Fig 2: Residual MAIT cells are highly activated during HFRS(A) Representative flow cytometry plots showing frequencies of CD38+, CD69+, granzyme B+, and Ki67+ MAIT cells in controls and individuals with HFRS during the acute phase (days 3–9, n = 22), intermediate phase (days 10–21, n = 18), and convalescent phase (conv.) (n = 24).(B) Frequencies of CD38+, CD69+, granzyme B+, and Ki67+ MAIT cells in controls and individuals with HFRS during the acute phase (n = 22), intermediate phase (n = 18), and conv. (n = 24). Bars represent median values. Kruskal-Wallis test, Friedman test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig 3: Transcriptomic differences of cis versus trans CD137 costimulation have implications in T cell proliferation and DNA repair.a, volcano plot of differentially expressed genes of cis-costimulated and trans-costimulated CD8+ T cells with mAb coated microbeads for 32 hours. Red dots indicate genes related to DNA damage/repair and blue dots indicate genes related to T cell activation. Dotted lines mark the t test p value cut-off of p < 0.01 and log2FC > 2. b, Top significantly affected pathways based on Ingenuity pathway analysis (IPA). The horizontal bars denote the different pathways based on the z-score. Orange color indicates upregulation, while red color indicates downregulation. c, top genes displaying altered expression in the DNA damage/repair pathway (left panel) and cell division (right panel) in cis- versus trans costimulation. d, representative dot plots of nuclear Ki67 expression in CD8+ T cells after costimulation with mAb coated beads. Cumulative data (n = 12) are shown as the difference between the value of cis and trans conditions to which the anti-CD3 background was subtracted in each case. e, CD8+ T cells were labeled with a violet proliferation dye (VPD) and stimulated with mAb coated beads for 96 h. Representative dot plot of proliferating CD8+ T cells and cumulative data (n = 12). Cumulative data are shown as the difference between the value of cis and trans conditions to which the anti-CD3 background was subtracted in each case. f, DNA synthesis was assessed in CD8+ T cells by cis-costimulation with wild-type HCT116 cells or trans costimulation with the alternatively EpCAM and 5T4 silenced variant combinations in the presence of CD3-EpCAM and ALG.APV-527 BsAbs (n = 9 for cis, n = 8 for trans1 and trans2). Co-cultures were treated with 10 µM EdU for 2 h and its incorporation to genomic DNA was detected according to the recommended staining protocol. VPD labeled CD8+ T cells were analyzed for DNA damage after stimulation with mAb coated beads. Intranuclear staining of phosphorylated gH2AX on total proliferating CD8+ T cells (g) (n = 10) and cells analyzed as per division cycle upon gating (h) (n = 5) was measured by flow cytometry. The division peaks are numbered from 1 to 3. Data are given as mean ± s.e.m. Statistical significance was determined with paired t test (two-sided) for d, e, g and h and one-way Anova (one-sided) with Holm-Sidak multiple comparison test for f. (*p < 0.05, **p < 0.01, ***p < 0.001). Source data can be retrieved under accession number GSE158041 and as Source Data file.

Fig 4: Analysis of the anti-proliferative effect of the expression of Nefmut/43M2 vector in HPV16-infected cells. (A) Western blot analysis of the expression of Nefmut-derived products in transfected SiHa cells. Filter was revealed by both anti-Nef and HRP-conjugated anti-β-actin Abs. Molecular mass markers in kDa are reported on the left. Results are representative of three independent experiments. (B) FACS analysis of SiHa cells after transfection with a GFP-expressing vector. As control, mock-transfected cells also were analyzed. The analysis was carried out on 7-AAD negative cells. Percentages of positive cells are indicated. Shown are results representative of six independent experiments. (C) Cell viability of SiHa cells 48 hrs after transfection with the indicated vectors. A total of 2 × 105 cells was transfected with 1 µg of each DNA vector, and cell viability was scored 48 hrs later by the trypan blue exclusion method. Whatever the DNA vector used, an average of 10–20% of the dead cells were detected 24 hrs after transfection. Shown are mean values + standard errors of data from four independent experiments carried out in duplicates. *p<0.05 by two-tailed Mann–Whitney U-Test. Inset, geometric mean-fluorescence intensity as calculated by FACS analysis conducted on SiHa cells transfected with the indicated vectors and labeled with anti-Ki67 mAb. Shown are the mean values calculated from an experiment performed with duplicate conditions. (D-G) Effects induced by the expression of the Nefmut/43M2 vector on the proliferation of cells expressing HPV18-E7. (D) ELISA for the binding of 43M2 to recombinant E7 from either HPV16 or HPV18 strains. The results are representative of three independent experiments carried out in quadruplicate conditions. (E) FACS analysis of HeLa cells 48 hrs after transfection with a GFP-expressing vector. Percentages of DNA vector-expressing cells are reported. The analysis was carried out on 7-AAD negative cells. Results are representative of two independent experiments. (F) Western blot analysis of the expression of Nefmut-derived products in transfected HeLa cells. Filter was revealed by both anti-Nef and HRP-conjugated anti-β-actin Abs. Molecular mass markers in kDa are reported on the left. Results are representative of three independent experiments. (G) Cell viability of both SiHa and HeLa cells 48 hrs after transfection as assessed by MTS-based cell proliferation assay. Transfection procedures were as described for panel C, except that, to perform the MTS assay, the cells from each well of the 24-well plates were split in four microwells of a 96-well plate 48 hrs after transfection. Shown are the mean values of absorbance at 490nm (OD) + standard errors of the results obtained from four independent experiments performed in quadruplicate. *p: 0.0002, **p: 0.0054 by two-tailed Student T Test.

Fig 5: MAIT cell activation correlates with disease severity markers(A and B) Plasma levels of (A) cytokines and (B) granzymes in controls (n = 18) and individuals with HFRS during the acute phase (days 3–9, n = 24), intermediate phase (days 10–21, n = 21), and convalescent phase (conv.) (n = 24).(C) Correlations between plasma IL-6 levels and the frequencies of Ki67+, granzyme B+, and PD-1+ MAIT cells during acute HFRS (n = 22).(D) Correlations between platelet counts and the frequencies of Ki67+ and CD38+ MAIT cells during acute HFRS (n = 22).Spearman‘s rank correlation coefficient. Bars represent median values. Kruskal-Wallis test, Friedman test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.