Mutant HSP70 reverses autoimmune depigmentation in vitiligo

Abstract

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.

Fig. 1

APCs in progressive human vitiligo reflect the phenotype observed in response to HSP70i. (A) PBMCs from non-vitiligo (N.V.) control (left flow plot) and vitiligo (right flow plot) patients were gated for expression of CD11b and CD11c after excluding T and B cells. (B) Quantification of the percentage of cells among R1 to R3 indicates an increase in R2 (CD11b⁺CD11c⁺) cells among the vitiligo patient samples. (C to E) Non-vitiligo (N.V.) patient (C) and vitiligo patient (D) nonlesional (N.L.) and (E) perilesional (P.L.) skin were stained for expression of APC markers CD11b (red staining and arrows) and CD11c (blue staining and arrows) with colocalization observed in purple (purple staining and arrows). (F) Quantification of double positive–stained cells indicates a significant increase in CD11b⁺CD11c⁺ cells within perilesional skin. Scale bars, 25 μm. n = 3. *P < 0.05, **P < 0.01.

Fig. 2

A peptide within the C terminus of HSP70i is required for inducing depigmentation. (A) To identify the region of HSP70i critical for inducing depigmentation, mice were vaccinated with DNA plasmids encoding wildtype (WT) N terminus, C terminus, or full-length HSP70i. Mice vaccinated with HSP70i N terminus (residues 1 to 377) expressed depigmentation similar to the empty vector control–treated animals. Mice vaccinated with the HSP70i C terminus displayed far greater levels of depigmentation than those exposed to full-length HSP70i plus hTRP-2 vaccination (n = 5). Representative images of C57BL/6 mice imaged 4 weeks after vaccination with DNA-encoding melanosomal antigen hTRP-2 and either full-length HSP70i, N-terminal (N-term) or C-terminal (C-term) HSP70i fragments, or empty vector (EV) are shown. Mice were vaccinated five times, every 7 days, with 5.6 μg of total DNA. (B) Schematic of the N-terminal ATP-binding domain (ABD) and C-terminal substrate–binding domain (SBD) of HSP70i and fragments (HSP70i_1–377 and HSP70i_320–641) and mutations introduced within the putative DC-activating region. (C) No depigmentation was observed in mice receiving vaccinations with hTRP-2 and several mutant versions of HSP70i compared to hTRP-2 and WT HSP70i, supporting the crucial involvement of the 11-mer with the exceptions of amino acids 446 and 447. Representative images of mice 5 weeks after vaccination (n = 10) are shown. Mice were vaccinated four times, every 7 days, with 4 μg of total DNA. (D) Three-dimensional representation showing the peripheral location of the mutant Q435A (magenta) residue (red arrow) ideally located at the interface of the ATP- and substrate-binding domains. *P < 0.05, **P < 0.01.

Fig. 3

WT HSP70i accelerates and mutant HSP70i prevents depigmentation in vitiligo-prone mice. (A) Tolerogenic DCs are induced in response to HSP70i_Q435A. (B) Representative flow cytometry plots and cumulative graphs of mice vaccinated three times in total, every 7 days, with 4 μg of WT, HSP70i_Q435A (Q435A), or empty vector (EV) control DNA. Splenocytes from Pmel-1 mice (n = 3) 6 months after vaccination were analyzed by flow cytometry. Gating for CD11b and CD11c expression among nonlymphocytes, we observed three distinct populations that vary in abundance in response to the treatments. Differential expression of CD11b and CD11c was found after vaccination with WT or HSP70i_Q435A plasmids. In contrast to WT HSP70i, the anti-inflammatory population (R1) displays increased, and the inflammatory population (R2) displays decreased, abundance in response to mutant HSP70i-encoding DNA. (C) Images were obtained 6 months after treatment. Pmel-1 mice depigmented within and distal to the site of vaccination. (D) Cumulative graphs of mice in (C). Pmel-1 mice receiving mutant HSP70i displayed significantly less depigmentation compared to WT HSP70i or empty vector–exposed C57BL/6 mice, confirming inhibition by mutant HSP70i. (E) To test the therapeutic effects of HSP70i_Q435A in rapidly depigmenting vitiligo-prone mice, tyrosinase-responsive TCR transgenic h3TA2 mice were vaccinated five times, every 6 days, with 5.6 μg of either empty vector– or HSP70i_Q435A-encoding DNA plasmid. Mice were imaged 4 weeks after the final vaccination at just over 9 weeks of age. (F) Cumulative data from (E) demonstrating that empty vector–vaccinated mice had significantly more depigmentation than the HSP70i_Q435A-vaccinated mice (n = 4). *P < 0.05, **P < 0.01.

