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Summary: In this work, Peng et al present the previously unsolved apo structure of the C-C motif chemokine receptor 8 (CCR8). CCR8 is a G-protein coupled receptor (GPCR) and a potential therapeutic target for treating some cancers and autoimmune diseases. Previous structural studies of this protein have resolved CCR8 in complex with an antagonist antibody as well as with its endogenous agonist CCL1; this study solves the ligand-free structure of CCR8 to gain insights into how a nonpeptidic agonist LMD-009 induces calcium flux. The authors demonstrate that two mutants of the CCR8 protein lead to diminished cellular ability to mediate calcium mobilization, indicating an interaction with LMD-009 and providing insights into CCR8 agonism and activation. The structure presented within the manuscript is of high quality and the conclusions of this work could be enhanced using a combination of in silico structural analysis and additional experiments that couple their structural insights to cellular function.

Major Points:

• The authors report that the structure of the apo CCR8 is similar to the structure of CCR8-CCL1, “indicating an active state” (line 27). Clarification of what this “active state” entails in the context of the experimental results presented would be useful in interpreting the results as well as understanding the impact of this structure.

• A significant portion of the background is dedicated to an explanation of the role of CCR8 on T helper 2 (Th2) and tumor-resident regulatory T (Treg) cells. At the least, some discussion tying the results presented in this manuscript to these details would make the story more coherent.

• A functional comparison of LMD-009 with the natural ligand, CCL1, would help contextualize the results presented in Figure 6, especially with the authors emphasizing the similarity of the apo and CCL1-bound forms of CCR8. Does CCL1 activate calcium flux in the same way for this assay? 

• The authors rely on previous studies to inform the design of their functional assays in Figure 6, including a study that makes use of molecular dynamics (MD) simulations to identify potential key interactions of CCR8 and an inhibitor. A brief in silico study (either modeling or, preferably, molecular simulation in line with the previous study) of this newly solved structure with these two key mutations introduced with and without LMD-009 present, would greatly bolster the mechanistic claims made by the authors.

• Figures 6H and 6L contain the experimental results for the mutations of interest, but other panels (6B, 6F, 6I) show decreases in total response (MAX-MIN ratio). While differences in EC50 are communicated, a discussion of why these mutations lead to decreased total response would be valuable. For similar reasons, results presented in Figures 6G and 6J, where the EC50 of LMD-009 has been improved by mutations, should be discussed. 

Minor Points:

• An explanation of the notation used when printing residues in the manuscript may be useful to readers without intimate knowledge of GPCR biochemistry.

• Authors may consider moving Figure 1 to the Supplemental Information.

• Authors should provide more details on the validation of reporter cell lines, including transfection protocols. 

• Line 184-185: “The structure of CCR8 in complex with LMD-009 is expected to be solved…” requires some clarification. Are the authors expecting to solve this structure as well, or is this speculation that someone else eventually will and will see the importance of these interactions?

Competing interests

The authors declare that they have no competing interests.