Jonathan M. Eby

Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

Vitiligo can be reversed through immune targeting with mutant heat shock protein 70. New Treatment Makes Vitiligo Beat It Whether your grant application is due, you have a paper that needs to be submitted, or your patient load is too high, medical science is not a relaxing profession. High stress is known to negatively affect your health at both the whole body and cellular level. One way the body responds to cellular stressors is through the induction of heat shock proteins (HSPs). Now, Mosenson et al. suggest that mutant HSP70 could be a potential treatment for autoimmune vitiligo. The authors noticed that mutant inducible HSP70 (HSP70i) could prevent T cell–mediated depigmentation in a mouse model of vitiligo, perhaps by shifting dendritic cells from an inflammatory to a regulatory phenotype. Moreover, a DNA vaccine of the mutant HSP70i could be used therapeutically to partially restore pigmentation in a second model of depigmentation. The authors then took these studies into ex vivo human skin, showing that their mutant HSP70i could prevent the disease-related shift from quiescent to effector T cell phenotype. Although these observations still need to be translated into the clinic, they form the basis for a new potential treatment for autoimmune vitiligo. 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.

A Quantitative Increase in Regulatory T Cells Controls Development of Vitiligo

T cell cytolytic activity targeting epidermal melanocyte is shown to cause progressive depigmentation and autoimmune vitiligo. Using the recently developed transgenic mice h3TA2 that carry T cell with a HLA-A2 restricted human tyrosinase reactive TCR and develop spontaneous vitiligo from an early age, we addressed the mechanism regulating autoimmune vitiligo. Depigmentation was significantly impaired only in IFN-γ knockout h3TA2 mice but not in TNF-α or perforin knockout h3TA2 mouse strains, confirming a central role for IFN-γ in vitiligo development. Additionally, the regulatory T cells (Treg) were relatively abundant in h3TA2-IFN-γ−/− mice, and depletion of Treg employing anti-CD25 antibody fully restored the depigmentation phenotype in h3TA2-IFN-γ−/− mice mediated in part through upregulation of pro-inflammatory cytokines as IL-17and IL-22. Further therapeutic potential of Treg abundance in preventing progressive depigmentation was evaluated by adoptively transferring purified Treg or using rapamycin. Both adoptive transfer of Treg and rapamycin induced lasting remission of vitiligo in mice treated at the onset of disease, or in mice with established disease. This leads us to conclude that reduced regulatory responses are pivotal to the development of vitiligo in disease-prone mice, and that a quantitative increase in the Treg population may be therapeutic for vitiligo patients with active disease.

A central role for inducible heat-shock protein 70 in autoimmune vitiligo

Inducible heat‐shock protein 70 (HSP70i) is a protein regulated by stress that protects cells from undergoing apoptosis. Such proteins are marvellously 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 funnelled through HSP70i, then blocking the stress protein from activating DCs can lend new treatment opportunities for vitiligo.

A Coreceptor-Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4+ T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2–restricted TCR was positively selected on both CD4+ and CD8+ single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3+CD4−CD8− double-negative T cells. TIL 1383I TCR transgenic CD4+, CD8+, and CD4−CD8− T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2+/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3+ T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.

Tumour necrosis factor-α inhibition can stabilize disease in progressive vitiligo

Tumour necrosis factor (TNF)‐α, a proinflammatory cytokine central to many autoimmune diseases, has been implicated in the depigmentation process in vitiligo. We review its role in vitiligo by exploring its pro‐ and anti‐inflammatory properties and examine the effects of blocking its actions with TNF‐α antagonist therapeutics in reports available in the literature. We found that TNF‐α inhibition halts disease progression in patients with progressive vitiligo but that, paradoxically, treatment can be associated with de novo vitiligo development in some patients when used for other autoimmune conditions, particularly when using adalimumab and infliximab. These studies reinforce the importance of stating appropriate outcomes measures, as most pilot trials propose to measure repigmentation, whereas halting depigmentation is commonly overlooked as a measure of success. We conclude that TNF‐α inhibition has proven useful for patients with progressive vitiligo, where TNF‐α inhibition is able to quash cytotoxic T‐cell‐mediated melanocyte destruction. However, a lingering concern for initiating de novo disease will likely prevent more widespread application of TNF inhibitors to treat vitiligo.

