Fig 1: Mechanisms of NK92 cellular immunity at genotyping levels via CIAMAP.(A) In nontransformed/wild-type cells (bottom right), TRAIL binds its receptor (1), resulting in the recruitment of CASP8 by FADD (2) and the formation of the death-inducing signaling complex (DISC) (3). DISC initiates the subsequent cascade of caspases via the activation of NF-kB (4), which induces apoptosis (5). In transformed cells (bottom left), the alternative set of TRAIL receptors blocks the formation of DISC and hence avoids apoptosis. (B) RNA sequencing to evaluate the leading mechanism of contact-triggered cellular immunity. PRF1 encodes perforin, and IFN series genes encode IFN families. A significant increase of IFNG, TNF, and FASLG gene expression in groups III and IV indicates that NK92 cell cytotoxicity is activated via intercellular interaction. Meanwhile, the PRF1 gene expression level has few changes. (C) Differential expression of the TRAIL-related pathway genes in samples with and without doxorubicin. (D) To characterize K562 cells, the differential expression of gene markers for K562 cells is analyzed. A dramatic down-regulation in groups III and IV displays a low tumor cell activity. (E) Volcano representation of the gene expression profile of NK92 cells treated with doxorubicin. About 18,824 genes were up-regulated, while 16,796 genes were down-regulated when doxorubicin was present. (F) Gene ontology (GO) analysis suggests that most of the genes identified in our study regulate the cytotoxicity of activated NK92 cells.
Fig 2: Validation of the enhanced CD8 TEMRA portion in another subcohort and discovery of heightened co-expression of CD8 cytotoxic molecules.a Scatter dot plots showing the frequency of total CD8 T cells among CD3 or total living lymphocyte singlets using the FCM analysis of cryopreserved PBMC from another subcohort of the Luxembourg Parkinson’s study (11 iPD vs 12 HC, all CMV+ except for two seronegative HC). Scatter dot plots showing the frequency of CD8 TEMRA (b) or TCM (c) among total CD8 T cells using different marker combinations. Of note, one sample was excluded from PD as it was identified as an outlier by the default setting of the ROUT method of Graphpad. d Scatter dot plots showing the ratios between CD8 TEMRA and CD8 TCM using different marker definitions. Scatter dot plots showing the frequency of GZMA+ (e), GZMB+ (f), perforin+/PRF1+ (g) or GZMK+ cells (h) among various CD8 T subsets. i Scatter dot plots showing the frequency of GZMA+GZMB+PRF1+GZMK- cells among total CD8 T cells or CD8 subsets. j Gating strategy was used to identify different CD8 subsets by FCM (BD FACSymphonyTM S6). k Representative FCM plots showing the expression of GZMA, GZMB, PRF1 and GZMK among CD8 TEMRA. l Scatter dot plots showing MFI of perforin among total CD8 cells or CD8 subsets. The results in a–i, l were analyzed using ordinary one-way ANOVA test with two-stage linear step-up procedure of Benjamini/Krieger/Yekutieli correction. q-values (FDR) were displayed. Data are presented as mean ± standard deviation (s.d.). Each symbol represents the measurement from one individual. all q-values are indicated. CMV Cytomegalovirus, FCM flow cytometry, GZMA/B/K Granzyme A/B/K, HC healthy controls, n = 12 including 5 males; PD patients with Parkinson’s disease, n = 11 including 8 males. Source data are provided as a Source Data file.
