Once-daily upadacitinib versus placebo in adults with extensive non-segmental vitiligo: a phase 2, multicentre, randomised, double-blind, placebo-controlled, dose-ranging study.

Background: Janus kinase (JAK) inhibition is a promising approach for treating vitiligo. We aimed to assess the efficacy and safety of upadacitinib, an oral selective JAK inhibitor, in adults with non-segmental vitiligo. Methods: This was a phase 2, multicentre, randomised, double-blind, placebo-controlled, dose-ranging study completed at 33 clinical centres in the United States, Canada, France, and Japan. Eligible patients were aged 18-65 years with non-segmental vitiligo and had a Facial Vitiligo Area Scoring Index (F-VASI) ≥0.5 and a Total Vitiligo Area Scoring Index (T-VASI) ≥5. Patients were randomly assigned (2:2:2:1:1) using an interactive response technology to receive upadacitinib 6 mg (UPA6), upadacitinib 11 mg (UPA11), upadacitinib 22 mg (UPA22), or placebo (PBO; preassigned to switch to either UPA11 or UPA22 in period 2) once daily for 24 weeks (period 1). For weeks 24-52 (period 2), patients randomly assigned to upadacitinib continued their treatment, and patients receiving PBO switched to their preassigned upadacitinib dose in a blinded fashion. The primary endpoint was the percent change from baseline in F-VASI at week 24. Efficacy was analysed in the intention-to-treat population, and safety was examined in all randomly assigned patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT04927975. Findings: Between June 16, 2021, and June 27, 2022, 185 patients (including 115 [62%] who were female and 70 [38%] who were male) were randomly assigned to UPA6 (n = 49), UPA11 (n = 47), UPA22 (n = 43), or PBO (n = 46). At week 24, the LS mean difference versus PBO in the percent change from baseline in F-VASI was -7.60 (95% CI -22.18 to 6.97; p = 0.3037) for UPA6, -21.27 (95% CI -36.02 to -6.52; p = 0.0051) for UPA11, and -19.60 (95% CI -35.04 to -4.16; p = 0.0132) for UPA22. The LS mean difference versus PBO in the percent change from baseline in T-VASI was -7.45 (95% CI -16.86 to 1.96; p = 0.1198) for UPA6, -10.84 (95% CI -20.37 to -1.32; p = 0.0259) for UPA11 and -14.27 (95% CI -24.24 to -4.30; p = 0.0053) for UPA22. Ongoing treatment with upadacitinib induced continuous skin repigmentation over time without reaching a plateau through week 52. The rates for study drug discontinuation and serious treatment-emergent adverse events (TEAEs) were higher in the UPA22 group than in the UPA11 and UPA6 groups. Eight serious TEAEs, including one death of unknown cause and one case of infiltrating lobular breast carcinoma, were reported through 52 weeks; only two serious TEAEs (coronary artery arteriosclerosis [UPA6 (n = 1)] and non-fatal ischemic stroke [UPA11 (n = 1)]) were deemed by the investigator to have a reasonable possibility of being related to study drug. The one case of breast cancer in the UPA11 group was deemed unrelated to study drug, and the one death of unknown cause in the UPA22 group was reviewed and adjudicated and was deemed to be unrelated to study drug. The most common TEAEs were COVID-19, headache, acne, and fatigue. No new safety signals were observed. Interpretation: Upadacitinib monotherapy led to substantial repigmentation of both facial and total body vitiligo lesions and may offer an effective treatment option for adults with extensive non-segmental vitiligo. Based on these findings, upadacitinib 15 mg is being investigated in adults and adolescents with non-segmental vitiligo in an ongoing phase 3 randomised controlled trial. Funding: AbbVie Inc.

Performing Suction Blister Skin Biopsies.

Traditional skin sampling methods include punch or shave biopsies to produce a solid tissue sample for analysis. These biopsy procedures are painful, require anesthesia, and leave permanent scars. This unit describes a suction blister skin biopsy method that can be used in place of traditional biopsy methodologies as a minimally invasive, non-scarring skin sampling technique. The induction of suction blisters uses an instrument with a chamber that applies negative pressure and gentle heat to the skin. Blister formation occurs within 1 hr, producing up to five blisters, each 10 mm in diameter per biopsy site. Blister fluid can be extracted and centrifuged to retrieve cells from the epidermis and upper dermis for flow cytometry, single-cell RNA sequencing, cell culture, and more without the need for digestion protocols. In addition, the blister fluid can be used to measure soluble proteins and metabolites. This unit describes the preparation of supplies and subjects, the suction blister biopsy procedure and blister formation, fluid extraction, and post-blistering care. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of supplies and subject Basic Protocol 2: Suction blister biopsy procedure and formation Basic Protocol 3: Blister fluid extraction Basic Protocol 4: Post-blister care and clean up.

