Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a) Primary BEC from healthy (n=7), COPD (n=6), or smoker (n=6) donors were incubated with the D426 G11 MAIT cell clone in an ELISPOT assay with IFN-γ production as the readout. Data points are the mean IFN-γ spot-forming units (SFU) of two technical replicates per donor. Statistical analysis was performed as described in the experimental procedures and is summarized in . b-c) BEC from a representative healthy and COPD donor were incubated with blocking antibodies to IL-12/IL-18 or MR1 five hours prior to addition of the MAIT cells in an IFN-γ ELISPOT assay. Results are presented as b) the mean of two experimental replicates and c) representative ELISPOT well images.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Incubation, Enzyme-linked Immunospot, Blocking Assay

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a) Primary BEC from healthy, COPD, or smoker donors were infected with media control, M. smegmatis (0.1μl/well), or S. pneumoniae (MOI 20) for one hour prior to addition of MAIT cells in an IFN-γ ELISPOT assay. Data points are the mean IFN-γ spot-forming units (SFU) of two technical replicates per donor. Statistical analysis was performed as described in the experimental procedures and is summarized in . b) BEC from a representative healthy and COPD donor were incubated with blocking antibodies to IL-12/IL-18 or MR1 one hour prior to infection with S. pneumoniae (MOI 20) and subsequent addition of the MAIT cell clones in an IFN-γ ELISPOT assay. Results are presented as the mean of two experimental replicates.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Infection, Control, Enzyme-linked Immunospot, Incubation, Blocking Assay, Clone Assay

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a-b) BEC from representative healthy, COPD, or smoker donors were infected with fluorescently labeled S. pneumoniae for three hours. Fixed cells were stained with DAPI and α-MHC-Ia antibody to label the cell surface. Approximately 20 fields per donor were selected without bias based on nuclear stain, and whole cells within these fields were then analyzed by Imaris to enumerate the number of bacteria associated with individual cells. a) Data points indicate individual cells, analyzed by one-way ANOVA statistical analysis. b) Representative images of S. pneumoniae -infected primary BEC. White = MHC-Ia surface staining. Red pseudocolor = fluorescent S. pneumoniae . Arrows indicate adherent S. pneumoniae (yellow) enumerated for analysis. c) IFN-γ SFU fold change between no-treatment control and M. smegmatis- or S. pneumoniae- infected primary BEC from healthy, COPD, or smoker donors. Raw data shown in Figure 2a. Statistical analysis was performed as described in the experimental procedures and is summarized in .

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Infection, Labeling, Staining, Bacteria, Control

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a-b) Primary BEC from healthy (n=7), COPD (n=6), or smoker (n=6) donors were incubated with media containing 0% or 30% CSE for three hours prior to the addition of MAIT cells in an IFN-γ ELISPOT assay. Statistical analysis was performed as described in the experimental procedures and is summarized in - . a) Data points are the mean IFN-γ spot-forming units (SFU) of two technical replicates, paired by individual donor. b) IFN-γ SFU fold change between 0% CSE- and 30% CSE-treated primary BEC from healthy, COPD, or smoker donors, calculated pairwise by donor.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Incubation, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Chronic obstructive pulmonary disease and cigarette smoke exposure lead to dysregulated MAIT cell activation by bronchial epithelial cells

