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Transcriptomics of vitiligo melanocytes after hydroxychloroquine treatment

ABSTRACT: Purpose: In order to explore the mechanism of hydroxychloroquine (HCQ) in treating vitiligo. We report the application of transcriptome profiling (RNA-seq) in human vitiligo melanocytes cell line PIG3V after HCQ treatment. Methods: The PIG3V cells were divided randomly in two groups: HCQ treatment (1 μg/mL, 24 hours) and the cells without treatment (only medium). RNA samples extracted from PIG3V cells were quantified by NanoDrop and Bioanalyzer. After 2 rounds of purification Poly A RNA was extracted then from total RNA by using Dynabeads Oligo. Small pieces of Poly A RNA were returned through Magnesium RNA Fragmentation Module under the condition of 94℃ for 5 minutes. Subsequently, SuperScript Reverse Transcriptase was used to reverse-transcribe the cleaved RNA pieces into cDNA. Then, U-labeled second-stranded DNAs was synthesized. After adding of A-base to each strand, preparing for indexed adapters, and heat-labile UDG enzyme treatment, PCR amplification was performed under (1) 95℃, 3 minutes; (2) 98℃, 15 seconds for 8 cycles; (3) 60℃, 15 seconds, 72℃, 5 minutes. Ultimately, RNA sequencing was performed according to protocol of Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China). After calculation, differential expressed genes were screened by fold change (FC)＞2 or FC＜0.5 and p value＜0.05. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample and identified 60612 genes and 229420 transcripts. Approximately 0.3% of the genes showed differential expression between the HCQ treated PIG3V cells and the cells without HCQ, with a fold change＞2 or＜0.5 , p value＜0.05. Conclusions: Our research showed the first detailed transcriptome analysis of human vitiligo melanocytes PIG3V, with or without HCQ treatment. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic mechanisms of HCQ in treating vitiligo.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188603 | GEO | 2021/11/13

REPOSITORIES: GEO

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Project description:Immunotherapy with checkpoint inhibitors is an efficient treatment for metastatic melanoma. Development of vitiligo upon immunotherapy represents a specific immune-related adverse event (irAE) diagnosed in 15% of patients and associated with a positive clinical response. Therefore, a detailed characterization of immune cells during vitiligo onset in melanoma patients would give insight into the immune mechanisms mediating both this irAE and the anti-tumor response. To better understand these aspects, we analyzed T cell subsets from peripheral blood of metastatic melanoma patients undergoing treatment with anti-programmed cell death protein (PD)-1 antibodies. Stratification of patients for developing or not developing vitiligo during therapy revealed an association between blood reduction of mucosal associated invariant T (MAIT), T helper (h) 17, natural killer (NK) CD56bright, and T regulatory (T-reg) cells and vitiligo onset. To deeply characterize the tumoral T cell response concomitant to vitiligo onset, we analyzed T cell content in skin biopsies collected from melanoma patients who developed vitiligo. Consistently with the observed blood reduction of Th17 cells in melanoma patients developing vitiligo during immunotherapy, we found an enrichment of Th17 cells in the vitiligo skin biopsy, suggesting a migration of Th17 cells from the blood into the autoimmune lesion. To further characterize T cells in vitiligo skin lesion of melanoma patients, we sequenced T cell receptor (TCR) of cells derived from biopsies of vitiligo and primary melanoma of the same patient. Interestingly, we found different TCR sequences between vitiligo and primary melanoma lesions, except for a few cases showing the same TCR sequences. In contrast, shared TCR sequences were identified between vitiligo and metastatic tissues of the same patient. These data indicate that T cell response against normal melanocytes, which is involved in vitiligo onset, is not mainly mediated by the reactivation of specific T cell clones infiltrating primary melanoma but may be elicited by T cell clones targeting metastatic tissues. Altogether, our data indicate that anti-PD-1 therapy induces a de novo immune response, composed of different T cell subtypes, whose role may be related to the development of vitiligo and the response against metastatic tumor.

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Transcriptomics of vitiligo melanocytes after hydroxychloroquine treatment

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