Lesional CD8+ T-Cell Number Predicts Surgical Outcomes of Melanocyte-Keratinocyte Transplantation Surgery for Vitiligo.

The melanocyte-keratinocyte transplantation procedure (MKTP) treats stable and recalcitrant vitiligo. Despite careful selection of candidates based on clinical stability, the success of the procedure is unpredictable. The aim of our study was to define the immunological profile of stable vitiligo lesions undergoing MKTP and correlate them with clinical outcomes. We included 20 MKTP candidates with vitiligo and a patient with piebaldism as a control. Prior to MKTP, T-cell subsets and chemokines in the recipient skin were measured by flow cytometry and ELISA. During MKTP, melanocytes in the donor skin were quantified by flow cytometry. After MKTP, patients were followed for 12 months and repigmentation was assessed clinically and by ImageJ analysis of clinical photographs. Baseline immunologic biomarkers, duration of clinical stability, and transplanted melanocyte number were correlated to postsurgical repigmentation scores. CD8+ T cells were elevated in 43% of the clinically stable vitiligo lesions. CD8+ T-cell number negatively correlated with postsurgical repigmentation scores (r = -0.635, P = 0.002). Duration of clinical stability, skin chemokines, and transplanted melanocyte number did not influence postsurgical repigmentation. This study demonstrates that CD8+ T-cell number correlates negatively with success of postsurgical repigmentation and can be a biomarker to identify ideal surgical candidates.

Adolescent extra-truncal progressive macular hypomelanosis.

Two adolescent females presented to outpatient clinic with isolated, non-scaly, asymptomatic hypopigmented macules and patches on the arm(s). Both cases had Wood's lamp exams notable for extralesional punctiform coral-red perifollicular fluorescence on the back and faint intralesional enhancement. In one case, biopsy was performed and deemed consistent with progressive macular hypomelanosis. The patient had complete response to antimicrobial therapy and sun exposure.

Activation of the NLRP1 inflammasome in human keratinocytes by the dsDNA mimetic poly(dA:dT).

The accrual of cytosolic DNA leads to transcription of type I IFNs, proteolytic maturation of the IL-1 family of cytokines, and pyroptotic cell death. Caspase-1 cleaves pro-IL1ß to generate mature bioactive cytokine and gasdermin D which facilitates IL-1 release and pyroptotic cell death. Absent in melanoma-2 (AIM2) is a sensor of dsDNA leading to caspase-1 activation, although in human monocytes, cGAS-STING acting upstream of NLRP3 mediates the dsDNA-activated inflammasome response. In healthy human keratinocytes, AIM2 is not expressed yet caspase-1 is activated by the synthetic dsDNA mimetic poly(dA:dT). Here, we show that this response is not mediated by either AIM2 or the cGAS-STING-NLRP3 pathway and is instead dependent on NLRP1. Poly(dA:dT) is unique in its ability to activate NLRP1, as conventional linear dsDNAs fail to elicit NLRP1 activation. DsRNA was recently shown to activate NLRP1 and prior work has shown that poly(dA:dT) is transcribed into an RNA intermediate that stimulates the RNA sensor RIG-I. However, poly(dA:dT)-dependent RNA intermediates are insufficient to activate NLRP1. Instead, poly(dA:dT) results in oxidative nucleic acid damage and cellular stress, events which activate MAP3 kinases including ZAKα that converge on p38 to activate NLRP1. Collectively, this work defines a new activator of NLRP1, broadening our understanding of sensors that recognize poly(dA:dT) and advances the understanding of the immunostimulatory potential of this potent adjuvant.

Efficacy and safety of oral ritlecitinib for the treatment of active nonsegmental vitiligo: A randomized phase 2b clinical trial.

BACKGROUND: Vitiligo is a chronic autoimmune disorder characterized by depigmented patches of the skin. OBJECTIVE: To evaluate the efficacy and safety of ritlecitinib, an oral JAK3 (Janus kinase)/TEC (tyrosine kinase expressed in hepatocelluar carcinoma) inhibitor, in patients with active nonsegmental vitiligo in a phase 2b trial (NCT03715829). METHODS: Patients were randomized to once-daily oral ritlecitinib ± 4-week loading dose (200/50 mg, 100/50 mg, 30 mg, or 10 mg) or placebo for 24 weeks (dose-ranging period). Patients subsequently received ritlecitinib 200/50 mg daily in a 24-week extension period. The primary efficacy endpoint was percent change from baseline in Facial-Vitiligo Area Scoring Index at week 24. RESULTS: A total of 364 patients were treated in the dose-ranging period. Significant differences from placebo in percent change from baseline in Facial-Vitiligo Area Scoring Index were observed for the ritlecitinib 50 mg groups with (-21.2 vs 2.1; P < .001) or without (-18.5 vs 2.1; P < .001) a loading dose and ritlecitinib 30 mg group (-14.6 vs 2.1; P = .01). Accelerated improvement was observed after treatment with ritlecitinib 200/50 mg in the extension period (n = 187). No dose-dependent trends in treatment-emergent or serious adverse events were observed across the 48-week treatment. LIMITATIONS: Patients with stable vitiligo only were excluded. CONCLUSIONS: Oral ritlecitinib was effective and well tolerated over 48 weeks in patients with active nonsegmental vitiligo.

AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma.

The STING and absent in melanoma 2 (AIM2) pathways are activated by the presence of cytosolic DNA, and STING agonists enhance immunotherapeutic responses. Here, we show that dendritic cell (DC) expression of AIM2 within human melanoma correlates with poor prognosis and, in contrast to STING, AIM2 exerts an immunosuppressive effect within the melanoma microenvironment. Vaccination with AIM2-deficient DCs improves the efficacy of both adoptive T cell therapy and anti-PD-1 immunotherapy for "cold tumors," which exhibit poor therapeutic responses. This effect did not depend on prolonged survival of vaccinated DCs, but on tumor-derived DNA that activates STING-dependent type I IFN secretion and subsequent production of CXCL10 to recruit CD8+ T cells. Additionally, loss of AIM2-dependent IL-1ß and IL-18 processing enhanced the treatment response further by limiting the recruitment of regulatory T cells. Finally, AIM2 siRNA-treated mouse DCs in vivo and human DCs in vitro enhanced similar anti-tumor immune responses. Thus, targeting AIM2 in tumor-infiltrating DCs is a promising new treatment strategy for melanoma.

Type I interferon signaling limits viral vector priming of CD8+ T cells during initiation of vitiligo and melanoma immunotherapy.

Vitiligo is an autoimmune skin disease in which epidermal melanocytes are targeted for destruction by CD8+ T cells specific for melanocyte/melanoma-shared antigens. IFNγ is the central cytokine driving disease, but the role of type I IFN in vitiligo remains unclear. We investigated the functional role of type I IFN during vitiligo progression using two different mouse models: one induced with a vaccinia virus (VV) vaccine and one induced with dendritic cells to prime autoimmune T cells. Induction of vitiligo by VV in IFNaR-deficient mice led to the development of severe vitiligo compared with wild-type (WT) mice and was characterized by a significantly enhanced effector CD8+ T-cell response. Severe vitiligo in this model was a result of VV persistence, because exacerbation of disease in IFNaR-deficient mice was not observed when antigen-pulsed dendritic cells were used to induce vitiligo instead of virus. Treatment of B16F10 melanoma-inoculated mice with VV vaccine therapy also induced a significantly enhanced anti-tumor response in IFNaR-deficient mice compared with WT. These results not only help define the pathways responsible for vitiligo progression but also suggest that blockade of type I IFNs following administration of a VV vaccine may provide increased immunogenicity and efficacy for melanoma immunotherapy.

Validation of a physician global assessment tool for vitiligo extent: Results of an international vitiligo expert meeting.

Currently, vitiligo lacks a validated Physician Global Assessment (PGA) for disease extent. This PGA can be used to stratify and interpret the numeric scores obtained by the Vitiligo Extent Score (VES). We investigated the interrater reliability of a 5-point PGA scale during an international vitiligo workshop. Vitiligo experts from five different continents rated photographs of non-segmental vitiligo patients with varying degrees of extent with the PGA score. Good interrater agreements (intraclass correlation coefficient >0.6) were observed between the raters overall and within each continent. All hypotheses to evaluate construct validity were confirmed. Median VES values per category were for limited 1.10 [IQR: 0.21-1.67], moderate 3.17 [IQR: 1.75-6.21], extensive 9.58 [IQR: 6.21-13.03] and very extensive 42.67 [IQR: 21.20-42.67]. Defined categories for vitiligo extent can be valuable for inclusion criteria and may impact future reimbursement criteria.

Fas ligand promotes an inducible TLR-dependent model of cutaneous lupus-like inflammation.

Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9-/- autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.

Suction blistering the lesional skin of vitiligo patients reveals useful biomarkers of disease activity.

