The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit -arrestin1 and -arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit -arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4. K E Y W O R D SACKR4, atypical chemokine receptor, CCL19, CCL20, CCL21, CCL25, chemokine scavenging, -arrestin
The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the β2-adrenergic receptor (β2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with β2AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38.
Secondary lymphoid organs (SLO) such as the spleen, lymph nodes and Peyer’s patches are strategically positioned to survey bodily surfaces and to support the generation of cellular and humoral immunity. The movement and interaction of antigens, antigen presenting cells, B and T lymphocytes within SLOs is coordinated by specialized fibroblastic reticular cells (FRCs) that form dedicated microenvironments and provide essential niche molecules such as the chemokine CXCL13. High-resolution transcriptomic analysis of Cxcl13-expressing cells in mouse models has previously enabled the molecular characterization of heterogenous B cell-interacting reticular cells (BRC) in lymph nodes. However, it remains unknown to what extent the molecular identity of niche-forming BRCs is conserved across SLOs. Here, we employed single cell RNA-sequencing of Cxcl13-expressing cells from murine lymph node, spleen and Peyer’s patch to compare the molecular identity of BRCs across SLOs. While structural and developmental genes dominated organ-specific gene signatures, we found conserved gene signatures reflecting crucial immunomodulatory functions. The highest conservation was observed in follicular dendritic cells, a BRC subset specialized in the capture and presentation of antigen. Moreover, immunomodulatory gene signatures were preserved in BRCs from human lymph nodes and palatine tonsils highlighting the important role of BRC-defined microenvironments in steering efficient immune responses in SLOs across species.
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