Fig 2: Validation of ISG screen hits in targeted CRISPRa and ectopic cDNA expression studies.(A) Representative flow cytometry histograms for SARS-CoV-2 N protein in A549-SunTag ACE2 and A549?STAT1-SunTag ACE2 transduced with indicated gRNAs, treated (red) or not treated (gray) with Dox, at 24 hours post-infection with SARS-CoV-2 (M.O.I. = 2). (B) Percent of infected (SARS-CoV-2 N protein positive) cells quantified across biological replicates (n = 3, n = 2 for CD74 gRNA in A549-SunTag ACE2 cells) for experiments described in (A). Values denote percent of infected cells in Doxon cultures relative to paired Doxoff cultures. Points represent individual biological replicates, black lines indicate mean values of biological replicates for each indicated ISG gRNA. Red points indicate statistical significance (p < 0.05) as determined by paired ratio Student’s t-test. (C) Representative immunofluorescence images for SARS-CoV-2 N protein and DAPI in A549-SunTag ACE2 and A549?STAT1-SunTag ACE2 transduced with indicated gRNAs and treated with Dox, at 72 hours post-infection with SARS-CoV-2 (M.O.I. = 2). (D) Percent of infected (SARS-CoV-2 N protein positive) cells quantified across biological replicates (n = 3, n = 2 for ADPRHL2 gRNA in A549-SunTag ACE cells) for experiments described in (C). Values denote percent of infected cells in Doxon cultures relative to paired Doxoff cultures. Points represent individual biological replicates, black lines indicate mean values of biological replicates for each indicated ISG gRNA. Statistical significance as in (B). (E) Representative flow cytometry histograms for SARS-CoV-2 N protein in A549 ACE2 and A549?STAT1 ACE2 transduced with expression constructs for indicated cDNAs (fLUC, firefly luciferase negative control), treated (red) or not treated (gray) with Dox, at 24 hours post-infection with SARS-CoV-2 (M.O.I. = 2). (F) Percent of infected (SARS-CoV-2 N protein positive) cells quantified across biological replicates (n = 3) for experiments in (E). Values denote percent of infected cells in Doxon cultures relative to paired Doxoff cultures. Points represent individual biological replicates, black lines indicate mean values of biological replicates for each indicated ISG cDNA. Red points indicate statistical significance (p < 0.05) as determined by paired ratio Student’s t-test.

Fig 3: The dynamic characteristics and functional changes in T cells from lung and intestinal tissues during SARS-CoV-2 infection(A) Subclustering of T cells in all lung tissue samples (n = 6).(B) Quantification of T cell subsets in 3 (n = 1), 7 (n = 1), and 10 (n = 1) dpi in lung. Three healthy animals were regarded as the control. For each T cell cluster, the average fraction of cells is shown at different infection time points.(C) Subclustering of T cells in the small intestine (n = 8).(D) The histogram represents the quantification of T cell cluster frequency at different infection time points (3, 7, and 10 dpi) in the small intestine.(E) The differential expression levels of the genes CD38, CD69, GZMB, CD74, IFI27, and IFNG at 3 (n = 1), 7 (n = 2), and 10 (n = 2) dpi in the small intestine. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Wilcoxon test.(F) Analysis of the expression of T cell activation-related factors by a confocal immunofluorescence assay. Top: T cells were detected by an anti-CD3 antibody followed by Alexa Fluor 488 dye-conjugated secondary antibody. The T cell activation-related molecules CD38 and CD69 were detected with anti-CD38 and anti-CD69 primary antibodies, followed by secondary antibodies conjugated with Alexa Fluor 555 and Alexa Fluor 647 dyes, respectively. Bottom: T cells were detected by an anti-CD3 antibody followed by Alexa Fluor 488 dye-conjugated secondary antibody. The T cell activation-related granzyme B and molecules CD74 were detected with anti-granzyme B and anti-CD74 primary antibodies, followed by secondary antibodies conjugated with Alexa Fluor 555 and Alexa Fluor 647 dyes, respectively. Nuclei were stained with DAPI (magnification, 630×). Scale bar, 100 μm. Control, normal tissues.(G) Percentage of CD3+CD38+, CD3+CD69+, CD3+CD74+, and CD3+GZMB+ cells were computed in total intestine cells (number of cells from three randomly selected fields captured by confocal fluorescence microscope) for normal controls and COVID-19 intestine samples.(H) Genes clustered by their expression pattern during the progression of SARS-CoV-2 infection (3, 7, 10 dpi). GO term analysis revealed the top five enriched biological processes in nine clusters from the lung.(I) Genes clustered by their expression pattern during the progression of SARS-CoV-2 infection (3, 7, 10 dpi). GO term analysis revealed the top five enriched biological processes in nine clusters from the small intestine. Cluster 4 does not contained a significantly enriched term of biological process, so it is not shown.(J) Signature scores of the GO term “regulatory T cell differentiation” genes in T cell subsets (Trm, Teff, and Naive_T or _Tcm) in the lung by Ucell package.(K) Signature scores of the GO term “response to virus” gene module in major immune cells, including B cells, DC cells, macrophages, NK cells, and T cells of the small intestine by Ucell package.∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Wilcoxon test.See also Figure S2.

