pe cy7 ifnγ  (Cytek Biosciences)

Journal: NPJ Vaccines

Article Title: A replication-deficient gammaherpesvirus vaccine protects mice from lytic disease and reduces latency establishment

doi: 10.1038/s41541-024-00908-x

Figure Lengend Snippet: C57BL/6 mice were either sham-vaccinated or vaccinated twice (prime+boost) with 1 × 10 6 PFU RDV-50.stop followed by mock challenge or IN challenge with 1 × 10 3 PFU WT MHV68 at d15 post-boost and analyzed d7 post-challenge. a Flow cytometric gating strategy to determine the frequency of CD44 hi CD8 T cells in the lungs that were reactive with viral p79 or p56 epitopes. b Total p79- or p56-tetramer+ CD8 T cells per lung of individual mice with the indicated vaccination and challenge regimen. c Left column, flow cytometric gating strategy to determine the frequency of CD44 hi CD8 T cells in the spleens that were reactive with viral p79. Middle column, p79-tetramer+ CD8 T cells were further analyzed for markers of short-lived effector cell (SLEC, KLRG + CD127 − ) and memory precursor effector cell subsets (MPEC, KLRG − CD127 + ). Right column, MPECs were further delineated into CD62L − effector and CD62L + central MPECs. d Total p79-tetramer+ CD8 T cells per spleen of individual mice with the indicated vaccination and challenge regimen. The percentage of p79-reactive CD8 T cells that were SLECs e , MPECs f , and effector vs memory MPECs g were enumerated based on the gating strategy for surface markers in c . Intracellular cytokine levels of effector cytokines TNFα and IFNγ were examined 6 h after stimulation with h p79 and i p56 peptides. j Percentage of CD44 hi CD8 T cells producing both TNFα and IFNγ. For each graph, symbols represent individual mice, ( N = 4–5); bars and whiskers are mean +/− SD. *, p < 0.05; **, p < 0.05; ***, p < 0.001; ****, p < 0.0001 in Sidak’s multiple comparisons test of one-way ANOVA between the indicated groups.

Article Snippet: Upon fixation and permeabilization with the BD Cytofix/Cytoperm kit (BD Biosciences), cells were stained with antibodies to IFNγ (dilution 1:100; clone XMG1.2, PE-Cy7, cat# 505826) and TNFα (dilution 1:100; clone MP6-XT22, BV650, cat# 506333, BioLegend).

Techniques:

Journal: NPJ Vaccines

Article Title: A replication-deficient gammaherpesvirus vaccine protects mice from lytic disease and reduces latency establishment

doi: 10.1038/s41541-024-00908-x

Figure Lengend Snippet: C57BL/6 mice were either sham-vaccinated or vaccinated twice (prime+boost) with 1×10 6 PFU RDV-50.stop followed by mock challenge or IN challenge with 1×10 3 PFU WT MHV68 at d15 post-boost. a Flow cytometric gating strategy to determine the frequency of CD44 hi CD8 T cells in the lungs of individual mice with the indicated vaccination and challenge regimen that were reactive with viral p79 at d16 post-challenge. b , c Total p79- or p56-tetramer+ CD44 hi CD8 T cells per lung of individual mice after initial prime and sequential boost. d Total p79-tetramer+ CD44 hi CD8 T cells per spleen of individual mice after initial prime and sequential boosts. Percentage of p79-tetramer+ CD44 hi CD8 T cells with markers of e short-lived effector cell (SLEC, KLRG + CD127 − ) and f memory precursor effector cell subsets (MPEC, KLRG − CD127 + ). g MPECs were further delineated into CD62L − effector and CD62L + central MPECs for p79-tetramer+ CD8 T cells. Intracellular cytokine levels of effector cytokines TNFα and IFNγ were examined 6 h after stimulation with h p79 and i p56 peptides. j Percentage of CD44 hi CD8 T cells producing both TNFα and IFNγ in response to viral peptide stimulation. For each graph, symbols represent individual mice, ( N = 3–5); bars and whiskers are mean +/− SD. *, p < 0.05; **, p < 0.05; ***, p < 0.001; ****, p < 0.0001 in Sidak’s multiple comparisons test of one-way ANOVA between the indicated groups.

Article Snippet: Upon fixation and permeabilization with the BD Cytofix/Cytoperm kit (BD Biosciences), cells were stained with antibodies to IFNγ (dilution 1:100; clone XMG1.2, PE-Cy7, cat# 505826) and TNFα (dilution 1:100; clone MP6-XT22, BV650, cat# 506333, BioLegend).