doi: 10.1101/2022.02.28.482383

Figure Lengend Snippet: a-b) Primary BEC from healthy (n=7), COPD (n=6), or smoker (n=6) donors were incubated with media containing 0% or 30% CSE for three hours. BEC were infected with M. smegmatis (a, 0.05μl/well) or S. pneumoniae (b, 20 MOI) for one hour prior to the addition of MAIT cells in an IFN-γ ELISPOT assay. Statistical analysis was performed as described in the experimental procedures and is summarized in - . c) Fold change of a) and b) IFN-γ SFU between 0% CSE- and 30% CSE-treated primary BEC infected with M. smegmatis or S. pneumoniae , calculated pairwise by donor. d-e) BEC from representative healthy, COPD, or smoker donors were incubated with medium containing 0% or 30% CSE for three hours, then infected with fluorescently labeled S. pneumoniae for three hours. Fixed cells were stained with DAPI and α-MHC-Ia antibody to label the cell surface. Approximately 20 fields per donor were selected without bias based on nuclear stain, and whole cells within these fields were then analyzed by Imaris to enumerate the number of bacteria associated with individual cells. d) Representative images of S. pneumoniae -infected healthy BEC treated with 0% or 30% CSE. White = MHC-Ia surface staining. Red pseudocolor = fluorescent S. pneumoniae . Arrows indicate adherent S. pneumoniae (yellow) enumerated for analysis or extracellular microbes (red) excluded for analysis. e) Data points indicate individual cells, analyzed by one-way ANOVA statistical analysis. 0% CSE results from are included as reference.

Article Snippet: The following antibodies were used: for ELISPOT assays: α-MR1 (26.5, Biolegend), α-IL-12p70 (MAB219100, R&D systems), α-IL-18 (D044-3, MBL International Corporation), α-IgG2A isotype (400224, Biolegend), α-human IFN-γ (7-B6-1, MabTech); for fluorescence microscopy: α-human HLA-A,B,C (W6/32, biotinylated, Biolegend), streptavidin-AlexaFluor-647 (Life Technologies); for flow cytometry: α-MR1 (26.5, conjugated to APC, Biolegend), α-human HLA-A,B,C (W6/32, conjugated to APC, Biolegend).

Techniques: Incubation, Infection, Enzyme-linked Immunospot, Labeling, Staining, Bacteria

Journal: Nature Communications

Article Title: KSHV infection of B cells primes protective T cell responses in humanized mice

doi: 10.1038/s41467-024-49209-w

Figure Lengend Snippet: A IgM serum levels of humanized NSG mice 4 weeks after i.p. infection with mock, KSHV only, EBV (wt or zko) only or EBV (wt or zko) and KSHV. 11 (EBVwt) independent experiments with serum from N = 11 (mock), N = 5 (KSHV), N = 21 (EBVwt), and N = 23 (KSHV+EBVwt) mice and 6 (EBVzko) independent experiments with N = 8 (mock), N = 1 (KSHV), N = 13 (EBVzko) and N = 15 (KSHV+EBVzko) mice. Data are presented as individual data points with mean and SD. MWU ** p < 0.01, *** p < 0.001. Significant p values: mock – Ewt 0.0099; mock – Ewt+K 0.0023; mock – Ezko+K 0.0008. B IgG serum levels of humanized NSG mice 4 weeks after i.p. infection with mock, KSHV only, EBV (wt or zko) only or EBV (wt or zko) and KSHV. 3 (EBVwt) independent experiments with serum from N = 3 (mock), N = 5 (KSHV), N = 7 (EBVwt), and N = 8 (KSHV+EBVwt) mice and 6 (EBVzko) independent experiments with N = 7 (mock), N = 1 (KSHV), N = 14 (EBVzko) and N = 11 (KSHV+EBVzko) mice. Data are presented as individual data points with mean and SD. Two sided MWU * p < 0.05. Significant p value Ewt – Ewt+K 0.0126 ( C ) Heatmap of KSHV-specific IgM antibodies in serum from humanized mice 4 weeks after i.p. infection with mock, KSHV, EBV (wt or zko) or EBV (wt or zko) and KSHV, measured by ELISA. Columns represent mice, rows represent antigens. Color intensity represents the background-substracted optical density (OD) on a logarithmic scale. D Ratio of median sero-reactivity (OD) of EBV + KSHV and Mock&EBV per antigen is indicated on the x-axis with the corresponding FDR-adjusted p -value on the y-axis. Labels indicate antigens with a significant ( p < 0.05) and greater than 1.5 fold sero-reactivity in EBV + KSHV + vs Mock&EBV + mice. C , D Composite data with 47 mice from 4 independent experiments, N = 10 (mock), N = 3 (KSHV), N = 6 (Ezko), N = 10 (Ezko+K), N = 1 (Ew), N = 17 (Ew+K) mice per group. Source data are provided as a Source data file.

