Comparing chemokine receptor CXCR4 signaling pathways in T cells and B cells

Summary: Abstract Body: Chemokine receptors are expressed broadly throughout different cells in the immune system. However, it is unclear if the signaling pathways used by the same receptor are similar or different between cell types. The purpose of this study was to test the hypothesis that the chemokine receptor CXCR4 utilizes different signaling partners in T cells compared to B cells. CXCR4 was chosen as the receptor of interest as it is highly expressed on both B and T cells and plays a critical role in appropriate bone marrow extravasation and coordinating immune responses. CXCR4 is also a HIV co-receptor and is thought to be involved in cancer metastasis. Utilizing CRISPR-Cas9 gene editing, a peroxidase tag, APEX2, was appended to the endogenous loci of CXCR4 at the C-terminus in B cells (Raji) and T cells (Jurkat). APEX2 labels proteins with biotin in a radius of approximately 200 angstroms following a pulse of hydrogen peroxide. Biotin-labeled proteins allow for enrichment by streptavidin beads and subsequent analysis by mass spectrometry. We validated efficient biotin labeling by anti-biotin western blotting. We confirmed that the APEX2 tag does not interfere with G protein activation by CXCL12 or prohibitively impair surface expression. We further probed downstream signaling events using RNA-seq following treatment of wild-type B cells or T cells with CXCL12 compared to CXCR4 knockout B cells or T cells. Our integrated multi-omics workflow allows for rigorous evaluation of key signaling mediators of a common chemokine receptor in different immune cells, illuminating fundamental principles of signal transduction. Emily M. Meara^{1, 2, 3}, Jeffrey S. Smith^{1, 2}, Ari S. Hilibrand², Julia M. Rogers², Andrew C. Kruse² 1. Dermatology, Brigham and Women's Hospital, Boston, MA, United States. 2. Dermatology, Harvard Medical School, Boston, MA, United States. 3. Dermatology, University of Massachusetts Chan Medical School, Worcester, MA, United States. Adaptive and Auto-Immunity