Fig 1: Design of therapeutic peptide‐producing T cell and its validation. a–c) Schematic illustration of mode of action of PC–Gla–TACE–MMP–TRAP (PTMT) T cell. During the blood circulation, serum‐abundant TACE and MMP1 cleave the peptide sequence, and the PC‐Gla and TRAP peptides are released. The PC‐Gla and TRAP bind to EPCR and PAR‐1, respectively, and, in turn, induce therapeutic effects. d) Vector design for PTMT. The therapeutic domains of PC‐Gla and TRAP, and TACE‐ and MMP1‐responsive cleavage sites are inserted. e) Transduction efficiency of PTMT‐T cell depending on the MOI. f) Western blot analysis of PTMT‐T cell‐secreted PC‐Gla depending on the culture time in vitro. g) Immunofluorescence images of PC‐Gla expressed in human CD8+ PTMT‐T cells. Scale bar, 75 µm. h) Scanning electron microscopy images of native and PTMT CD8+ T cells. Scale bar, 1 µm. i) Western blot analysis of MMP1, TACE, and PC‐Gla expressed in PTMT‐T cells when they were exposed to infectious disease patients’ plasma (n = 3/each group). The infectious diseases include septic shock and COVID‐19. j) High‐performance liquid chromatography (HPLC) analysis of gPC‐Gla expressed in PTMT‐T cells upon the exposure to infectious disease patients’ plasma. k) Enzyme‐linked immunosorbent assay (ELISA) analysis of PC‐Gla expressed in PTMT‐T cells upon the exposure to infectious disease patients’ plasma. Statistics, significance: The experiment was performed at least three times with replicates. Data are presented as mean ± SEM. P‐values are calculated using an ANOVA (j,k). *p < 0.05, ** p < 0.01.
Fig 2: PTMT‐T cell protects endothelial protein C receptor (EPCR) of an infected blood vessel. a) The expression level of soluble EPCR (sEPCR) depending on the severity of COVID‐19 infectious diseases. (normal [n = 20], mild COVID‐19 [n = 70], and severe COVID‐19 [n = 30]). b) The expression level of sEPCR in survived and deceased individuals. c) The computed tomography images of COVID‐19 patients’ lung tissues. The degree of inflammation is related to the expression level of sEPCR. Representative images from each group are shown (n = 5). d) Western blot analysis of EPCR and PAR‐1 in severe COVID‐19 patients’ peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVECs) when cocultured with PTMT‐T‐ cells. As the number of PTMT‐T cell increases, the expression levels of EPCR and PAR‐1 increase. e) Concentration of sEPCR cleaved by severe COVID‐19 patients’ PBMCs when cocultured with PTMT‐T cells. f) Concentration of sEPCR cleaved by HUVECs when cocultured with PTMT‐T cells. g) PAR‐1 cleavage activity (**p < 0.01). h) Recovery of the damaged engineered blood vessel by the PTMT‐T cells. The engineered blood vessel, which was formed in organ‐on‐a‐chip platform was damaged by the severe COVID‐19 patients’ plasma, and recovered after the administration of PTMT‐T cell (24 h coculture). Scale bar, 200 µm. i) Quantification of transendothelial permeability (n = 6/each group, *p < 0.05). Statistics, significance: The experiment was performed at least three times with replicates. Data are presented as mean ± SEM. P‐values are calculated using an ANOVA (a,b,e,f,g,i). *p < 0.05, ** p < 0.01 (PBS (n = 6) and native and PTMT‐T cell (n = 4).
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