Article Snippet: 100’000 or 200’000 cells were co-cultured with equal volume of R10, PMA/Iono or equal amount of autologous E-CLs or KE-CLs or KE-CLs pulsed with K6-peptide pool (37 °C, 1 h, 1 mM peptide) over night and IFNγ ELISA was performed on the supernatant according to manufacturer protocol (MabTech).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: KSHV infection of B cells primes protective T cell responses in humanized mice

doi: 10.1038/s41467-024-49209-w

Figure Lengend Snippet: A IFNγ ELISpot of CD19-negative cells isolated from humanized mice after overnight culture with medium (R10), E-CL or KE-CL. Shown as spot forming units per one million CD19-negative cells; data are presented as individual values for R10 and mean values from duplicates for E-CL and KE-CL, with mean and SD. 1 experiment with N = 6 (Mock), N = 4 (EBVzko) and N = 5 (EBVzko+KSHV) animals. B . Experimental outline of the T cell isolation protocol from KSHV/EBV co-infected huNSG mice. CD19-negative splenocytes from KSHV + /EBVzko + mice were co-cultured with autologous KE-CLs for 24 h, IFNγ producing cells were isolated by IFNγ capture assay and cultured in limiting dilution. C IFNγ levels (pg/ml) in supernatant of isolated CD4 + and CD8 + T cell subpopulations after co-culture with E-CL or KE-CL were determined by ELISA upon growth after single cell dilution. N = 1. D Degranulation measured by % CD107a positivity on the isolated T cell subpopulations after co-culture with R10, E-CL or KE-CL. N = 2 (10-1, 10-14 and 1-9), N = 4 (01-1) independent experiments, means of technical duplicates and SD are shown. Two sided unpaired t -test, * p < 0.05; Significant p values 10-1 0.0393; 10-14 0.0235; 01-01 0.0369. E Specific killing (% dead cells compared to target cell without T cell condition) measured for CD8 + T cell subpopulation 01-1 after co-culture with E-CL or KE-CL at different effector to target cell ratios. 1 independent experiment, Shown are means of 2 technical replicates with SD. F Specific killing measured for CD4 + and CD8 + T cell subpopulations after co-culture with E-CL or KE-CL at an effector-target ratio of 2.5 to 1. 2 (10-1, 10-14, 1-9) or 3 (01-01) independent experiments, individual values represent means of technical replicates, bars show means, error bars represent SD. Two sided MWU, ** p < 0.01. exact p -value 01-01 0.0043 ( G ) Number of spot forming units per one million cells measured via IFNγ KSHV proteome wide ELISpot of KSHV-reactive T cells (isolated from the IFNγ capture assay) after stimulation with overlapping peptide pools for the indicated KSHV or control antigens. Figure shows the individual values from all wells with a signal (all data can be found in Supplementary Table ) with mean and SD. H Relative IFNγ production of the T cell subpopulation 01-1 after co-culture with KE-CL or KE-CL pulsed with the K6 peptide pool. N = 2 independent experiments, n = 3 (KE-CL + T cells) and n = 6 (KE-CL + T cells + K6 peptide mix) replicates per experiment. IFNγ production relative to the mean of the T cell only condition is shown for each replicate, SD is displayed. Two sided Unpaired t -test, ** p < 0.01. exact p -value 0.0033. Source data are provided as a Source data file.

Article Snippet: 100’000 or 200’000 cells were co-cultured with equal volume of R10, PMA/Iono or equal amount of autologous E-CLs or KE-CLs or KE-CLs pulsed with K6-peptide pool (37 °C, 1 h, 1 mM peptide) over night and IFNγ ELISA was performed on the supernatant according to manufacturer protocol (MabTech).

