stm.sciencemag.org/cgi/content/full/11/511/eaav7561/DC1

Supplementary Materials for

IFN-γ enhances cell-mediated cytotoxicity against keratinocytes via JAK2/STAT1 in

lichen planus

Shuai Shao, Lam C. Tsoi, Mrinal K. Sarkar, Xianying Xing, Ke Xue, Ranjitha Uppala, Celine C. Berthier, Chang Zeng, Matthew Patrick, Allison C. Billi, Joseph Fullmer, Maria A. Beamer, Bethany Perez-White, Spiro Getsios, Andrew Schuler,

John J. Voorhees, Sung Choi, Paul Harms, J. Michelle Kahlenberg, Johann E. Gudjonsson*

*Corresponding author. Email: johanng@med.umich.edu

Published 25 September 2019, Sci. Transl. Med. 11, eaav7561 (2019)

DOI: 10.1126/scitranslmed.aav7561

The PDF file includes:

Fig. S1. Transcriptomic profiling analysis and tissue immunofluorescence of IFN expressions in LP/HLP lesions. Fig. S2. IFN-γ increases keratinocyte susceptibility to cell-mediated cytotoxicity. Fig. S3. Keratinocyte death in in vitro coculture system is mediated by apoptosis and necroptosis. Fig. S4. Epidermal keratinocyte death in LP skin lesions is both apoptosis and necroptosis. Fig. S5. Cytotoxic responses to IFN-γ–primed keratinocytes are MHC class I-dependent. Fig. S6. IFN-γ induces keratinocytes to express MHCs through JAK2/STAT1 signaling. Fig. S7. Chromatograms and validation for JAK1, JAK2, STAT1, and STAT2 KO cell lines. Fig. S8. Targeting JAK signaling protects keratinocytes from cell-mediated cytotoxicity. Fig. S9. Epidermal keratinocyte death in CLE and LP, but not psoriasis (PV), is characterized by both apoptosis and necroptosis. Legends for tables S1 and S2 Table S3. Skin donor demographics.

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/11/511/eaav7561/DC1)

Table S1 (Microsoft Excel format). The list of DEGs based on microarray data in skin lesions. Table S2 (Microsoft Excel format). Drug target enrichment analysis among the genes differentially expressed in LP. Data file S1 (Microsoft Excel format). Primary data

Fig. S1. Transcriptomic profiling analysis and tissue immunofluorescence of IFN

expressions in LP/HLP lesions. (A, B) Type I IFN (A) and Type III IFN (B) expressions in LP,

HLP, and healthy control skin in the microarray dataset. (C) Representative image of co-

localization of IFN-γ with CD3, CD4, and CD8 in LP lesions (n=4) and healthy controls (NC,

n=4). Nuclei were stained with DAPI. (D) Representative image of IFNα/β/κ expression in LP

lesions and (n=4) healthy controls (NC, n=4). Scale bars, 100 μm.

Fig. S2. IFN-γ increases keratinocyte susceptibility to cell-mediated cytotoxicity.

(A) The schematic outline of the keratinocytes and PBMCs co-culture model used in this study.

This system utilizes keratinocytes from one individual and mixes in with lymphocytes from a

second individual analogous to what is done in a mixed lymphocyte reaction. (B) Representative

flow cytometry data of unstained, PI stained, and FITC Annexin-V stained keratinocytes. (C)

The statistical analysis of keratinocyte apoptosis in several conditions of co-culture as shown in

the right legends. Fractions on right side of labels refer to ratio of KCs/PBMCs. (D)

Keratinocytes primed with IFN-γ, IFN-α, or IFN-β for 24 h were then co-cultured with

CD3/CD28 microbeads-activated PBMCs for 72 h, and cell death was evaluated by Annexin-V

PI staining. (E) Representative flow cytometry data of analysis in Fig. 2C. One-way ANOVA.

