Migration of monocytes into tissues is driven by activation of the G protein-coupled receptor CCR2 in response to inflammatory chemokine CCL2 and is critical to normal immune responses. In addition to cell migration, CCR2 has a largely unappreciated scavenging function, by which it sequesters excess chemokine from the extracellular environment. We believe that this scavenging function allows CCR2 to control its own chemokine gradient, while its ability to recycle rather than downregulate allows it to maintain responsiveness in the presence of high chemokine concentration. Similarly, this function may be responsible for the difficulty observed when attempting to pharmacologically inhibit CCR2 activity. Therefore, an understanding of the molecular drivers underlying this chemokine scavenging is essential for developing effective therapeutics.Chemokine scavenging was widely regarded as a function of only atypical chemokine receptors (ACKRs), while scavenging by G protein coupled chemokine receptors has largely been ignored. In our soon to be published study (Chapter 1), we show that scavenging by CCR2 occurs in a manner independent of G proteins, G protein receptor kinases, arrestins, or clathrin, suggesting that a yet unknown non-canonical mechanism is largely responsible for this function. We therefore took a discovery-based approach using proximity labeling proteomics to further define the interactome of CCR2 along G protein-dependent and -independent pathways (Chapter 2). Finally, we discuss speculative, yet exciting, explanations of observations made in our latest findings and consider possible future experimental directions (Chapter 3). Collectively, the work presented herein highlights the complexity of CCR2 function, and significantly contributes to understanding and identifying the molecular drivers involved in chemokine scavenging.

Despite promising clinical responses to programmed cell death-1 (PD-1) blockade in less than 20% of head and neck squamous cell carcinoma (HNSCC) patients, advances are needed to extend these benefits to patients with resistant tumors. Resistance is attributed to immune suppression within the tumor microenvironment (TME), which restricts the intratumoral (IT) recruitment and activation of tumor-killing CD8+ T cells, CD4+ T cells, and NK cells. CXCR3, a chemokine receptor for the interferon-inducible chemokines, CXCL9, CXCL10, and CXCL11, is highly expressed on activated CD8+ and CD4+ T cells as well as NK cells and plays a critical role in directing their migration. Chapter 1 establishes the correlation with increased expression of CXCR3 and its ligands, CXCL9 and CXCL10, with early tumor stage, increased immune infiltration, inflammatory gene signatures, and improved overall outcomes in patients with HNSCC. In chapter 2, syngeneic murine models of tobacco-associated HNSCC are utilized to show that IT delivery of recombinant CXCL10 drives tumor elimination through the recruitment of CD8+ T cells, CD4+ T cells, and NK cells. Furthermore, we demonstrate that by combining IT CXCL10 treatment with PD-1/PD-L1 checkpoint blockade inhibition, we achieve further suppression of tumor growth and complete remittance. Chapter 3 highlights that T cells recruited to tumors via CXCL10 ligand display enhanced activation, decreased markers of early T cell exhaustion, and increased tumor antigen specificity, indicating that CXCL10 not only directs cell migration, but may also enhance T cell function. Finally, in chapter 4, we explore additional projects and future directions aimed at enhancing the effectiveness of CXCL10-treatment through combinational therapies and improved delivery.

The past several years have seen a rapid advancement in our understanding of how CXCR4 transmits the extracellular CXCL12 signal into intracellular signaling, which ultimately causes cell migration along with other signaling outcomes critical in both immune cell function and various cancers. We began this project uncertain as to the stoichiometry of CXCL12:CXCR4 binding, and through various functional and structural studies were able to settle on an early complex model with 1:1 stoichiometry (chapters 2&3). An improvement in our ability to model the CXCL12:CXCR4 complex was enabled by the solution of the vMIP-II:CXCR4 crystal structure by our laboratory soon after, and other landmarks in chemokine receptor structural determination (chapter 4) have allowed for a global understanding of chemokine:receptor structure to emerge. At this point the structural link between CXCL12 N-terminus binding and the more conserved intracellular aspects of CXCR4 activation is finally coming into view. In our latest soon to be submitted study, we provide functional evidence decisively supporting the orientation proposed in our current model, as well as revealing previously unrecognized complexity in the nature of CXCR4 activation (chapter 5). In chapter 6, we comprehensively discuss tentative but exciting possible explanations for evidence in our latest findings of greater complexity in chemokine:chemokine receptor signaling than has been appreciated.

We have progressed from a rough understanding of how CXCL12 activates CXCR4 to an experimentally validated full-length CXCL12:CXCR4 signaling complex model. Ultimately, we have made significant contributions to understanding the structure of CXCR4 bound to CXCL12, the precise mechanism whereby CXCL12 stabilizes CXCR4’s active state(s), and the complexity of the downstream functional consequences of receptor activation.