Techniques: Enzyme-linked Immunospot, Isolation, Cell Isolation, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of primary TCR-transduced T cell generation for downstream assays. B IFNɣ levels of primary TCR-transduced T cells or normalized luminescence of TCR-transduced Jurkat Lucia NFAT cells co-cultured overnight with HLA-matched LCLs pulsed with decreasing peptide concentrations. Graph shows mean ± SD of n = 2 (Jurkat Cl33), n = 3 (Jurkat Cl156/12, primary Cl114) or n = 4 (rest) independent experiments. C IFNɣ ELISA of TCR-transduced T cells co-cultured with cognate peptide-pulsed or unpulsed HLA-matched LCLs. Boxplots represent 5 (Cl114, Cl33), 4 (Cl12) or 3 (Cl156) independent experiments, using T cells from 8 (Cl114, Cl33, Cl12) or 6 (Cl156) different donors, tested against 9 (Cl114), 6 (Cl33), 4 (Cl156), 3 (Cl12) different LCLs. D Frequency of CD107a + T cells after 6 h of co-culture with cognate peptide-pulsed or unpulsed HLA-matching LCLs. Boxplots represent 3 (Cl114, Cl33, Cl12) or 1 (Cl156) independent experiments, using T cells from 6 (Cl12), 4 (Cl114, Cl33) or 2 (Cl156) different donors, tested against 6 (Cl114), 5 (Cl33), 4 (Cl156) or 3 (Cl12) different LCLs. E Specific killing of HLA-matched pulsed or unpulsed LCLs after co-culture with TCR-transduced T cells. Boxplots represent 3 (Cl156, Cl33, Cl12) or 2 (Cl114) independent experiments, using T cells from 6 (Cl156, Cl33), 5 (Cl12) or 3 (Cl114) different donors, tested against 5 (Cl114, Cl156, Cl33) or 2 (Cl12) different LCLs. C–E Paired two-tailed t-test. Intracellular cytokine staining (6 h) and surface marker assessment (18 h) of CD4 + ( F ) and CD8 + ( G ) T cells after stimulation with PMA/Ionomycin, cognate peptide-pulsed or unpulsed LCLs or medium only as a negative control. Mean frequency of cells positive for the indicated marker (y-axis) in each condition (x-axis) is plotted from 3 (CD4 + ) or 1 (CD8 + ) independent experiments with 5 (CD4 + ) or 2 (CD8 + ) different T cell donors. Numerical means are shown for LCL-stimulated conditions. Asterisks indicate significance from paired two-sided t-tests comparing pulsed versus unpulsed LCLs. A–E * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 from left to right (2D) 0.00015; (2E) p = 0.00236, p = 0.00019.

Article Snippet: IFNɣ or TNF was measured in the supernatant according to the manufacturer’s instructions (Human IFNɣ (HRP), Mabtech, 3420-1H; Human TNF (HRP), Mabtech, 3512-1H) and the signal was detected on an Infinite 200 PRO (Tecan).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Two Tailed Test, Staining, Marker, Negative Control

