. Based on the calcium responsein T cells and cell-cell contact status in vivo, we found that ICOSL facilitated the calciumsignaling in T cells and then promoted Tfh cells to entangle with wild type, but not ICOSLdeficient GC B cells. Then the transcriptional analysis indicated that wild type GC B cellsacquired much more CD40signaling from Tfh cells while competing with ICOSLdeficient GC B cells. In addition, we further cultured B cells with different stimulation invitro and discovered that CD40signaling specifically upregulated ICOSL expression onGC B cells as the feedback. At last, the BCR mutation analysis shown that ICOSL can directly regulate affinity maturation of B cells, beside the plasma cell differentiationprocess.In summary, based on the discovery of novel functions of ICOS and ICOSLmolecules, we clarified the mechanisms under how ICOS regulate T cell motility and Tfhcells differentiation, and demonstrated that Tfh cell could promote the GC reaction andaffinity maturation through the positive feedback loop composed with ICOS-ICOSL andCD40L-CD40pathway.