Fig. 4

Decreased T cell infiltration is observed after HSP70i_Q435A vaccination. (A) Pmel-1 mice vaccinated with empty vector (EV) DNA showed abundant skin infiltration by CD3⁺ T cells (arrows) localized mostly to hair follicles (asterisks). (B) CD3⁺ T cell infiltrates seen in (A) were decreased after HSP70i_Q435A (Q435A) administration. (C and D) T cell infiltrates in skin of h3TA2 mice in response to empty vector and (D) HSP70i_Q435A. (E) Cumulative data showing changes in skin-infiltrating T cells in treated mice. (F) Quantification of T cell infiltrates in h3TA2 mice. (G) Most of the skin-infiltrating T cells were cytotoxic. (H) In the Pmel-1 mice, CD8⁺ infiltrates in response to empty vector decreased after HSP70i_Q435A exposure. (I) Reduced number of melanocyte-reactive T cells was observed in response to HSP70i_Q435A. T cells were quantified on the basis of expression of the tyrosinase transgene–reactive TCR V_β12 subunit. (J) A decrease in V_β12⁺ cells was observed after vaccination with HSP70i_Q435A. (K) Quantification of cytotoxic T cell infiltrates in C57BL/6 and Pmel-1 mice. (L) Quantification of melanocyte-reactive T cells in h3TA2 mice. Arrows mark stained cells. Scale bars, 50 μm. *P < 0.05.

Fig. 5

HSP70i-treated mice display a similar effector T cell phenotype as vitiligo patient tissue. (A) A decreased percentage of splenocytes from the WT HSP70i-exposed Pmel-1 mice (Fig. 3) expresses the T cell transcription factor Eomes, supporting an enhanced effector T cell phenotype. (B and C) Combined with T-bet expression (C), the resulting Eomes/T-bet ratio further indicates increased effector responses in mice exposed to HSP70i (n = 3). (D and E) Representative histograms of human PBMCs from non-vitiligo (N.V.) control and vitiligo patients similarly analyzed for Eomes and T-bet expression. (F) The Eomes/T-bet ratio indicates an effector response in vitiligo peripheral blood compared to non-vitiligo control patient samples. (G and H) Nonlesional (N.L.) (G) and perilesional (P.L.) (H) vitiligo skin was stained for T-bet (red staining and arrows) and CD8 (blue staining and arrows) expression, with the cytotoxic T cells shown in purple (purple staining and arrows). A significantly larger number of CD8⁺/T-bet⁺–expressing cells were observed in the dermis of perilesional skin. (I and J) Nonlesional (I) and perilesional (J) skin stained for Eomes (red staining and arrows) and CD8 (blue staining and arrows). Eomes-expressing CD8 cells were significantly increased in non-vitiligo control and nonlesional skin from vitiligo patients. (K) The ratio of Eomes/T-bet–expressing CD8 cells indicates an effector response in perilesional vitiligo skin, similar to that seen in the periphery. n = 3. *P < 0.05, **P < 0.01.

Fig. 6

Mutant HSP70i suppresses human DC activation. (A) Representative histogram displaying HLA-ABC expression from human PBMCs differentiated into immature DCs via GM-CSF and IL-4 and exposed to His-tagged WT or HSP70i_Q435A (Q435A) protein (1 μg/ml). Commercially purchased HSP70i [WT(C)], LPS (1 μg/ml), and complete media served as controls. CD11c⁺ cells were analyzed for expression of CD80, CD83, CD86, or HLA-ABC and analyzed by flow cytometry. (B to E) Changes in mean fluorescence intensity (MFI) between treatments indicating increased expression of activation markers after addition of His-tagged isolated WT HSP70i and commercially purchased HSP70i [WT(C)] and reduced expression of all activation markers after addition of HSP70i_Q435A compared to control media. (F) ImageStream images of DCs after the addition of His-tagged WT or HSP70i_Q435A (Q435A), or media alone. Cells were stained with antibodies (Ab) toward HSP70i (SPA-820) or the His tag. Single and overlaid channel images are displayed. (G) Similar MFIs using the anti-His antibody show that DCs bind both mutant and WT HSP70i.

Fig. 7

WT and mutant HSP70i differentially activate human skin T cells. Skin explants were maintained submerged in control media supplemented with or without 4 μg of WT or HSP70i_Q435A (Q435A) protein for 6 days, and T cell profiles were analyzed after gating for CD3 expression. (A) Eomes expression was significantly up-regulated in T cells from WT and mutant HSP70i-treated skin T cells. (B) T-bet expression is reduced among T cells in response to HSP70i_Q435A. (C) Together, the Eomes/T-bet ratios reveal an effector versus memory phenotype in response to WT and mutant HSP70i, respectively. (D) The T cell–homing receptor CLA is highly expressed among T cells in response to WT HSP70i. n = 2. *P < 0.05, **P < 0.01.

Fig. 8

Transfected human skin expresses HSP70i. Skin biopsies were biolistically DNA-transfected with WT HSP70i (WT), HSP70i_Q435A (Q435A), or empty vector and analyzed by flow cytometry 4 days later for expression of HSP70i. Histograms display the relative expression and MFI for each vaccination. Note marked expression of HSP70i as well as its mutant variant among cells collected from adult skin with the respective constructs. n = 3.