Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo.

SUMMARY HSP70i and other stress proteins have been used in anti-tumor vaccines. This begs the question whether HSP70i plays a unique role in immune activation. We vaccinated inducible HSP70i (Hsp70-1) knockout mice and wild-type animals with optimized TRP-1, a highly immunogenic melanosomal target molecule. We were unable to induce robust and lasting depigmentation in the Hsp70-1 knockout mice, and in vivo cytolytic assays revealed a lack of CTL activity. Absence of T cell infiltration to the skin and maintenance of hair follicle melanocytes was observed. By contrast, depigmentation proceeded without interruption in mice lacking a tissue specific constitutive isoform of HSP70 (Hsp70-2) vaccinated with TRP-2. Next, we demonstrated that HSP70i was necessary and sufficient to accelerate depigmentation in vitiligo-prone Pmel-1 mice, accompanied by lasting phenotypic changes in dendritic cell subpopulations. In summary, these studies assign a unique function to HSP70i in vitiligo, and identify HSP70i as targetable entity for treatment.

Muscle stem cells (MuSCs) hold great therapeutic potential for muscle genetic disorders, such as Duchenne muscular dystrophy (DMD). The CRISP/Cas9-based genome editing is a promising technology for correcting genetic alterations in mutant genes. In this study, we used fibrin-gel culture system to selectively expand MuSCs from crude skeletal muscle cells of mdx mice, a mouse model of DMD. By CRISP/Cas9-based genome editing, we corrected the dystrophin mutation in expanded MuSCs and restored the skeletal muscle dystrophin expression upon transplantation in mdx mice. Our studies established a reliable and feasible platform for gene correction in MuSCs by genome editing, thus greatly advancing tissue stem cell-based therapies for DMD and other muscle disorders.

Inducible Heat Shock Protein 70 (HSP70i) is a protein regulated by stress that protects cells from undergoing apoptosis. Such proteins are marvelously well conserved throughout evolution, which has placed them in the spotlight for helping to understand the intriguing relationship between infection and immunity. In the presence of stress proteins, dendritic cells (DCs) will sense this alarm signal and respond by recruiting immune cells of different plumage to fit the occasion. In times of stress, melanocytes will secrete antigen bound HSP70i to act as an alarm signal in activating DCs, that comes equipped with an address of origin to drive the autoimmune response in vitiligo. Here we pose that if the autoimmune response is funneled through HSP70i, then blocking the stress protein from activating DCs can lend new treatment opportunities for vitiligo.

SUMMARY Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.