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Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435-445 (HSP70i Q435A ) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70i Q435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70i Q435A in mice affects anti-tumor responses. We found that HSP70i Q435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70i Q435A -encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70i Q435A -encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70i Q435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy., (© 2021. Cell Stress Society International.)

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.

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.

Vitiligo is characterized by progressive skin depigmentation resulting from an autoimmune response targeting epidermal melanocytes. Melanocytes are particularly immunogenic by virtue of the contents of their melanosomes, generating the complex radical scavenging molecule melanin in a process that involves melanogenic enzymes and structural components, including tyrosinase, MART-1, gp100, TRP-2 and TRP-1. These molecules are also prime targets of the immune response in both vitiligo and melanoma. The immunogenicity of melanosomal proteins can partly be explained by the dual role of melanosomes, involved both in melanin synthesis and processing of exogenous antigens. Melanocytes are capable of presenting antigens in the context of MHC class II, providing HLA-DR+ melanocytes in perilesional vitiligo skin the option of presenting melanosomal antigens in response to trauma and local inflammation. Type I cytokine-mediated immunity to melanocytes in vitiligo involves T cells reactive with melanosomal antigens, similar to T cells observed in melanoma. In vitiligo, however, T cell tuning allows T cells with higher affinity for melanocyte differentiation antigens to enter the circulation after escaping clonal deletion in primary lymphoid organs. The resulting efficacious and progressive autoimmune response to melanocytes provides a roadmap for melanoma therapy.

Autoimmune depigmentation of the skin, vitiligo, afflicts a considerable number of people, yet no effective therapeutic modalities have been developed to treat it. In part, this can be attributed to the obscure etiology of the disease, which has begun to reveal itself only recently. It is known that pigment is lost as a function of reduced melanocyte numbers in the epidermis, and that depigmentation is accompanied by T cell influx to the skin in the vast majority of patients. Characterizing such infiltrating T cells as type 1 proinflammatory cytokine-secreting cells reactive with melanocyte-specific antigen is a major step toward effective therapy. Melanoma research has shown that differentiation antigens, also expressed by normal melanocytes, can be immunogenic when expressed in the melanosomal compartment of the cell. Similar reactivity to melanosomal antigens is apparent for T cells infiltrating vitiligo skin. It may eventually be possible to treat patients with decoy antigens that anergize such Tcells, or to prevent recruitment of the T cells to the skin altogether. In this respect, it is important that T cells are recruited to the skin as a function of dendritic cell activation and that dendritic cells are likely activated at sites of epidermal trauma as a consequence of stress proteins that spill over into the microenvironment. Stress proteins chaperoning antigens representative of the cells from which they were derived are then processed by dendritic cells and contribute to their activation. Activated dendritic cells not only migrate to draining lymph nodes to recruit T cells but may execute cytotoxic effector functions as well. The contribution of the effector functions to actual depigmentation of the skin remains to be investigated.

In vitiligo, CD8+ T cells and IFN-γ production drive disease activity, representing potential targets to control depigmentation. The persistence of resident memory T cells (T RMs) in patient skin causes relapses, highlighting the significance of IL-15-targeted management strategies. Future treatment avenues potentially involve pairing oral JAK inhibitors with phototherapy and repurposing medications such as metformin and rapamycin for managing vitiligo. Local targeting of Janus kinase activity mitigates inflammation to halt CD8+ T cell activity, and nucleotide-based treatments serve as a viable option for skin conditions. IL-2 mutein preferentially targets Tregs to enhance their activity and proliferation and manage depigmentation. The autoimmune condition vitiligo, characterized by skin depigmentation, presents challenges for effective treatment design, with Janus kinase (JAK) inhibitors and other repurposed drugs offering a promising strategy for symptom management. This review explores advantages and shortcomings of current therapies, while presenting the urgent need for further innovative approaches. We emphasize the growing understanding of autoimmune involvement in vitiligo, highlighting several novel treatment avenues including relieving melanocyte stress, preventing dendritic cell activation, halting T cell migration, and suppressing inflammation and autoimmunity. Integrating psychodrama therapy to remediate stress alongside medical interventions marks a holistic approach to enhance patient well-being. The molecular underpinnings of vitiligo care are covered, emphasizing exciting advances revolutionizing vitiligo treatment and improving the quality of life for affected individuals. [ABSTRACT FROM AUTHOR]

Depigmentation in vitiligo occurs by progressive loss of melanocytes from the basal layer of the skin, and can be psychologically devastating to patients. T cell-mediated autoimmunity explains the progressive nature of this disease. Rather than being confronted with periods of rapid depigmentation and bouts of repigmentation, patients with long-standing, treatment-resistant vitiligo can undergo depigmentation treatment. The objective is to remove residual pigmentation to achieve a cosmetically acceptable result - that of skin with a uniform appearance. In the United States, only the use of mono-benzyl ether of hydroquinone ( MBEH) is approved for this purpose. However, satisfactory results can take time to appear, and there is a risk of repigmentation. MBEH induces necrotic melanocyte death followed by a cytotoxic T-cell response to remaining, distant melanocytes. As cytotoxic T-cell responses are instrumental to depigmentation, we propose that combining MBEH with immune adjuvant therapies will accelerate immune-mediated melanocyte destruction to achieve faster, more definitive depigmentation than with MBEH alone. As Toll-like Receptor ( TLR) agonists - imiquimod, CpG, and Heat Shock Protein 70 ( HSP 70) - all support powerful Th1 responses, we propose that using MBEH in combination with these agents can achieve superior depigmentation results for vitiligo patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 cytotoxic T-lymphocyte activity. Absence of T-cell infiltration to the skin and maintenance of hair follicle melanocytes were 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 a targetable entity for treatment. [ABSTRACT FROM AUTHOR]

The authors reflect on melanocyte toward the understanding of the incidence of vitiligo. Melanocytes are immunologic entities within individuals' skin that contribute to the realization that they are ideal targets for an autoimmune response. The authors note the significance of noting the development of a regulatory response as well as the factors of immunosuppressive cytokines in recognizing vitiligo as an autoimmune disease of the skin. A discussion on melanocytes is offered.