Sample preparation
Skin punch biopsies were taken from for histopathological evaluation were taken from all patients (n=12), skin punch biopsies for research purposes were available only for patients in Zurich: COVID-MDR- (n=4), MDR- (n=7) and DRESS (n=4), all obtained from the trunk. Skin from HC was obtained as discarded tissue from cutaneous surgery (n=5). Skin samples were formalin-fixed and paraffin-embedded (FFPE). Blood samples were obtained from COVID-MDR- (n=5), DRESS- (n=5), MDR-patients (n=3) and HC (n=4). Blood, collected using serum tubes, was processed immediately after collection and stored at -80 Celsius until further processing.
Blinded histopathological assessment
Slides with hematoxylin/eosin (HE)-stained skin sections (4 COVID-MDR, 4 MDR and 4 DRESS, Figure S1) were scanned and blindly evaluated by a board-certified dermatopathologist. For further details, see the Supplementary Materials and Methods.
Immunohistochemistry (IHC) stainings and quantification of CD3+ cells
FFPE tissue sections (4 COVID-MDR, 4 MDR, 3 DRESS and HC) were stained with an anti-ACE2 antibody (Thermofisher, cat. no. MA5-31395, mouse IgG1, clone CL4035, 1:2000) and an anti-CD3 antibody (Dako, cat. No. M7254, clone F7.2.38, mouse IgG1, 1:50). Randomly selected images were obtained per scanned CD3-stained skin section of each donor. Representative photos are depicted in Figure S2. For further details, see the Supplementary Materials and Methods.
Imaging Mass Cytometry (IMC)
All antibodies used for IMC were titrated and validated by immunofluorescence for specific staining patterns and in IMC for co-staining with other known markers. Some antibodies were additionally tested with an antigen-binding fragment (Fab) labelling kit, used as previously described19. We designed an IMC panel consisting of 36 antibodies covering both non-leukocytic and leukocytic, mostly T cell- and antigen presenting cell-related, antigens (Table 2). We stained and processed COVID-MDR (n=4), DRESS (n=4), MDR (n=4), and HC (n=4) skin sections. For further details, see the Supplementary Materials and Methods.
IMC data analysis
Pre-processing and single-cell segmentation was performed following the instructions on the Bodenmiller Github repository (https://github.com/BodenmillerGroup/ImcSegmentationPipeline).
After single-cell generation all subsequent analysis were performed using R bioconductor. For cell-type annotation we manually gated major cell-types of interest using the cytomapper R package20. The following markers were used to define cell-types: CD8+T cells I (CD3+, CD8+), CD4+ T cells I (CD3+, CD8- CD4), keratinocytes I(E-cadherin+, Filaggrin-), keratinocytes II (Filaggrin+), Langerhans cells (LC; E-cadherin+, Langerin+), macrophages (CD163+), neutrophils (polymorphonuclear leukocytes (PMN); MPO+), plasmacytoid dendritic cells (pDC; CD303+), vasculature (CD31+). Roughly, half of all cells were manually gated using the markers from above. For the remaining cells in the dataset we used a random forest classifier to assign cell-types based on uniquely labelled cells21. Therefore, all labelled cells were spit in training and test data (70:30). A random forest model was trained on the training-set (10 fold cross-validation, mtry parameter optimization) and the model performance validated on the test-set. After prediction of a cell-type for all unlabeled cells the classification results were inspected on images and additional rounds of cell labelling performed if needed. We excluded 1 HC sample, since it contained highly increased numbers of CD3+ T cells and was obtained from an excision of peritumoral tissue.
Cellular interactions were quantified using our published neighborhood algorithm22 and a R implementation thereof (https://github.com/BodenmillerGroup/neighbouRhood). All R code used for IMC data analysis in this study is available via our github (https://github.com/BodenmillerGroup/Skin_rash).
RNA extraction and sequencing of skin biopsies
RNA was extracted from FFPE skin biopsies of 5 HC skin samples and lesional skin biopsies of MDR (n=7) and COVID-MDR (n=3) patients with a Qiagen® RNeasy FFPE Kit. Library preparation for RNA-seq was performed by using the TruSeq Stranded RNA library preparation kit (Illumina) from total RNA. Sequencing was performed on the Illumina NextSeq 500 platform. For further details, see the Supplementary Materials and Methods.