Data are presented as the mean ± SD of measurements obtained in triplicate experiments. *P <

0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. S3. Keratinocyte death in in vitro coculture system is mediated by apoptosis and

necroptosis. Representative images showing the expression of cleaved-caspase3, p-RIP3, and p-

MLKL in keratinocytes from in vitro co-culture system. The experiment was repeated for three

times. Scale bars, 100 μm.

Fig. S4. Epidermal keratinocyte death in LP skin lesions is both apoptosis and necroptosis.

Representative images of cleaved-caspase3, p-RIP3, and p-MLKL staining in LP skin lesions

(n=5) and healthy controls (NC, n=5). Scale bars, 100 μm.

Fig. S5. Cytotoxic responses to IFN-γ–primed keratinocytes are MHC class I-dependent.

(A) The mRNA expression of MHC I molecules in keratinocytes stimulated with IFN-γ, IFN-α,

or IFN-β for 24 h. One-way ANOVA. Data are presented as the mean ± SD of measurements

obtained in triplicate experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001. (B)

Representative flow cytometry data as shown in Fig. 3D.

Fig. S6. IFN-γ induces keratinocytes to express MHCs through JAK2/STAT1 signaling.

(A) Representative immunofluorescence staining of STAT2 and pSTAT2 in LP/HLP skin lesions

and controls (n=4). The nuclei were stained with DAPI. Scale bar, 100µm. (B) The

quantification of p-JAK1/JAK1, p-JAK2/JAK2, p-STAT1/STAT1, p-STAT2/STAT2 in IFN-γ-

stimulated keratinocytes from one representative experiment. (C) The mRNA expression of IFN-

γ-induced genes including MX1, OASL, IRF7, IRF9 in JAK1, JAK2, STAT1, and STAT2 KO cells

after IFN-γ (10 ng/ml) induction for 24 h. One-way ANOVA. Data are presented as the mean ±

SD of measurements obtained in triplicate experiments. **P < 0.01, ***P < 0.001, ****P <

0.0001.

Fig. S7. Chromatograms and validation for JAK1, JAK2, STAT1, and STAT2 KO cell lines.

(A) Homozygous or heterozygous compound KO was validated by Sanger Sequencing for each

of the JAK1, JAK2, STAT1 and STAT2 KO keratinocytes. (B) The validation of JAK1, JAK2,

STAT1, and STAT2 KO keratinocytes using western blot.

Fig. S8. Targeting JAK signaling protects keratinocytes from cell-mediated cytotoxicity.

(A) JAK1 and STAT2 KO cells were primed with IFN-γ and co-cultured with CD3/CD28

activated PBMCs. The representative flow cytometry data (left panel) and analysis of

keratinocyte cell-death (right panel) in the co-culture model were shown (n=3). Two-way

ANOVA. (B, C) Western blot was used to validate the protein expression of MHC I molecule in

IFN-γ-treated keratinocytes (n=3) (B) and in skin lesions (n=2) (C) and quantification of MHC I

molecule expression (right panel). One-way ANOVA. Data are presented as the mean ± SD of

measurements obtained in quadruplicate experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig. S9. Epidermal keratinocyte death in CLE and LP, but not psoriasis (PV), is

characterized by both apoptosis and necroptosis. Representative images of cleaved-caspase3,

p-RIP3, and p-MLKL staining in HLP, CLE, and PV skin lesions (n=5) and healthy controls

(NC, n=5). Scale bars, 100 μm.

Table S1. The list of DEGs based on microarray data in skin lesions.

(shown in Excel labeled table S1).

Table S2. Drug target enrichment analysis among the genes differentially expressed in LP.

(shown in Excel labeled table S2).

Table S3. Skin donor demographics.

Diagnosis Average age (+/- stdev) Gender male vs female

Lichen planus 52.4±14.1 7/13

Hypertrophic

lichen planus

64.41±15.36 5/12

Healthy controls 52±14.72 15/9