Journal: Discovery Immunology

Article Title: Triphasic production of IFN γ by innate and adaptive lymphocytes following influenza A virus infection

doi: 10.1093/discim/kyad014

Figure Lengend Snippet: IAV-specific CD4 and CD8 T cells are activated and increase Ifn γ expression following challenge infection. GREATxSMART mice were either infected i.n. with IAV WSN (H1N1) for 40 days (memory) or re-infected with X31 (H3N2) for 3 days. Antigen-specific CD4 and CD8 T cells from the lung, spleen, and Med LN were examined. (A) Representative flow plots of IA b /NP 311-3250 -specific CD4+ T cells from the Med LN in memory (top) and recall infection (bottom) and expression of (B) Ifn γ , CD69, and ICOS from IA b /NP 311-325 -specific CD4+ T cells in the spleen, Med LN, and lung. (C) Representative flow plots of D b /NP 368-374 -specific CD8+ T cells from the lung in memory (top) and recall infection (bottom) and expression of (D) Ifn γ , CD69, and PD1 from D b /NP 368-374 -specific CD8+ T cells in the spleen and lung of memory and recall. Each point represents an individual mouse from 9 to 11 mice combined from two independent experiments; error bars are SEM. Significance tested via an unpaired t test, * P < 0.05, ** P < 0.01, *** P < 0.001. Some samples were removed from these groups for technical reasons: too few MHC tetramer + cells, thymus contamination in the Med LN, or loss of cells during analysis.

Article Snippet: Antibodies used were: anti-CD3 BV785 (BioLegend; clone: 17A2), anti-CD4 BUV805 (BD Bioscience; clone: RM4-5), anti-CD8 BV421 (ThermoFisher; clone: 53-6.7), anti-CD44 BUV395 (BD; clone: IM7), anti-CD45.2 BV605 (BioLegend; clone: 104), anti-CD69 PE-Cy7 (ThermoFisher; clone: H1.2F3), anti-CD127 APC (ThermoFisher; clone: A7R34), anti- γ δ TCR PE-Cy7 (BioLegend; clone: GL3), anti-ICOS BV785 (BioLegend; clone: C398.4A), anti-NK1.1 APC-Cy7 (BioLegend; clone: PK136), anti-PD1 BV711 (BioLegend; clone: 29F,1A12), and ‘dump’ antibodies: B220 (RA3-6B2), F4/80 (BM8), and MHC II (M5114) all on eFluor-450 (ThermoFisher) or PerCP-Cy5.5 (ThermoFisher; B220 and F4/80, and BioLegend; MHCII).

Techniques: Expressing, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TCR affinity biases Th cell differentiation by regulating CD25, Eef1e1, and Gbp2

doi: 10.4049/jimmunol.1801609

Figure Lengend Snippet: (A) Tfh differentiation by B3K506 (filled circle, n=6) and B3K508 (empty circle, d 1 d 2 n=6, d 3 n=8) cells in the initial three d following Lm-P5R infection. (B) Experimental model and resulting mean frequencies ± SD of CD69+ CD25+ B3K508 cells after one d of co-culture with SIRP⍺+ (square, n=3) or XCR1+ (circle, n=3) DCs from Lm-P5R infected mice two d post-infection. (C) Frequency of Tfh, Th1, or uncommitted cells among B3K506 or B3K508 TCR Tg cells in DT-treated WT (filled circle) or Clec4a4DTR (empty circle) mice three d after Lm-P5R (B3K506 WT n=8, Clec4a4DTR n=8; B3K508 WT n=6, Clec4a4DTR n=8) or Lm-P2A (B3K508 WT n=4, Clec4a4DTR n=6) infection. (D) Frequency of Tfh, Th1, or uncommitted cells among B3K506 or B3K508 cells in WT (filled circle) or Batf3−/− (empty circle) mice three d after Lm-P5R (B3K506 WT n=11, Batf3−/− n=7; B3K508 WT n=8, Batf3−/− n=7) or Lm-P2A (B3K508 WT n=5, Batf3−/− n=7) infection. The bars in A, C, and D represent the mean. Pooled data from two or three independent experiments are shown. Spleens were analyzed for the depicted experiments. Two-way ANOVA was used to determine significance for B and one-way ANOVA with Sidak’s multiple comparison test was used to determine significance for A, C and D. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: For identification of surface markers, the sample was stained on ice with various combinations of the following antibodies: APC-ef780-labeled B220 (RA3–6B2; ThermoFisher Scientific), APC-ef780-labeled CD11b (M1–70; ThermoFisher Scientific), APC-ef780-labeled CD11c (N418; ThermoFisher Scientific), BV786-labeled CD4 (GK1.5; BD biosciences), AF700-labeled CD44 (IM7; ThermoFisher Scientific), FITC-labeled CD45.1 (A20; Biolegend), BUV395-labeled CD45.2 (104; BD biosciences), BUV395-labeled CD25 (PC61; BD biosciences), PE-labeled CD69 (H1.2F3; ThermoFisher Scientific) and FITC-labeled CD90.1 (HIS51; ThermoFisher Scientific).

Techniques: Infection, Co-Culture Assay

Journal: Scientific Reports

Article Title: Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding

doi: 10.1038/s41598-019-40147-y

Figure Lengend Snippet: T cell activation marker expression is unaffected in cells processed via µVS . Flow cytometry was used to quantify the surface level expression of key T cell activation markers (CD69, CD154, CD44, CCR7, CD45RA and CD25) in T cells processed with µVS (processed cells, red dashed) compared to non-processed cells (handling control, blue solid) 24 hours post transfection. Histogram overlays represent expression levels as a percent of maximum value of each marker for a representative sample for each condition. Experiment was performed in triplicate.

Article Snippet: Samples were rinsed and re-suspended in flow buffer containing 25 μL mL −1 of each of the following monoclonal anti-human antibodies per sample: anti-human CD3-AF700 (56-0037-42, ThermoFisher), -CD40L/CD154-FITC (11-1548-42, ThermoFisher), -CD25-PE (120257-42, ThermoFisher), -CCR7-APC-eF780 (47-1979-42, ThermoFisher) -CD44-APC-eF780 (47-0441-80, ThermoFisher), -CD69-eF450 (48-0699-42, ThermoFisher), and -CD45RA-SB702 (67-0458-42, ThermoFisher).

Techniques: Activation Assay, Marker, Expressing, Flow Cytometry, Transfection