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of EBV&KSHV co-infected LCL (EK LCL) generation. B Flow cytometry histograms of GFP expression in different LCLs. C Western blot of whole-cell lysates from iSLK.219 and Brk.219 (± lytic induction) and from EBV-only or puromycin-selected EK LCLs (4 donors) stained for LANA and Tubulin. Tubulin-normalized LANA quantification indicated below blots. D IFNɣ production by TCR-transduced T cells co-cultured with HLA-matched LCLs ( ± KSHV infection), normalized to untransduced controls. Boxplots representing five (Cl33), four (Cl114, Cl12) or three (Cl156) independent experiments using T cells from eight (Cl114, Cl33, Cl12) or six (Cl156) donors, tested against six (Cl114, Cl33), four (Cl156) or three (Cl12) LCLs. Paired two-tailed t-test. E Frequencies of proliferating (CTV-diluted) or mouse CD19 + CD8 + T cells after seven-day co-culture of Cl12-transduced CD8 + T cells with irradiated HLA-matched or mismatched LCLs (± KSHV infection, ± peptide-pulsed), normalized to untransduced T cells. Boxplots representing two independent experiments with four T-cell and two target-cell donors. Linear mixed model fit by REML, T cell and LCL donor as a random effect, Holm-adjusted Tukey test. F IFNɣ secretion by T cells co-cultured with peptide-pulsed, KSHV-infected, or untreated MC116 B cells, or medium only. IFNɣ levels are shown relative to PMA/Ionomycin controls. Median ± SD of 2 independent experiments using T cells from three (Cl12) or four (rest) donors. Two-way ANOVA, Tukey’s multiple comparisons test, multiplicity adjusted p -values. G IFNɣ ELISA of TCR-transduced T cells co-cultured with autologous B cells infected with EBV or EBV&KSHV on days 1, 5, 14 post-infection. Mean of one experiment with two donor pairs. H IFNɣ ELISA of untransduced T cells incubated 72 h with conditioned media from freshly infected B cells. Mean ± SD of one experiment with three donor pairs. One-way ANOVA on log 10 -transformed data, Tukey’s multiple comparisons test. ( A–H ) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 (left to right, top to bottom): (3D) p = 0.03852; (3E) p = 0.00034; (3 F) Cl156: p = 0.0078, p = 0.0013, p = 0.0107; Cl33: p = 0.0224; Cl114: p = 0.0402, p = 0.0472; Cl12: p = 0.0016; (3H) p = 0.0037.

Article Snippet: IFNɣ or TNF was measured in the supernatant according to the manufacturer’s instructions (Human IFNɣ (HRP), Mabtech, 3420-1H; Human TNF (HRP), Mabtech, 3512-1H) and the signal was detected on an Infinite 200 PRO (Tecan).

Techniques: Infection, Flow Cytometry, Expressing, Western Blot, Staining, Cell Culture, Two Tailed Test, Co-Culture Assay, Irradiation, Enzyme-linked Immunosorbent Assay, Incubation, Transformation Assay

Journal: Nature Communications

Article Title: Chemically modified tRNA enhances the translation capacity of mRNA rich in cognate codons

doi: 10.1038/s41467-025-62981-7

Figure Lengend Snippet: a Left, schematic diagram showing that LNP formulation consisted of tRNA, Spike-mRNA, ionizable lipid, PEG2000-DMG, DSPC and cholesterol. Right, abbreviation of LNPs and tRNAs. T1 (Lst-UUA) was used as the benchmark vaccine. Placebo (blank LNP) was used as a negative control. Figure was generated using BioRender. b , c Dynamic changes of RNA levels and antigen concentrations of LNP formulations transfected in HEK293T cells ( n = 3 biological replicates). d Schematic diagram of the mRNA-tRNA vaccination, antibody detection, and splenic lymphocytes detection in BALB/c mice. Figure was generated using BioRender. e Measurement of the IgG antibody endpoint GMTs elicited by the mRNA-tRNA vaccines using ELISA ( n = 8 mice in each group). f Quantification of the NT50 of mRNA-tRNA immunized mouse sera by Spike B.1.1.529 pseudovirus neutralizing assay. The sera were collected 2 weeks after the booster injection (T1 ~ T7, n = 8 mice in each group; placebo, n = 3 mice). g ELISpot assay measurement of the SARS-CoV-2 Spike-specific IFN-γ, TNF-α, IL-2, and IL-4 responses of splenocytes from immunized mice. Mouse spleens were isolated and dissociated 5 weeks post boost-injection (T1 ~ T7, n = 8 mice in each group; placebo, n = 3 mice). h FACS analysis results showing the percentages and cell counts of CD4 + Tem cells producing IL-2, and IL-4 after stimulation with Spike-peptide pool ( n = 8 mice in each group). i FACS analysis results showing the percentages and cell counts of CD8 + Tem cells producing IFN-γ and TNF-α after stimulation ( n = 8 mice in each group). Statistical significance was analysed using one-way ANOVA with Dunnett’s correlation ( e – i ) or two-way ANOVA with Dunnett’s correlation ( c ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. Data are presented as mean ± s.d. Source data are provided as a Source Data file.

