and CXCL11 bind to the canonical receptors CXCR4 and CXCR3, respectively. Here, we present the engineering of a chemokine-like chimera, which selectively binds to ACKR3. The addition of a ybbR13 tag at the C-terminus allows site specific enzymatic labeling with a plethora of fluorescent dyes. The chimera is composed of the N-terminus of CXCL11 and the main body and C-terminus of CXCL12 and selectively interacts with ACKR3 with high affinity, while not interfering with binding of CXCL11 and CXCL12 to their cognate receptors. We further provide evidence that the chimera can be used to study ACKR3 function in vivo.

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3GFP/+, ACKR4GFP/+, GPR182mCherry/+, and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.

Highlights d ACKR3 marks two equal-sized marginal zone B cell (MZB) populations d ACKR3-negative MZBs can differentiate into ACKR3-positive cells, but not vice versa d Transferred ACKR3 ko follicular B cells fail to populate an empty marginal zone d ACKR3-deficient MZBs are impaired in mounting an early immune response