No abstract

The lymph node periphery is an important site for many immune functions, from pathogen containment to T helper differentiation, yet the cues that position cells in this region are largely undefined. Here, using a sphingosine 1-phosphate (S1P) reporter, we found that cells sensed higher concentrations of S1P in the medullary cords than in the T zone, and the S1P transporter SPNS2 on lymphatic endothelial cells generated this gradient. Natural killer (NK) cells are located at the periphery of the lymph node, predominantly in the medulla, and we found that expression of SPNS2, S1P receptor 5 on NK cells, and CXCR4 were all required for NK cell localization during homeostasis and rapid interferon-γ production by NK cells after challenge. Our findings elucidate the spatial cues for NK cell organization, and reveal a novel role of S1P in positioning cells within the medulla.

The lipid chemoattractant sphingosine 1-phosphate (S1P) guides cells from the low-S1P environment of tissues into the high-S1P environment of circulatory fluids( 1 ). Notably, S1P directs T cell exit from lymph nodes (LN), where T cells are initially activated, into lymph, from which T cells reach blood and ultimately inflamed tissues( 1 ). T cells follow S1P gradients primarily using S1P receptor 1 (S1PR1)( 1 ). While recent work has described how S1P gradients are established at steady-state, little is known about S1P distribution in disease, or about how changing S1P levels may affect immune responses. Here, we find that S1P concentrations increase in LN during an immune response. Hematopoietic cells, including inflammatory monocytes (iMo), are an important source of this S1P, an unexpected finding as endothelial cells provide lymph S1P( 1 ). iMo require the early activation marker CD69 to supply this S1P, in part because CD69 expression is associated with reduced levels of S1pr5 . CD69 acts as a “stand-your-ground” signal, keeping immune cells at a site of inflammation by regulating both S1P receptors and S1P gradients. Finally, increased S1P prolongs T cell residence time in LN, and exacerbates the severity of experimental autoimmune encephalomyelitis. This finding suggests the hypothesis that LN residence time regulates T cell differentiation, and points to novel uses of drugs targeting S1P signaling.

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell–derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.