Fig 2: S-acylation of ZAP-70 is required for T cell activation.A, IL-2 production by P116 (ZAP-70 -/-) Jurkat T cells stably rescued with WT or acylation-deficient C564R ZAP-70. IL-2 concentrations were measured by ELISA in supernatants from resting cells or cells stimulated for 24 h with plate-bound anti-CD3 antibody. Data shown are representative of three independent biological repeats and represented as mean ± SEM. B and C, Expression of CD25 and CD69 T cell surface activation markers by P116 stably rescued with WT or C564R ZAP-70. Cells were stimulated for 24 h with plate-bound anti-CD3 antibody and analyzed by flow cytometry. D and E, Quantification of CD25 and CD69 expression measured by flow cytometry. Data shown are pooled from three independent biological repeats and represented as mean ± SEM.

Fig 3: miR-1468-5p directly targets HMBOX1 in HDLECs to suppress CD8+ T cell immunity(A) Target gene prediction of miR-1468-5p with two bioinformatics tools. (B) qRT-PCR analysis of predicted genes expression in HDLECs transfected with miR-1468-5p mimics or NC. (C) Western blot of HMBOX1 expression in HDLECs transfected with miR-1468-5p mimics or NC. (D) The wild type (WT) and a mutated type (MT) of binding site between miR-1468-5p and HMBOX1. (E) Relative luciferase activity of HDLECs with indicated treatments. (F and G) Flow cytometry analysis of PD-L1 expression (F) and quantification of tube formation (G) in HDLECs transfected with siHMBOX1 or siRNA. (H and I) Flow cytometry analysis of PD-L1 expression (H) and quantification of tube formation (I) in HDLECs treated with miR-1468-5p mimics or NC in the presence of HMBOX1 or vector. Scale bar, 10 µm. (J-M) Flow cytometry analysis of IFN-? (J), CD69 (K), PD-1 (L), and Annexin V (M) expression on CD8+ T cells co-cultured with HDLECs treated with miR-1468-5p mimics or NC in the presence of HMBOX1 or vector. The numeric values under the western blot bands represent the protein relative expression (the indicated protein/GAPDH). Error bars represent the mean ± SD of three independent experiments. *p < 0.05; ***p < 0.001.

Fig 4: RSV-specific CD8+ T cells in BAL display a distinctive resident memory phenotype.Tetramer+ CD8+ T cells in blood and BAL were co-stained for markers to assess their differentiation status. (a) CD69 and CD103 as canonical markers of resident memory CD8+ T cells are shown in blood (n=9) and BAL (n=5) from infected volunteers. Significant P values for two-tailed Wilcoxon matched pairs tests in blood compared with baseline are shown (day 7, P=0.0313; day 10, P=0.0039; day 14, P=0.0313; and day 28, P=0.0313). (b) Memory markers CD45RA and CCR7 are shown in blood (n=19) and BAL (n=8). Mean±significant P values for two-tailed Wilcoxon matched pairs tests compared with baseline are shown in blood for T-effector/effector memory cells (day 7, P=0.0034; day 10, P=0.0002; day 14, P=0.0002; day 28, P=0.0067) and effector memory T cells re-expressing CD45RA (day 7, P=0.0443; day 10, P=0.0025; day 14, P=0.0003; day 28, P=0.0135). (c) Proliferation and activation markers Ki-67 and CD38 are shown in blood (n=19) and BAL (n=8). Significant P values for two-tailed Wilcoxon matched pairs tests are shown compared with baseline in blood (day 7, P=0.0025; day 10, P=0.0001; and day 14, P=0.0005) and BAL (day 7 versus day 10, P=0.0444; and day 10 versus day 28, P=0.0022 as no Ki-67+CD38+ cells were found in any baseline samples). Throughout, representative plots from a single subject at day 0, 10 and 28 post infection are shown with tetramer+ cells as red dots and total CD8+ T cells in grey contours. Summarized data are shown for all RSV-infected subjects (who could be analysed using tetramers) with median frequencies shown in red. Abbreviated P values are shown: NS (not significant)=P>0.05, *P<0.05, **P<0.01.

Fig 5: iNKT cells activation status and cytokine production in PBMCs of psoriasis patients. Intracellular IFN-? and IL-4 production of circulating iNKT cells was analyzed after stimulation with Cell Stimulation Cocktail for 5 h. (A) Representative FACS dot plots for iNKT cell CD69 expression in psoriasis patients and healthy controls. (B) Summary plots showing individual results of the frequency of CD69 + iNKT cells in psoriasis patients versus healthy controls. (C) Representative histogram and summary plots showing individual results of the MFI of IFN-?-producing iNKT cells in psoriasis patients versus healthy controls. (D) Representative histogram and summary plots showing individual results of the MFI of IL-4-producing iNKT cells in psoriasis patients versus healthy controls. (E) Representative histogram and summary plots showing individual results of the MFI of IL-17-producing iNKT cells in psoriasis patients versus healthy controls. (F) Summary plots showing individual results of IL-4-producing iNKT cells/IFN-?-producing iNKT cells and IL-17-producing iNKT cells/IFN-?-producing iNKT cells in psoriasis patients versus healthy controls. Data show mean +SEM. p-values were determined by paired Student’s t-test. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.