TNF-α, a proinflammatory cytokine central to many autoimmune diseases, has been implicated in the depigmentation process in vitiligo. We reviewed its role in vitiligo by exploring its pro- and anti-inflammatory properties and examined 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 to treat other autoimmune conditions, particularly when using adalimumab and infliximab. These studies reinforce the importance of stating appropriate outcomes measures as most pilot trials proposed to measure repigmentation, whereas halting depigmentation was commonly overlooked as a measure of success. We conclude that TNF inhibition was 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 a more widespread application of TNF inhibitors to treat vitiligo.

Vitiligo is a T-cell mediated skin disorder characterized by progressive loss of skin color. In individuals genetically predisposed to the disease, various triggers contribute to the initiation of vitiligo. Precipitating factors can stress the skin, leading to T-cell activation and recruitment. Though hereditary factors are implicated in the pathogenesis of vitiligo, it is unknown whether precipitating, stressful events play a role in vitiligo. To understand this, we utilized a validated perceived stress scale (PSS) to measure this parameter in vitiligo patients compared to persons without vitiligo. Additionally, we probed a clinical database, using a knowledge linking software called ROCKET, to gauge stress-related conditions in the vitiligo patient population. From a pool of patients in an existing database, a hundred individuals with vitiligo and twenty-five age-and sex-matched comparison group of individuals without vitiligo completed an online survey to quantify their levels of perceived stress. In parallel, patients described specifics of their disease condition, including the affected body sites, the extent, duration and activity of their vitiligo. Perceived stress was significantly higher among vitiligo individuals compared to those without vitiligo. ROCKET analyses suggested signs of metabolic-related disease (i.e., 'stress') preceding vitiligo development. No correlation was found between perceived stress and the stage or the extent of disease, suggesting that elevated stress may not be a consequence of pigment loss alone. The data provide further support for stress as a precipitating factor in vitiligo development.

Human HSP70i carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells in vitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70i into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70i-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70i.

Vitiligo is an autoimmune skin disease characterized by melanocyte destruction. Regulatory T cells (Tregs) are greatly reduced in vitiligo skin, and replenishing peripheral skin Tregs can provide protection against depigmentation. Ganglioside D3 (GD3) is overexpressed by perilesional epidermal cells, including melanocytes, which prompted us to generate GD3-reactive chimeric antigen receptor (CAR) Tregs to treat vitiligo. Mice received either untransduced Tregs or GD3-specific Tregs to test the hypothesis that antigen specificity contributes to reduced autoimmune reactivity in vitro and in vivo. CAR Tregs displayed increased IL-10 secretion in response to antigen, provided superior control of cytotoxicity towards melanocytes, and supported a significant delay in depigmentation compared to untransduced Tregs and vehicle control recipients in a TCR transgenic mouse model of spontaneous vitiligo. The latter findings were associated with a greater abundance of Tregs and melanocytes in treated mice versus both control groups. Our data support the concept that antigen-specific Tregs can be prepared, used, and stored for long-term control of progressive depigmentation.

T regulatory cells (Treg) avert autoimmunity but their increased levels in melanoma confer a poor prognosis. To explore the basis for Treg accumulation in melanoma, we evaluated chemokine expression in patients. A 5-fold increase was documented in the Treg chemoattractants CCL22 and CCL1 in melanoma-affected skin versus unaffected skin, as accompanied by infiltrating FoxP3+ T cells. In parallel, there was a ~2-fold enhancement in expression of CCR4 in circulating Treg but not T effector cells. We hypothesized that redirecting Treg away from tumors might suppress autoimmune side-effects caused by immune checkpoint therapeutics now used widely in the clinic. In assessing this hypothesis, we observed a marked increase in skin Treg in mice vaccinated with CCL22, with repetitive vaccination sufficient to limit Treg accumulation and melanoma growth in the lungs of animals challenged by tumor cell injection, whether using a prevention or treatment protocol design. The observed change in Treg accumulation in this setting could not be explained by Treg conversion. Overall, our findings offered a preclinical proof of concept for the potential use of CCL22 delivered by local injection as a strategy to enhance the efficacious response to immune checkpoint therapy while suppressing its autoimmune side-effects.

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Summary We isolated gp100-reactive T cells from perilesional skin of a patient with progressive vitiligo with superior reactivity toward melanoma cells compared with tumor-infiltrating lymphocytes 1520, a melanoma-derived T-cell line reactive with the same cognate peptide. After dimer enrichment and limited dilution cloning, amplified cells were subjected to reverse transcription and 5′ RACE to identify the variable TCRα and TCRβ subunit sequences. The full-length sequence was cloned into a retroviral vector separating both subunits by a P2A slippage sequence and introduced into Jurkat cells and primary T cells. Cytokine secreted by transduced cells in response to cognate peptide and gp100-expressing targets signifies that we have successfully cloned a gp100-reactive T-cell receptor from actively depigmenting skin.

An immunotherapeutic strategy is discussed supporting anti-tumor activity toward malignancies overexpressing ganglioside D3. GD3 can be targeted by NKT cells when derived moieties are presented in the context of CD1d. NKT cells can support anti-tumor responses by secreting inflammatory cytokines and through cytotoxicity toward CD1dGD3 tumors. To overexpress GD3, we generated expression vector DNA and an adenoviral vector encoding the enzyme responsible for generating GD3 from its ubiquitous precursor GM3. We show that DNA encoding α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (SIAT8) introduced by gene gun vaccination in vivo leads to overexpression of GD3 and delays tumor growth. Delayed tumor growth is dependent on CD1d expression by host immune cells, as shown in experiments engaging CD1d knockout mice. A trend toward greater NKT cell populations among tumor-infiltrating lymphocytes is associated with SIAT8 vaccination. A single adenoviral vaccination introduces anti-tumor activity similarly to repeated vaccination with naked DNA. Here, greater NKT tumor infiltrates were accompanied by marked overexpression of IL-17 in the tumor, later switching to IL-4. Our results suggest that a single intramuscular adenoviral vaccination introduces overexpression of GD3 by antigen-presenting cells at the injection site, recruiting NKT cells that provide an inflammatory anti-tumor environment. We propose adenoviral SIAT8 (AdV-SIAT8) can slow the growth of GD3 expressing tumors in patients.