CCL22 to activate Treg migration and suppress depigmentation in vitiligo

In vitiligo, gradual cutaneous depigmentation and cytotoxic T cell activity against melanocytes is accompanied by a paucity of regulatory T cells (Tregs) in vitiligo patient skin, indicating that autoimmune responses are not adequately held in check. Thus we sought a means to repopulate patient skin with Tregs. We hypothesized that enhanced expression of CCL22 can promote Treg skin homing to suppress depigmentation. The mouse Ccl22 gene was cloned into an expression vector and resulting DNA was used for gene gun treatment. Two spontaneous depigmentation models with different kinetics of melanocyte loss were utilized, expressing tyrosinase-reactive and gp100-reactive T cell receptor transgenes. Mice were subjected to 5 gene gun treatments 6 days apart, scanned for depigmentation weekly thereafter and monitored for activation and proliferation of relevant T cells and for Treg infiltration to the skin. Significantly reduced depigmentation 2 weeks after treatment was accompanied by a markedly increased abundance of Tregs in the skin at the expense of melanocyte reactive, TCR transgenic T cells as well as by reduced proliferation and reduced IFN-γ production in response to cognate peptide. Continued treatment may be necessary for sustained, local immunosuppression. These findings suggest that topical CCL22 may be used for the treatment of vitiligo.

A current viewpoint of lymphangioleiomyomatosis supporting immunotherapeutic treatment options.

Lymphangioleiomyomatosis (LAM) leads to hyperproliferation of abnormal smooth muscle cells in the lungs, associated with diffuse pulmonary parenchymal cyst formation and progressive dyspnea on exertion. The disease targets women of child-bearing age. Complications include pneumothoraces and chylous pleural effusions. Ten-year survival is estimated at 70%, and lung transplantation remains the only validated treatment. It has been observed that LAM cells express markers associated with melanocytic differentiation, including gp100 and MART-1. Other melanocytic markers have also been observed. The same proteins are targeted by T cells infiltrating melanoma tumors as well as by T cells infiltrating autoimmune vitiligo skin, and these antigens are regarded as relatively immunogenic. Consequently, vaccines have been developed for melanoma targeting these and other immunogenic melanocyte differentiation proteins. Preliminary data showing susceptibility of LAM cells to melanoma derived T cells suggest that vaccines targeting melanosomal antigens can be successful in treating LAM.

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress

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.

Immune Responses in A Mouse Model of Vitiligo with Spontaneous Epidermal De- and Repigmentation

To generate a mouse model of spontaneous epidermal depigmentation, parental h3TA2 mice, expressing both a human‐derived, tyrosinase‐reactive T‐cell receptor on T cells and the matching HLA‐A2 transgene, were crossed to keratin 14‐promoter driven, stem cell factor transgenic (K14‐SCF) mice with intra‐epidermal melanocytes. In resulting Vitesse mice, spontaneous skin depigmentation precedes symmetrical and sharply demarcated patches of graying hair. Whereas the SCF transgene alone dictates a greater retinoic acid receptor‐related orphan receptor gamma (RORγt)+ T‐cell compartment, these cells displayed markedly increased IL‐17 expression within Vitesse mice. Similar to patient skin, regulatory T cells were less abundant compared with K14‐SCF mice, with the exception of gradually appearing patches of repigmenting skin. The subtle repigmentation observed likely reflects resilient melanocytes that coexist with skin‐infiltrating, melanocyte‐reactive T cells. Similar repigmenting lesions were found in a different TCR transgenic model of vitiligo developed on an SCF transgenic background, supporting a role for SCF in repigmentation.

α1‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

Background Recently, we provided evidence that α1‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α1‐ARs is unknown. Methods and Results We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D‐AR:ACKR3, CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b‐AR in β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries. Conclusions α1‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D‐AR function, and activation of ACKR3 negatively regulates α1‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1‐ARs function within a network of hetero‐oligomeric receptor complexes.