The Atypical Chemokine Receptor ACKR3: Function in vivo in Immune Responses and Coupling to Signal Transduction Pathways

In jawed vertebrates throughout development cells migrate along predefined cues to find their destination. In adults most prominent is the continuous movement of immune cells engaged in surveillance and defense of the host. Some tumors have adopted the migratory scheme to escape immune reactions and to spread in metastasis. The chemokine system is known as essential regulator in directing cell migration, where locally produced chemokines form patterns on which cells can migrate through the activation of G-protein coupled chemokine receptors. However, the maintenance of spatial gradients requires the presence of sinks in apposition to the source of attractant. Recently grouped separately and renamed as atypical chemokine receptors, the ACKRs define a group of structurally related receptors for chemokines which mainly act as sinks and therefore support their function in orchestrating cell migration.ACKR3 binds and scavenges the chemokines CXCL12 and CXCL11. Several reports indicate that the receptor contributes to the overall activity of the CXCR4, the chemokine receptor for CXCL12. Its prominent mRNA expression levels in B cells subsets has prompted us to investigate its role in immune responses. We have recently performed in vitro studies showing that ACKR3 is transiently upregulated in plasmablasts during B cell maturation and that the expression is required to license the cells to egress from CXCL12 rich germinal centers. Preliminary data from in vivo studies using pharmacological inhibitors of ACKR3 point in the direction that ACKR3 is required for the prolonged generation of vaccine directed IgG. The finding is consistent with production of IgG from bone marrow resident plasma blasts, which appear absent after inhibition of ACKR3. We intend now to confirm the observations in vivo using appropriate mouse models where conditional ACKR3fl/fl is selectively deleted in the B cell compartment (CD19cre) or in maturing plasmablast (BLIMP-1cre and IgHg1cre). The data shall reveal whether therapeutic inhibition of ACKR3 can be used to suppress undesired immune responses. Using a ACKR3 reporter mouse with an allele of the receptor replaced with green fluorescent protein (GFP) showed a marked expression in marginal zone B (MZB) cells consistent with the reported high mRNA levels. FACS analysis of MZBs surprisingly unveiled that only about 50% of the cells express GFP. The two populations are well separated and we have used these criteria to sort the cells and extracted mRNA for transcriptome analysis. Part of the proposal is to functionally characterize the two populations.ACKR3 like all atypical chemokine receptors does not couple to heterotrimeric G-protein, but can recruit arrestin in response to ligand binding. Although the receptor displays ligand independent cycling addition of chemokine enhances the intracellular trafficking. CXCR4, which couples to Gi-proteins, also binds arrestin and can presumably activate downstream signaling pathways. It was proposed that the equilibrium between G-protein and arrestin-dependent signaling pathways can be modulated by ligands as well as cellular components, a process called biased signaling. We propose here to analyze the receptor proteome of ACKR3 and CXCR4 by APEX2 proximity labeling. Peptides of affinity purified proteins from receptor proteomes obtained under different (biased) stimulatory conditions will be analyzed by mass spectrometry (B. Wollscheid, ETHZ). Quantitative comparison shall provide insight into intracellular signaling networks downstream of ACKR3 and CXCR4. We hope to find markers of biased signaling cascades that may be later used to specifically target the pathways in therapeutic settings.