BACKGROUND: Vitiligo is an autoimmune disease of the skin with limited treatment options; there is an urgent need to identify and validate biomarkers of disease activity to support vitiligo clinical studies. OBJECTIVE: To investigate potential biomarkers of disease activity directly in the skin of vitiligo subjects and healthy subjects. METHODS: Patient skin was sampled via a modified suction-blister technique, allowing for minimally invasive, objective assessment of cytokines and T-cell infiltrates in the interstitial skin fluid. Potential biomarkers were first defined and later validated in separate study groups. RESULTS: In screening and validation, CD8+ T-cell number and C-X-C motif chemokine ligand (CXCL) 9 protein concentration were significantly elevated in active lesional compared to nonlesional skin. CXCL9 protein concentration achieved greater sensitivity and specificity by receiver operating characteristic analysis. Suction blistering also allowed for phenotyping of the T-cell infiltrate, which overwhelmingly expresses C-X-C motif chemokine receptor 3. LIMITATIONS: A small number of patients were enrolled for the study, and only a single patient was used to define the treatment response. CONCLUSION: Measuring CXCL9 directly in the skin might be effective in clinical trials as an early marker of treatment response. Additionally, use of the modified suction-blister technique supports investigation of inflammatory skin diseases using powerful tools like flow cytometry and protein quantification.

The Vitiligo Working Group recommendations for narrowband ultraviolet B light phototherapy treatment of vitiligo.

BACKGROUND: Treatment of vitiligo with narrowband ultraviolet B light (NBUVB) is an important component of the current standard of care. However, there are no consistent guidelines regarding the dosing and administration of NBUVB in vitiligo, reflected by varied treatment practices around the world. OBJECTIVE: To create phototherapy recommendations to facilitate clinical management and identify areas requiring future research. METHODS: The Vitiligo Working Group (VWG) Phototherapy Committee addressed 19 questions regarding the administration of phototherapy over 3 conference calls. Members of the Photomedicine Society and a group of phototherapy experts were surveyed regarding their phototherapy practices. RESULTS: Based on comparison and analysis of survey results, expert opinion, and discussion held during conference calls, expert recommendations for the administration of NBUVB phototherapy in vitiligo were created. LIMITATIONS: There were several areas that required further research before final recommendations could be made. In addition, no standardized methodology was used during literature review and to assess the strength of evidence during the development of these recommendations. CONCLUSION: This set of expert recommendations by the VWG is based on the prescribing practices of phototherapy experts from around the world to create a unified, broadly applicable set of recommendations on the use of NBUVB in vitiligo.

CXCR4 identifies transitional bone marrow premonocytes that replenish the mature monocyte pool for peripheral responses.

It is well established that Ly6Chi monocytes develop from common monocyte progenitors (cMoPs) and reside in the bone marrow (BM) until they are mobilized into the circulation. In our study, we found that BM Ly6Chi monocytes are not a homogenous population, as current data would suggest. Using computational analysis approaches to interpret multidimensional datasets, we demonstrate that BM Ly6Chi monocytes consist of two distinct subpopulations (CXCR4hi and CXCR4lo subpopulations) in both mice and humans. Transcriptome studies and in vivo assays revealed functional differences between the two subpopulations. Notably, the CXCR4hi subset proliferates and is immobilized in the BM for the replenishment of functionally mature CXCR4lo monocytes. We propose that the CXCR4hi subset represents a transitional premonocyte population, and that this sequential step of maturation from cMoPs serves to maintain a stable pool of BM monocytes. Additionally, reduced CXCR4 expression on monocytes, upon their exit into the circulation, does not reflect its diminished role in monocyte biology. Specifically, CXCR4 regulates monocyte peripheral cellular activities by governing their circadian oscillations and pulmonary margination, which contributes toward lung injury and sepsis mortality. Together, our study demonstrates the multifaceted role of CXCR4 in defining BM monocyte heterogeneity and in regulating their function in peripheral tissues.

Understanding autoimmunity of vitiligo and alopecia areata.

PURPOSE OF REVIEW: Vitiligo and alopecia areata are common, disfiguring skin diseases. Treatment options are limited and include nontargeted approaches, such as corticosteroids, topical calcineurin inhibitors, narrow band ultraviolet B phototherapy, and other immune-modifying agents. The purpose of this article is to review shared, novel mechanisms between vitiligo and alopecia areata, as well as discuss how they inform the development of future targeted treatments. RECENT FINDINGS: Vitiligo and alopecia areata are both autoimmune diseases, and striking similarities in pathogenesis have been identified at the level of both the innate and adaptive immune system. Increased reactive oxygen species and high cellular stress level have been suggested as the initiating trigger of the innate immune system in both diseases, and genome-wide association studies have implicated risk alleles that influence both innate and adaptive immunity. Most importantly, mechanistic studies in mouse models of vitiligo and alopecia areata have specifically implicated an interferon (IFN)γ-driven immune response, including IFNγ, IFNγ-induced chemokines, and cytotoxic CD8 T cells as the main drivers of disease pathogenesis. These recent discoveries may reveal an effective strategy to develop new treatments, and several proof-of-concept clinical studies support this hypothesis. SUMMARY: The identification of IFNγ-driven immune signaling pathways has enabled discoveries of potential new treatments for vitiligo and alopecia areata, and supports initiation of larger clinical trials.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2.

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.