Objective: Human leukocyte antigen (HLA) class I molecules activate CD8 T cells by presenting peptides from viruses and other non-self proteins. Loss of HLA class I is associated with reduced CD8 T cell activation. We have previously shown that tobacco extracts reduce HLA class I surface expression by disrupting biosynthesis through a decrease in the transporter associated with peptide processing (TAP)1. Tobacco contains over 6,000 substances; preliminary studies have shown that nicotine and two well characterized carcinogens do not reduce HLA class I or TAP1. Here we use fractionation to partially identify the compound(s) that reduce HLA class I expression. Methods: Snuff extract was partitioned into four (4) fractions according to Swain et al. 1) polar compounds and strong acids 2) ether soluble weak acids 3) water soluble organic bases, and 4) ether soluble organic neutral compounds. Multiple concentrations of extracts were tested using mAb W6/32 to assess HLA class I expression by flow cytometry. As a control for toxicity, the surface expression of Ig was also assessed by antibody. Results: As before, whole extract reduced HLA class I expression by ~40%. Fraction 1 showed similar reductions, while the other fractions (2,3, and 4) had no effect. Solvent alone also had no effect on either HLA class I expression or cell viability (as assessed by propidium iodide). None of the extracts affected the expression of Ig, demonstrating that extract 1 acted on HLA class I and was not a non-specific effect. Conclusions: The whole snuff extract was generated in water and the fraction with similar activity is extract 1, a water soluble extract, consistent with our original findings. Future experiments will involve subfractionation of extract 1 to narrow down the possible compound(s) responsible for reducing the HLA class I surface expression.

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