Article Snippet: ELISpot assay of splenocytes was performed using the mouse IFN-γ, TNF-α, IL-2, and IL-4 ELISpot PLUS Kit (MabTech) according to the manufacturer’s instructions.

Techniques: Formulation, Negative Control, Generated, Transfection, Vaccines, Enzyme-linked Immunosorbent Assay, Neutralizing Assay, Injection, Enzyme-linked Immunospot, Isolation

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: IFN-γ responses of PBMCs from FMDV-infected pigs to synthetic peptide pools. PBMCs were isolated from pigs infected with FMDV O/BY/2010, stimulated in vitro with peptide pools composed of 75 conserved peptide segments of FMDV O/BY/2010, and stained for intracellular cytokines to detect the IFN-γ reactivity of CD4 + and CD8 + T cells. ( A ) Schematic diagram showing the number of pigs infected with serotype O FMDV and sample collection. ( B and C ) Kinetics of IFN-γ responses in PBMCs from five infected pigs stimulated with FMDV peptide pools. Measurements were performed using PBMCs obtained at 0, 7, 14, and 28 dpc. Note: due to the absence of epitope-specific IFN-γ response detected at day 0 of infection, data at this time point are not displayed. ( B ) Representative dot plots; ( C ) percentage of peptide-specific IFN-γ-producing CD4 + and CD8 + T cells. Data are presented as the mean ± standard deviation ( n = 5). The data were assessed by two-way ANOVA, ns: not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Infection, Isolation, In Vitro, Staining, Standard Deviation

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Screening of T-cell epitopes in FMDV NSP 2B and 2C by IFN-γ ELISpot. PBMCs were isolated from pigs #67, #55, and #68 at 14 or 28 days post-infection. Peptide pools or individual peptides were used to stimulate PBMCs for 36 h, followed by the ELISpot assay to detect IFN-γ secretion. ( A and C ) PBMCs from pigs #67, #55, and #68 at 14 and 28 days post-infection were stimulated with 19 peptide pools. A mixture of the 19 peptide pools and inactivated FMDV (iFMDV) served as positive controls; Dimethyl sulfoxide and DMEM served as negative controls. ( B and D ) Twenty-four individual peptides identified by cross-screening as potentially reactive were used to stimulate PBMCs from pigs #67, #55, and #68 at 14 or 28 days post-infection. A mixture of the 24 individual peptides and iFMDV served as positive controls; Dimethyl sulfoxide and DMEM served as negative controls, #55 pig in the 22 days post-infection was dead. ( E and F ) Statistics of individual peptides showing IFN-γ reactivity and recognition at 14 or 28 days post-infection. ( G ) Representative ELISpot images of reactive individual peptides, negative controls, and positive controls. Repeat three times for each sample ( n = 3), and the data are presented as the mean ± standard deviation. The data were assessed by one-way ANOVA, ns: not significant, * P < 0.05, significantly different from the negative control.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Enzyme-linked Immunospot, Isolation, Infection, Standard Deviation, Negative Control

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Detection of peptide-induced T-cell phenotypes by ICS and CFSE staining. PBMCs were isolated from FMDV-infected or uninfected pigs at 14 days post-infection. PBMCs were stimulated with individual peptides that showed IFN-γ reactivity as identified by ELISpot, followed by ICS and CFSE staining. ( A and B ) Representative dot plots of peptide-induced IFN-γ secretion in CD4 + and CD8 + T cells. ( C and D ) Representative dot plots of peptide-induced proliferation of CD4 + and CD8 + T cells. ( E and F ) Percentages of IFN-γ⁺ CD4⁺ and CD8⁺ T cells following in vitro peptide stimulation. ( G and H ) Percentages of proliferating CD4⁺ and CD8⁺ T cells in response to peptide stimulation, determined by CFSE. ICS and CFSE results from the FMDV O challenge group are presented as the mean ± SD ( n = 3). Statistical significance was assessed by one-way ANOVA, ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, significantly different from the Irrelevant control.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Staining, Isolation, Infection, Enzyme-linked Immunospot, In Vitro, Control