Fig 3: scRNA-seq reveals enhanced cytotoxic pathways in CD8 TEMRA of early-to-mid stage iPD.a Schematic for CD8 scRNA-seq. The other gates are in Supplementary Fig. 6a. b UMAP showing distribution of n = ~25,000 cells. Violin plots of selected genes defining CD8 naive/memory subsets (c), signifying cytotoxicity (d) and involving in top-ranked pathways enriched among upregulated genes in CD8 TEMRA (m). e Balloon plot showing the percentages of cells co-expressing the indicated markers. f, h UMAP showing joint density of GZMA+GZMB+PRF1+ (f) and GZMA+GZMB+PRF1+IFNG+ (h) in all CD8 cells. UMAP showing the individual GZMA+GZMB+PRF1+ (g) or GZMA+GZMB+PRF1+IFNG+ (i) cells among CD8 subsets. For visual comparability, random downsampling was employed to display the same number of cells for different conditions and subsets. j Volcano plots showing the expression changes in CD8 TEMRA. The selected top up- or downregulated DEGs are labeled in red or blue. Vertical dashed line indicates the log2FC value of 0.5 or −0.5, while the horizontal red line indicates –log10(0.05). k Top-ranked enriched KEGG pathways among upregulated DEGs in CD8 TEMRA of iPD vs HC. l Top-ranked GSEA pathways in upregulated DEGs in CD8 TEMRA. The lower part showing the rank distribution of the genes involved in the indicated pathway. The list on the right showing the leading-edge genes. n UMAP showing the unsupervised clustering analysis for n = ~7200 CD8 TEMRA cells. The clustering results were split for iPD or HC cells. Cells were randomly down-sampled to the same number for iPD and HC. o Violin plots of selected markers distinguishing clusters. p Heatmap of selected top DEGs in the clusters of CD8 TEMRA. P-values in j and p were analyzed using two-tailed nonparametric Wilcoxon Rank Sum test adjusted based on Bonferroni correction. In p, only the genes with adjusted P-value ≤0.05 were considered. P-values in k and l were analyzed using the one-tailed Fisher’s exact test and the one-tailed empirical phenotype-based permutation test, respectively. DEG differentially expressed genes, FACS fluorescence-activated cell sorting, FC fold change, GZMA/B/K Granzyme A/B/K, IFNG interferon gamma, UMAP uniform manifold approximation and projection. Panel a was created with BioRender.com.
Fig 4: Resazurin‐based cell viability assay reveals a dose‐dependent cell viability loss in in adherent breast cancer MDA‐MB‐231 cells upon NK‐EV treatment correlating with levels of cytotoxic factors. (a) Breast cancer MDA‐MB‐231 cells were treated for 5 hours with various E: T ratios of NK‐EVs or negative control 293F‐EVs, followed by endpoint resazurin‐based cell viability assay. The results are presented in (i) RFU (black dashed line represents lysed cell control) and (ii) EC50 curve analysis with variable slope for NK‐EV treatment with 95% confidence interval/prediction bands (red dashed line represents 50% response). Data are shown as mean ± S.E.M. from six independent experiments, each with technical quadruplets. (b) Spearman correlations of the selected NK‐EVs corona cytotoxic factors (GzmB, GNLY, PFN, FasL and IFN‐γ) in relation to cell viability assay responses in MDA‐MB231 cancer cells (n = 14 correlations pairs where one pair (a dot in the figure) represents the correlation between the level of a cytotoxic factor and the cell viability assay readout for a given NK‐EV dose).
Fig 5: Resazurin‐based cell viability assay reaffirms a dose‐dependent cell viability loss in leukaemia cells after NK‐EV correlating with levels of cytotoxic factors. (a) Leukaemia K562 cells were treated for 5 hours with various E: T ratios of NK‐EVs or negative control 293F‐EVs, followed by endpoint resazurin‐based cell viability assay. The results are presented in (i) RFU (Relative Fluorescence Units) readouts (black dashed line represents lysed K562 cell control; detergent‐treated) and (ii) EC50 curve analysis with variable slope for NK‐EV treatment with 95% confidence interval/prediction bands (red dashed line represents 50% response). Data are shown as mean ± S.E.M. from seven independent experiments, each with technical quadruplets. (b) Spearman correlations of selected NK‐EV's corona cytotoxic factors (GzmB, GNLY, PFN, FasL and IFN‐γ) detected by ELISA in relation to cell viability assay using leukaemia K562 cells (n = 14 correlations pairs where one pair (a dot in the figure) represents the correlation between the level of a cytotoxic factor and the cell viability assay readout for a given NK‐EV dose). (c) NK‐EVs corona cytotoxic factors (schematically shown) were assessed by ELISA and presented as the protein‐normalised levels (ng/mg of protein) of a given cytotoxic factor (GzmB, GNLY, PFN, FasL and IFN‐γ) using total protein levels (mg) detected by Qubit Protein Assay. Data are shown as mean ± S.D. from three independent experiments, each with technical duplicates.
Supplier Page from Abcam for Human Perforin ELISA Kit (PRF1)