Loss of Sphingosine-1-Phosphate Receptor 4 weakens innate mechanisms leading to defective lymph node hypertrophy and germinal center formation

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen-surveillance and identification of cognate lymphocytes. Sphingosine-1-phosphate (S1P) and its receptors (S1PR1-5) regulate various aspects of immunity, however, the role of S1PR4 in adaptive responses is not well understood. Here we use a localized TH1 footpad immunization model to carefully monitor changes in immune populations within circulation, the immunization site, and the dLN. We found that mice lacking S1PR4 ultimately developed attenuated GC and activation pathways due in part to upstream defects in neutrophil activity. Within hours of immunization, failed neutrophil mobilization and infiltration into the tissue of S1PR4-/- mice resulted in reduced local inflammation and vascular changes. Using depletion antibodies, we show that neutrophils were necessary to facilitate the early edema as well as the redirection of circulating lymphocytes into the dLN, thereby contributing to reduced dLN hypertrophy in S1PR4-/-. Using in vivo adoptive transfer, we demonstrated that these homing deficiencies were not lymphocyte-intrinsic but instead due to microenvironment failures within the S1PR4-/- host. Consistent with the known role of lymphocyte entry promoting DC emigration, the dLN of these mice exhibited defects in the prolonged but not early accumulation of DC. This was accompanied by inadequate growth of the vascular network, and reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule (HEV) regions, all of which can contribute to LN expansion. Overall, our study reveals that S1PR4 plays an important amplifying role during the early innate response that affects the magnitude of a downstream GC reaction. Overall design: Differential expression of wild type and S1PR4 knockout in Tfh cells, non-Tfh CD4 T-cells, germinal center B-cells, and non-germinal center B-cells.