Fig 4: Identification of keratinocyte subpopulations in rosacea.a UMAP plots showing keratinocyte subclusters and sample conditions. b Bar graph showing subpopulation percentage. c Feature plot showing CD74 expression in keratinocytes. d The percentage of CD74+ keratinocytes (n = 3 samples for each group). e Immunohistochemistry of KRT14, CD74 in HS, normal skin of patients (NS), and lesional skin of ETR, PPR, PhR. White arrows indicate CD74+ keratinocytes. Scale bar, 50 μm. f Quantification of CD74+ keratinocytes (n = 6/6/6/6/5 samples for HS/NS/ETR/PPR/PhR group used in e). g, h Top-ranked enriched pathways in CD74+ keratinocytes of L versus HS (g), L versus N (h). NES indicates enrichment scores. The color keys from red to blue indicate the P-value range. Two-sided permutation test without multiple comparison adjustments was used. i Heatmap of skin barrier-related genes in CD74+ keratinocytes. j Heatmap of IFN-related genes in CD74+ keratinocytes. k Immunohistochemistry of IRF1, KRT14. Scale bar, 100 μm. l Quantification of IRF1+ keratinocytes in epidermis n = 6/6/6/6/5 samples for HS/NS/ETR/PPR/PhR group used in k). m The back skins of IgG and anti-IFNγ antibody-treated mice intradermally injected with LL37 or control vehicle. Images were acquired 48 h after the first LL37 injection. Below panels, magnified images of yellow boxed areas. n The severity of the rosacea-like symptoms was determined with the redness area and score (n = 6). o Dermal infiltrating cells were quantified (n = 6). p The relative mRNA levels of Il1β, Il6, Tnfα in mice skins (n = 6). q The relative mRNA levels of Cldn4, Cldn10, Cldn23 in mice skins (n = 6). r Immunohistochemistry of CLDN4, KRT14 in mice skins. Scale bar, 50 μm. Epi, Epidermis. Der, Dermis. s Quantification of relative fluorescence intensity for CLDN4 in epidermis (n = 6). t Quantification of TEWL (n = 6). All results are representative of at least three independent experiments. Data are presented as mean ± SEM, and P-values were determined by one-way ANOVA with Tukey’s post hoc test (f, l, n, o, p, q, s, t) and two-tailed unpaired (L vs HS) or paired (L vs N) Student’s t-test (d).

Fig 5: Validation of the predictive value of CD74 expression in ICB treatment response in esophageal cancer. (A) mIHC staining of FFPE specimens from gastric cancer patients demonstrates that the expression of CD74 is co-localized with CD80, a marker for macrophage. White arrows indicate colocalized cells. (B) The tumor expression of CD74 in responders is significantly higher than the non-responders who were treated with ICB. (C) The expression of CD74 before and after ICB treatment in responders and non-responders. In the responders, the CD74 expression is significantly elevated after treatment, which is not observed in the non-responders. (D, E) Representative figures of CD74 expression in specimens before and after treatment in responders (D) and non-responders (E). (F) ROC curve analysis demonstrates that CD74 expression has good performance in discriminating the responders from the non-responders. (G) Response rates to ICB therapy in the CD74 high and low expression groups. (H-I) Kaplan–Meier curve analysis of the correlation between CD74 protein expression and overall survival (H) and disease-free survival (I) in esophageal cancer patients with ICB treatment. FFPE, formalin-fixed and paraffin-embedded; mIHC, multiplex immunohistochemistry; ROC, receiver operating characteristic.