Techniques:

Journal: NPJ Vaccines

Article Title: A replication-deficient gammaherpesvirus vaccine protects mice from lytic disease and reduces latency establishment

doi: 10.1038/s41541-024-00908-x

Figure Lengend Snippet: C57BL/6 mice were either sham-vaccinated or vaccinated (prime+boost) with 1 × 10 6 PFU RDV-50.stop or heat-inactivated (HI) WT MHV68. a Virus-specific IgG from RDV-50.stop or HI vaccinated mice at d31 post-boost measured by ELISA. b Virus neutralization in serum at d31 post-boost determined by a plaque reduction assay. The plaque reduction neutralization titer 50 (PRNT 50 ) value is the dilution of serum to reach 50% neutralization of plaques. For a-b , symbols denote individual mice ( N = 5); bars and whiskers represent mean +/− SD. ***, p < 0.001 in unpaired two-tailed t -test. c-f Following prime+boost vaccination, mice were challenged IN with 1 × 10 3 PFU WT MHV68 at d14 post-boost. c Acute replication at d7 post-challenge determined by measuring infectious particles per ml lung homogenate. Dotted line indicates limit of detection at 50 PFU/ml. d Total p56-tetramer+ CD44 hi CD8 T cells per spleen of individual mice at d7 post-challenge. e Percentag e of CD44 hi CD8 T cells producing both TNFα and IFNγ in response to viral p56 and p79 peptide dual stimulation at d7 post-challenge. For c – e , symbols denote individual mice ( N = 5–10); bars and whiskers represent mean +/− SD. ***, p < 0.001; **** , p < 0.0001; in Dunnett’s c or Sidak’s d , e multiple comparisons test of one-way ANOVA between the indicated groups. f Splenomegaly determined by spleen weights at d16, d25-31 and d42 post-challenge, respectively. Symbols denote individual mice ( N = 3–); bars and whiskers represent mean +/− SD. *, p < 0.05; **, p < 0.001; ***, p < 0.005; in Sidak’s multiple comparisons test of one-way ANOVA between the sham, RDV-50.stop and HI MHV68 vaccinated groups. g The frequency of genome-positive splenocytes determined by limiting dilution nested PCR of intact splenocytes at d16, d25-31 and d42 post-challenge. h The frequency of explant reactivation determined by limiting dilution coculture of intact viable splenocytes on a monolayer of primary MEFs d16, d25-31 and d42 days post-challenge. Disrupted splenocytes plated in parallel did not reveal preformed infectious virus in the vaccinated animals.

Article Snippet: Upon fixation and permeabilization with the BD Cytofix/Cytoperm kit (BD Biosciences), cells were stained with antibodies to IFNγ (dilution 1:100; clone XMG1.2, PE-Cy7, cat# 505826) and TNFα (dilution 1:100; clone MP6-XT22, BV650, cat# 506333, BioLegend).

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Neutralization, Two Tailed Test, Nested PCR

Journal: NPJ Vaccines

Article Title: A replication-deficient gammaherpesvirus vaccine protects mice from lytic disease and reduces latency establishment

doi: 10.1038/s41541-024-00908-x

Figure Lengend Snippet: a C57BL/6 mice were either sham-vaccinated or vaccinated twice (prime+boost) with 1 × 10 6 PFU RDV-50.stop followed by IN challenge with 1 × 10 3 PFU WT MHV68 at 90 days post-boost. b Acute replication at d7 post-challenge determined by measuring infectious particles per ml lung homogenate. Symbols denote individual mice ( N = 5); bars and whiskers represent mean +/− SD. ****, p < 0.0001 in one-tailed unpaired t test of log-transformed values. Dotted line indicates limit of detection at 50 PFU/ml. c Virus-spe c ific IgG from sham or RDV-50.stop vaccinated mice at d7 post-challenge measured by ELISA. Symbols denote individual mice ( N = 4); bars and whiskers represent mean +/− SD. d Virus neutralization in serum as determined by a plaque reduction assay. The plaque reduction neutralization titer 50 (PRNT 50 ) value is the dilution of serum to reach 50% neutralization of plaques. Symbols denote individual mice ( N = 4–5); bars and whiskers represent mean +/− SD. * , p < 0.05 in two-tailed unpaired t -test. e , f Total p56-tetramer+ CD44 hi CD8 T cells per lung e or spleen f of individual mice. g MPECs were delineated into CD62L − effector and CD62L + central MPECs for p56 − tetramer+ CD44 hi CD8 T cells. h Percentage of CD44 hi CD8 T cells producing both TNFα and IFNγ in response to viral p56 peptide stimulation. For d – h , symbols represent individual mice, ( N = 3-5); bars and whiskers are mean +/− SD. *, p < 0.05; **, p < 0.05; ***, p < 0.001; ****, p < 0.0001 in Dunn’s e or Sidak’s f,h multiple comparisons test of one-way ANOVA betwe e n the indicated groups. i Splenomegaly visualized for whole spleens and quantitated by spleen weights. Symbols represent individual mice, ( N = 5); bars and whiskers represent mean +/− SD. ****, p < 0.0001 in Tukey’s test of one-way ANOVA. j The frequency of latency determined by limiting dilution nested PCR of intact splenocytes for the viral genome. k The frequency of explant reactivation determined by limiting dilution coculture of intact viable splenocytes on a monolayer of primary MEFs. Disrupted splenocytes plated in parallel did not detect preformed infectious virus in the vaccinated animals. Error bars represent SEM. Symbols denote the average of one experiment with five mice per experiment.

