Soluble LAG-3 binding to cancer cells - influence of cell types, MHC class II and lipid rafts

Smith, Kendra Laura (2024) Soluble LAG-3 binding to cancer cells - influence of cell types, MHC class II and lipid rafts. Masters thesis, Memorial University of Newfoundland.

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Abstract

Interaction of immunoinhibitory molecules, lymphocyte activating gene-3 (LAG-3) and programmed cell death protein-1 (PD-1) on tumor infiltrating lymphocytes with their respective ligands, contributes to an exhausted immune phenotype. However, soluble LAG-3 (sLAG-3) enhances immunity by inducing dendritic cell maturation by binding MHC-II associated with lipid rafts. The first part of this thesis assessed the importance of MHC-II/lipid rafts versus peptide/MHC-II complexes (pMHC-II) as sLAG-3 ligands. Analysis of sLAG-3 binding to B-cell lines was done using flow cytometry. Results indicated stable pMHC-II play a larger role in sLAG-3 binding than incorporation of pMHC-II in lipid rafts. sLAG-3 binding, MHC-II, CD59 and PD-L1 were assessed by flow cytometry on IFN-γ treated melanoma cell line (MDA-MB-435) and different breast cancer cell lines (BCCL). Lipid raft disruptor, methyl-B-cyclodextrin was used to treat cell lines to determine if LAG-3 binding was affected by lipid rafts. Cell lines were MHC-II⁺ and PD-L1⁺, but only MDA-MB-435 bound sLAG3. Deficient LAG-3 binding to BCCL could not be explained by HLA-DM or lipid raft deficiency. Lipid raft disruption increased sLAG-3 binding, this could not be explained by MHC-II or CD59. This suggests that lipid raft disruption may expose MHC-II and other ligands to promote sLAG-3 binding. Altogether, results indicate LAG-3 binding is cell-context dependent and more complex than simply binding p/MHC-II complexes, preferentially located in lipid rafts.

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