HSP interaction with individual CD91 ligand binding domains 2255685

Abstract Introduction Heat Shock Proteins (HSPs) are a structurally diverse class of proteins that respond to cellular stress and naturally function to chaperone proteins and peptides throughout the cell. Upon release into the extracellular space, HSPs can act to potentiate immune responses against their chaperoned antigens. HSPs bind the receptor CD91on the surface of antigen presenting cells (APCs) which results in internalization of the HSP+antigen and downstream signaling resulting in APC activation. Interestingly, HSPs that bind CD91 do not share structural homology and result in different patterns of cytokine secretion by APCs following activation. CD91 is comprised of an alpha chain which is responsible for ligand binding and a beta chain which is responsible for signal transduction. Within the alpha chain, 4 separate ligand binding domains have been identified. In these studies, we aim to determine which ligand binding domain(s) are responsible for binding individual HSPs and if differences in binding domain usage promote different patterns of APC activation. Methods To investigate ligand binding, we have expressed individual mini-genes comprised of a single ligand binding domain tethered to the full-length beta chain in multiple cell lines. These mini-gene expressing cell lines will be used in conduction with fluorescently labeled HSPs to assess binding and downstream signaling pathways. Results We have generated minigene constructs spanning the 4 ligand binding domains of the extracellular portion of CD91. These minigenes have been stably expressed in 293T cells and iBMDMs using a lentiviral expression system. We have also subsequently knocked out endogenous CD91 in iBMDM. These cells will be used in binding and competition assays to establish the interaction of various immunogenic HSPs with the respective binding domains of CD91. Conclusion These results will be used to inform and improve HSP based immunotherapeutics. Funding Source NIH Topic Categories Vaccines and Immunotherapy (VAC)