Figure. CD8+ T-cell-driven pathology in alemtuzumab-treated patients with relapsing-remitting multiple sclerosis (RRMS) developing vitiligo.

(A) Patient 1: circumscribed, symmetrical white macules and patches typical for vitiligo (A.a). Exemplary skin biopsy from the same patient: melanocyte staining (A.b), panMel antibody cocktail directed to tyrosine, melanA, HMB45. Positive stainings for CD3 (A.c) and CD8 (A.d). (B) Patient 3: typical skin lesions for vitiligo (B.a). Exemplary skin biopsy from the same patient: melanocyte staining (B.b). Positive stainings for CD3 (B.c) and CD8 (B.d). Scale bars represent 100 μM in A and B. (C) Proportions of CD8⁺ T cells and CD8⁺ T-cell subsets (defined by the displayed markers) within peripheral blood mononuclear cells (PBMC) isolated from alemtuzumab-treated patients with RRMS (n = 30 for baseline, n = 29 at 6 months, n = 25 for 12 months, n = 17 for 18 months) and patient 2 (red triangle) were determined by flow cytometry. For the analysis of cytokine production, cells were treated with leukocyte activation cocktail (BD) for 4 hours. The red arrow indicates the timepoint of vitiligo onset. Boxplots show median, 25% and 75% percentile, whiskers represent 5% and 95% percentile. (D) Unique clones, clonality, as well as maximum frequency of distinct clones and top 5 clones in the CD8 T-cell receptor (TCR) repertoire was analyzed by deep sequencing. Prior to analysis, CD8⁺ T cells were magnetic-activated cell sorted from PBMC of healthy controls (ctrl, n = 10), treatment-naive patients with RRMS (MS naive, n = 11), and patient 2 at baseline (vitiligo). RNA of at least 2 × 10⁶ CD8⁺ T cells from patient 2 at baseline was purified and cDNA was sequenced by Adaptive Biotechnologies, resulting in a range from 1.5 to 2.5 × 10⁶ total reads in each analyzed sample. (E) 3D histograms show expansion of single clones in patient 2 in comparison to a representative treatment-naive patient with RRMS (i.e., sex- and age-matched and exhibiting a similar mean number of relapses and mean EDSS progression in the last 2 years as compared to patient 2). The x axis lists all Vβ genes, the z axis the Jβ genes, and the column height indicates the total reads of this specific V/J combination. (F) Clone tracking of the top 10 expanded clones (highest number of total reads) of patient 2 before and 18 months after alemtuzumab treatment. The proportion of clones in the whole TCR repertoire is depicted. HLA = human leukocyte antigen; IFN-γ = interferon-γ; TNF-α = tumor necrosis factor–α.