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Analysis of SLA restriction and conservation of T-cell epitopes. ( A ) Inhibitory effect of porcine SLA I/II Abs on the proliferative response to peptides 2B and 2C. PBMCs were isolated from FMDV-infected pigs at 14 days post-infection, treated with SLA I/II Abs (15 μg/well), and the responses were quantified by IFN-γ ELISpot. An irrelevant peptide was used to evaluate the specificity of peptide-induced responses. An isotype control Ab was used to examine the specificity of restriction by anti-SLA Abs, compared to the isotype Ab in the presence of anti-SLA Abs. ( B ) PBMCs from FMDV-infected pigs #67 and #68 were magnetically sorted with anti-CD8β monoclonal Ab, and flow cytometry was used to determine the purity of CD8β. ( C ) Monocyte-derived dendritic cell culture; black arrows indicate cells with dendrites. ( D ) CD8β + T cells and antigen-presenting cells were added to IFN-γ ELISpot plates at a ratio of 10:1, and T-cell epitopes of 2C and 2B were added for IFN-γ detection. ( E ) Reactivity of different peptides in inducing CD4⁺ T cells and CD4⁻CD8⁺ T cells. ( F ) Conservation analysis of 2B and 2C peptides; the logo plot was generated by aligning sequences of 2B and 2C from seven FMDV serotypes. Letters represent amino acid codes, and heights are proportional to frequencies. Repeat three times for each sample ( n = 3), and the data are presented as the mean ± standard deviation. The data were assessed by one-way ANOVA, ns: not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, significantly different from the isotype or irrelevant peptide control.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Isolation, Infection, Enzyme-linked Immunospot, Control, Flow Cytometry, Derivative Assay, Cell Culture, Generated, Standard Deviation

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Construction and characterization of CD40L-fused multi-epitope trimeric proteins. ( A ) Trimer-40L-TB expresses a trimeric fusion protein containing the extracellular domain of CD40L and TB epitopes. 5B-TB encodes a fusion protein lacking CD40L. 5B-B encodes a fusion protein lacking CD40L and T epitopes. Monomer TB encodes a monomeric vaccine, and 5B serves as a trimerization motif without antigenic epitopes. Numbers indicate the expected molecular weights of proteins in each construct. ( B ) SDS-PAGE and western blotting analysis of protein expression, identified using FMDV-positive serum. ( C ) ELISA detection of the reactivity of five proteins. Equal concentrations of the five proteins were coated, serially diluted, and detected using biotinylated O8 single-domain Ab. Repeat three times for each sample ( n = 3), and data are presented as the mean ± standard deviation. ( D ) SPR analysis of the binding affinity of trimeric and monomeric proteins. O8 single-domain Ab was immobilized on an M5 sensor chip, and four proteins were applied as analytes. Note: B-cell epitopes: VP1: 137–156 (O/BY; O/Panasia; A/GDMM; A/HW); sCD40L: residues 113–261; T-cell epitopes: 2C [157–174], 2C [151–168], 2C [109–126], and 2B [31–48]; AP: RRRWCKPPP; 5B: artificially designed protein self-assembling trimerization motif.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Construct, SDS Page, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Binding Assay

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Animal experiment design and activation analysis of T-cell epitopes. Four groups of pigs were immunized with Trimer-CD40L-TB ( n = 8), Trimer-TB ( n = 8), Trimer-B ( n = 8), and Trimer ( n = 4), followed by a booster immunization on 14 dpv and a challenge protection experiment on 35 dpv. PBMCs were isolated from each group at 35 dpv and stimulated with or without 2B and 2C peptides for 36 h, and IFN-γ-producing T cells were identified with the ELISpot assay. ( A ) Schematic diagram of the animal experiment design and sample collection. ( B ) Comparison of the total number of IFN-γ-secreting cells per million PBMCs in each group. ( C ) Representative IFN-γ spots in each group. Data are presented as the mean ± standard deviation ( n = 4). The data were assessed by two-way ANOVA, ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, significantly different from the Trimer-TB group.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Activation Assay, Isolation, Enzyme-linked Immunospot, Comparison, Standard Deviation

