Mentor Name: Hui Feng

Neuroblastoma accounts for approximately 15% of cancer-associated deaths in children. Furthermore, approximately 50% of the high-risk neuroblastoma cases are marked by MYCN gene amplification, driving aggressive disease. The Feng Lab previously reported that MYCN transcriptionally upregulates chemokine-like factor (CKLF), which has been shown to induce immunosuppression and tumor aggressiveness. The secreted isoform of CKLF, CKLF1, binds CCR4 receptors on CD4+ cells, recruiting them to the tumor microenvironment (TME). Functionally, CD4+ T-regulatory cells (Tregs) suppress the immune response by suppressing cytotoxic T and natural killer cells. However, CCR4 is not uniquely expressed on Tregs and is present on many beneficial immune cells, leading to off-target consequences. This highlights the need for a broader understanding of the CKLF-CCR4 interaction. I hypothesize that the CKLF1-CCR4 signaling induces immunosuppression through the recruitment of CD4+ cells to the TME. This research will characterize the CKLF1-CCR4 signaling on CD4+ cells upon CKLF1 treatment or CCR4 modulation through two aims. Aim 1 will study how CKLF1 supplementation promotes the CKLF1-CCR4 signaling in T cells by western blotting and the immunoproperties of these cells by flow cytometry analysis. I have already successfully cloned and produced CKLF1 protein via E. Coli bacteria, and have produced a pure protein sample via Ni-NTA column purification. I will use the purified protein to accomplish Aim 1. In Aim 2, I will determine how the CKLF-CCR4 signaling is impacted upon genetic overexpression or depletion of CCR4 in JURKAT T cells by western blotting and flow cytometry analysis. Current drugs focus on targeting the CCR4 receptor on CD4+ Tregs. Understanding the mechanisms by which CKLF1 induces immunosuppression will facilitate the development of therapeutic strategies to treat high-risk neuroblastoma.