Regulation of anti-tumor immunity by CX3CL1

Cancer immunotherapy has become a pillar of cancer treatment, but fails to deliver benefit for many patients. There is thus a critical need to identify and understand the mechanisms that limit the efficacy of cancer immunotherapy, and how they can be overcome. Our lab previously set out to identify mechanisms by which tumor cells can impede the anti-tumor T cell response, as T cells are critical for the success of nearly all immunotherapy strategies. We identified tumor-produced CX3CL1 as one such mechanism, and showed it has a “dominant-negative” effect on tumor immunity: tumors comprised of as little as 10% CX3CL1high tumor cells exhibit a blunted T cell response. We further carried out a pan-cancer analysis of CX3CL1 across 12 human cancer types and found that, in patients, high CX3CL1 is associated with immune microenvironments that are low in T cells and rich in macrophages; features that also correlate with poor responses to ICIs. Mechanistically, we showed that ablation of CX3CL1 in tumor cells resulted in a tumor microenvironment (TME) that is rewired to be “hotter”, characterized by more T cells and fewer CD206high macrophages. Interestingly, dendritic cell (DC) numbers are also increased in both tumors and tumor-draining lymph nodes of CX3CL1KO tumors. Further, knock-out of CX3CL1 sensitizes tumors to ICI. We have additionally found, and recently published, that CX3CL1high tumor cells have a particularly pronounced effect on the TME in their immediate vicinity, creating spatial niches of immunosuppression within the TME. These CX3CL1high regions are characterized by increased CD206high macrophages, fewer T cells and diminished T cell functional activity, and we hypothesize these niches underlie the impaired anti-tumor immunity we observe even when CX3CL1 is heterogeneously expressed. In this proposal, we will use genetic tools, a novel series CX3CL1KO cell lines established by our lab, and spatial analysis of human tumors to dissect the local and systemic effects of CX3CL1 on the TME and on anti-tumor immune control of tumors. Aim 1 will interrogate the mechanisms by which a CX3CL1high TME impairs DC infiltration and activity in tumors, combining functional assays of DCs with genetic tools to define the cell types and signaling axes responsible for impairing DCs in CX3CL1high tumors. Aim 2 will interrogate how CX3CL1high tumor cells create local suppressive niches, using a combination of our novel mouse model of tumor heterogeneity and spatial transcriptomics analysis of human bladder cancer and melanoma samples. Aim 3 will test the ability of CX3CR1:CX3CL1 therapeutic blockade to remodel the TME of CX3CL1high tumors as well as of heterogenous CX3CL1high:CX3CL1low tumors, and to sensitize these tumors to ICI. Together, these experiments will provide critical insights into how CX3CL1 impairs anti-tumor immunity, providing both a foundational for future work targeting the CX3CR1:CX3CL1 axis therapeutically as well as a detailed understanding of how local CX3CL1high tumor regions create immunosuppressive niches that impact systemic anti-tumor immune control. Project Number: 1R37CA304560-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Melissa Reeves | Institution: UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH, SALT LAKE CITY, UT | Award Amount: $518,090 | Activity Code: R37 | Study Section: Basic Cancer Immunobiology Study Section[BCIB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11368781