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.