Overexpression of inducible HSP70 (HSP70i) in tumors is exploited as a target for immunotherapy of melanoma and other cancers. HSP70i can also funnel external stress into an autoimmune response. As reported in Science Translational Medicine, we identified a peptide within HSP70i responsible for activating human dendritic cells (DCs) and developed HSP70iQ435A, a mutant DNA isoform with a single amino acid modification. The resulting gene product supports a tolerizing DC phenotype while preventing and even reversing autoimmunity in T cell receptor transgenic mice. T cells from treated mice, however, retain the ability to control melanoma once removed from the tolerizing environment and adoptively transferred into tumor bearing animals. DNA vaccinated mice also develop robust humoral responses to HSP70i that contain tumor growth. Protective responses were observed in CD8 knockout mice, and after adoptive B cell transfer to tumor challenged, wild-type mice. Our exciting preliminary data strongly suggest that HSP70iQ435A will suppress autoimmunity while supporting anti-tumor effects. The 'stressed' microenvironment within tumors leads to HSP70i overexpression that drives T cell recruitment and susceptibility to anti-HSP70 antibodies, whereas mutant HSP70 supports immune tolerance in the skin to prevent loss of normal pigment cells. We hypothesize that HSP70iQ435A polarizes DCs to support B cell responses to HSP70i surface expressed by tumor cells while preventing migration of MAA reactive T cells to normal tissues. Thus tumors are targeted by a combination of antibodies targeting the HSP70i c-terminus protruding from the membrane, and infiltrating T cells, while normal tissue cells are undisturbed through lack of HSP70i surface expression and reduced CTL recruitment. We will measure the ability of HSP70iQ435A to interfere with severe side effects of immunotherapy including graft versus host disease, and unravel the immune mechanism underlying divergent responses to HSP70iQ435A, addressing the consequences of treatment for the physiology and function of immune cells and their targets. A spontaneous swine model of melanoma and vitiligo is included to further the application of HSP70iQ435A. PUBLIC HEALTH RELEVANCE: Under the current application we propose to identify the mechanism by which a variant of inducible heat shock protein 70 (HSP70iQ435A) limits autoimmune responses that can develop in response to immunotherapy of melanoma, while sustaining a powerful anti-tumor response. Meanwhile, a DNA application of HSP70iQ435A will be put to the test in a swine model with spontaneous melanoma tumors as well as autoimmune side effects (vitiligo, uveitis) that develop as tumors regress.