Article Snippet: Upon fixation and permeabilization with the BD Cytofix/Cytoperm kit (BD Biosciences), cells were stained with antibodies to IFNγ (dilution 1:100; clone XMG1.2, PE-Cy7, cat# 505826) and TNFα (dilution 1:100; clone MP6-XT22, BV650, cat# 506333, BioLegend).

Techniques: One-tailed Test, Transformation Assay, Virus, Enzyme-linked Immunosorbent Assay, Neutralization, Two Tailed Test, Nested PCR

Journal: Nature Immunology

Article Title: Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis

doi: 10.1038/s41590-024-01814-z

Figure Lengend Snippet: a) Gating strategy for flow cytometry analysis of colon LPLs. b–d) Percentages from live cells of b) total lymphocytes and c-d) total CD4 + T cells (Live CD90.2 + TCR-β + CD4 + CD8-)) producing IFN-γ, GM-CSF, IL-17A and IL-10. Flow plots of CD4 + T-cells for IFN-γ production versus e) IL-10, f) GM-CSF and g) IL-17A. h) Gating of total CD4 + GM-CSF+ lymphocytes with corresponding flow plots of IFN-γ versus IL-17A. i) Gating of total GM-CSF+ lymphocytes with corresponding flow plots of IFN-γ versus IL-17A with bar plots of j) percentages (from live population) of lymphocytes producing GM-CSF alongside IL-17A, IFN-γ, or both. All flow analyses were performed on from H. hepaticus infected mice. k) Gene expression of Il12rb2, Il12rb1, Il23r, Stat4, Il18r1, Cxcr3 and Csf2 in bulk tissue total colon LPL isolated from uninfected and H. hepaticus infected mice. Differentially expressed genes in each condition against uninfected Prdm1 fl/fl Maf fl/fl mice were marked as follows: *=BH adjusted p value ≤ 0.05, **= BH adjusted p value ≤ 0.01, ***= BH adjusted p value ≤ 0.001, ****= BH adjusted p value ≤ 0.0001.

Article Snippet: LPLs were then fixed for 15 min at room temperature with 2% (v/v) formaldehyde (Sigma-Aldrich) and permeabilized for 30 min at 4 °C, using permeabilization buffer (eBioscience) and stained with the following cytokine antibodies for 30 min at 4 °C: IL-17A (eBio17B7, FITC, Invitrogen), IFNγ (XMG1.2, PE-Cy7, BD), IL-10 (JES5-16E3, APC, Invitrogen) and GM-CSF (MP1-22E9, BV421, BD).

Techniques: Flow Cytometry, Infection, Expressing, Isolation

Journal: Nature Immunology

Article Title: Blimp-1 and c-Maf regulate immune gene networks to protect against distinct pathways of pathobiont-induced colitis

doi: 10.1038/s41590-024-01814-z

Figure Lengend Snippet: Cell-to-cell communication networks inferred using CellChat software from gene expression of ligands and their receptors in immune cell clusters of interest from the colonic LPL scRNA-seq dataset. a) Number of interactions of cell-to-cell interactions, represented in the edge width, in and H. hepaticus infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1 , Maf , or both Prdm1 and Maf . b) Plot of “Outgoing interaction strength“ against “Incoming interaction strength” across all H. hepaticus infected conditions. c-d) Cell-to-cell communication networks underlying the c) Type II IFN (IFN-γ) and d) M-CSF pathways across all H. hepaticus infected Prdm1 fl/fl Maf fl/fl mice and mice with Cd4 Cre -mediated deletion of either Prdm1 , Maf , or both Prdm1 and Maf . The chord plot has receiver cells at the top (incoming signaling) and transmitter cells (outgoing signaling) the bottom. The edges are colored based on the cell clusters expressing the outgoing signals. In a,c-d) node size is proportional to the number of cells in each experimental group, and the edges are colored based on the cell clusters expressing the outgoing signals.

Article Snippet: LPLs were then fixed for 15 min at room temperature with 2% (v/v) formaldehyde (Sigma-Aldrich) and permeabilized for 30 min at 4 °C, using permeabilization buffer (eBioscience) and stained with the following cytokine antibodies for 30 min at 4 °C: IL-17A (eBio17B7, FITC, Invitrogen), IFNγ (XMG1.2, PE-Cy7, BD), IL-10 (JES5-16E3, APC, Invitrogen) and GM-CSF (MP1-22E9, BV421, BD).

Techniques: Software, Expressing, Infection