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: FMDV-specific IFN-γ-producing T cells produced by vaccinated pigs and the development of memory T cells. PBMCs were isolated from each group at 14 and 35 dpv, restimulated with or without 2B and 2C peptides for 10 h, and IFN-γ-producing T cells were detected by intracellular cytokine staining. ( A ) Representative dot plots of IFN-γ-producing CD4 + and CD8 + T cells in each group after restimulation with or without peptide pool (2C 157–174, 2B 31–48, 2C 109–126, and 2C 151–168). ( B ) Comparison of the percentages of FMDV-specific IFN-γ-producing CD4 + and CD8 + T cells in each group ( n = 5). ICS assay is presented as the mean ± standard deviation ( n = 5). ( C ) Effector and memory CD4 + and CD8 + T cells in PBMCs of each group were identified by multicolor flow cytometry, and the percent changes among 0, 14, and 35 dpv were compared. The left panel shows representative dot plots of CD4 + T CM (CD4 + CD27 + CD8α + ) and CD8 + T CM (CD45RA⁻CD27 + ) detected in each group. The right panel shows bar graphs depicting the percentage differences between CD4 + T CM and CD8 + T CM . T CM assay is presented as the mean ± standard deviation ( n = 4 or 5). All data were assessed by two-way ANOVA, ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Produced, Isolation, Staining, Comparison, Standard Deviation, Flow Cytometry

Journal: mBio

Article Title: T cell responses to nonstructural proteins promote cross-serotype immunity to foot-and-mouth disease virus

doi: 10.1128/mbio.03586-25

Figure Lengend Snippet: Analysis of Trimer-CD40L-TB vaccine-induced FMDV GH-Loop-specific GC B-cell counts, IgG Abs, neutralizing Abs, and accompanying broad-spectrum protection against FMDV serotypes O and A. Two pigs from each of the Trimer-CD40L-TB, Trimer-TB, and control groups were euthanized at 35 dpv, and lymphoid tissues were collected. ( A ) Isolating single cells was assessed by flow cytometry. Representative dot plots of GH-Loop-specific GC B cells in the Trimer-CD40L-TB, Trimer-TB, and control groups ( n = 2). ( B ) Bar graph analyzing the differences in the number of GH-Loop-specific GC B cells among the three groups ( n = 2/group). ( C ) Fluorescence staining analysis of GC B cells in tissue sections, with BCL-6 (red) and IgG (purple) used to localize B cells in GC. ( D ) Analysis of differences in the number of IgG + BCL-6 + fluorescent cells among the three groups ( n = 2/group). ( E ) PBMCs were isolated from pigs at 35 dpv. After coating with anti-porcine IgG Ab, PBMCs were added and incubated for 24 h, then biotinylated Trimer-B protein was used to detect the number of IgG antibodies secreted by B cells specific to B epitopes ( n = 4/group). ( F ) Serum samples were collected from each group of pigs, and FMDV NAb titers were detected by a neutralization test. FMDV NAb titers among the four groups were compared at 7, 14, 21, 28, and 35 dpv ( n = 4/group). All data assay is presented as the mean ± standard deviation ( n = 2 or 4). All data were assessed by one or two-way ANOVA, ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After washing with PBST (PBS containing 0.05% Tween 20), plates were incubated for 2 h at room temperature with biotinylated anti-porcine IFN-γ mAb (P2C11, Mabtech, Sweden) at 0.5 μg/mL.

Techniques: Control, Flow Cytometry, Fluorescence, Staining, Isolation, Incubation, Neutralization, Standard Deviation