OBJECTIVES/GOALS: The effect of immunosuppressive metabolites on anti-tumor immunity in human papillomavirus (HPV)-associated vs carcinogen-driven head and neck cancer is unknown. The objective of this study is to define the extent to which metabolites impair this response and identify novel metabolic targets for enhancing anti-tumor immunity. METHODS/STUDY POPULATION: HPV-associated and carcinogen-driven head and neck squamous cell carcinoma specimens were frozen following surgical excision, and tumor sections were cut onto glass slides. Slides were coated in alpha-cyano-4-hydroxy-cinnamic acid (CHCA) matrix and subjected to mass spectrometry imaging using matrix-assisted laser desorption ionization (MALDI) on a Bruker SolariX XR 12T Hybrid QqFT-ICR mass spectrometer run in positive mode. Slides were then stained for immunohistochemistry (IHC) using markers of CD8 T cells, macrophages (CD163), B cells (CD20), and tumor cells (panCK). Mass spectrometry imaging and IHC spatially resolved data will be co-registered and metabolite intensity in regions of interest (cell types) quantified. RESULTS/ANTICIPATED RESULTS: A total of seven HPV-associated (three metastatic lymph nodes and four primary tumors) and six carcinogen-driven (primary tumors) HNSC specimens were subjected to MALDI and IHC. Metabolites significantly enriched in HPV-associated HNSC relative to carcinogen-driven HNSC include 2,3-diphosphoglyceric acid, xanthine, 2,3,5-Trichloromaleylacetate, and indole-3-carboxyaldehyde. Metabolites significantly enriched in carcinogen-driven HNSC relative to HPV-associated HNSC include hesperetin 3'-O-sulfate, hypoxanthine, phosphorylcholine, and L-homocysteine sulfonic acid. In ongoing analyses, we anticipate identifying a relationship between CD8+ T cell enriched vs depleted regions and immunosuppressive metabolites (e.g., kynurenine, adenosine monophosphate). DISCUSSION/SIGNIFICANCE: Defining the extent to which CD8+ T cells interact with the metabolic milieu of the microenvironment will provide a foundation for metabolic Precision Medicine. Strategically targeting metabolic pathways to enhance the anti-tumor immune response will be leveraged for the design and implementation of immune modulatory metabolic therapy.