Journal: The Journal of Experimental Medicine

Article Title: T Cell Receptor β Chain Lacking the Large Solvent-exposed Cβ FG Loop Supports Normal α/β T Cell Development and Function in Transgenic Mice

doi:

Figure Lengend Snippet: The mutant TCR β chain is functionally expressed on the cell surface. (A) Cells transfected with TCR α chain together with a control β (αβ) or mutant β chain (αβ-loop − ) were stained with biotinylated anti-CD3ε mAb followed by streptavidin-APC. Staining of cells transfected with mutant β chain only is shown as negative control. Numbers represent the mean of fluorescence intensity of CD3 staining. (B and C) Functional response of 10 5 TCR transfectants (αβ, •; αβ-loop − , ○) cultured with 10 5 irradiated antigen-presenting cells (B cell lymphomas A20) and the indicated concentrations of influenza hemagglutinin peptide HA 110-119 (B) or superantigen SEC 3 (C). After 20 h, the culture supernatant was collected and tested for the presence of lymphokines using the IL-2–dependent proliferation assay of HT2 cell lines.

Article Snippet: The reagents used were mAbs biotinylated 145-2C11 (anti-CD3ε), PE-labeled RM4-5 (anti-CD4) and FITC-labeled H57-597 (anti-Cβ) , B20.1 (anti-Vα2), RR3-16 (anti-Vα3.2), B21-14 (anti-Vα8), and RR8-1 (anti-Vα11.1, 2) (all seven mAbs purchased from PharMingen ), Cy5-labeled 53-6.7 (anti-CD8), fluorescein-succinimidyl-ester (FLUOS)- labeled F23.1 (anti-Vβ8.1, 2, 3) , and second-step reagent streptavidin-allophycocyanin (APC) (Molecular Probes, Inc.).

Techniques: Mutagenesis, Transfection, Control, Staining, Negative Control, Fluorescence, Functional Assay, Cell Culture, Irradiation, Proliferation Assay

Journal: Nature Communications

Article Title: T cell cholesterol efflux suppresses apoptosis and senescence and increases atherosclerosis in middle aged mice

doi: 10.1038/s41467-022-31135-4

Figure Lengend Snippet: Ldlr −/− and T-Abc dko Ldlr −/− mice ( a – e , i – j ) and control ( Ldlr +/+ ) and T-Abc dko mice ( f – h ) were fed a chow diet for 12–13 months, and wild-type mice for 3 months (young) or 24 months (aged) ( i – j ). a – j Spleens were collected and CD4 + and CD8 + T cells were isolated. a , b CD4 + and CD8 + T cells were stimulated with αCD3/IL-2 for 12 h with concomitant staining for cleaved caspase3/7. a Representative flow cytometry plots of cleaved caspase 3/7 in CD4 + ( p = 0.015) and CD8 + ( p = 0.007) T cells gated as in Supplementary Fig. and ( b ) quantification. n = 6 Ldlr −/− and n = 5 T-Abc dko Ldlr −/− mice. c – h T cells were labeled with CFSE and stimulated with αCD3/αCD28 beads. CFSE dilution was measured by flow cytometry at 72 h after stimulation. c , f Representative CFSE dilutions gated as in Supplementary Fig. . The number of divisions were quantified for ( d ) CD4 + (division 0, p = 0.000017; 2, p = 0.00002; 3, p < 0.000001; 4, p < 0.000001) and ( e ) CD8 + (division 0, p = 0.0029; 1, p = 0.00095; 3, p = 0.0023; 4, p = 0.0000097; 5, p = 0.008) T cells. n = 7 Ldlr −/− and n = 6 T-Abc dko Ldlr −/− mice. The number of divisions were quantified for ( g ) CD4 + (division 0), p = 0.0002; 1, p = 0.013; 2, p = 0.017; 3, p = 0.00067; 4, p = 0.0031 and ( h ) CD8 + T cells (division 0, p = 0.0004; 1, p = 0.0056; 3, p = 0.012; 4, p = 0.0003; 5, p = 0.02). n = 4 control and n = 4 T-Abc dko mice. i – j T cells were isolated and RNA was extracted. Cyclin dependent kinase inhibitor 1a ( Cdkn1a) mRNA expression was measured in ( i ) CD4 + ( Ldlr −/− or aged wild-type vs T-Abc dko Ldlr −/− , p < 0.0001) and ( j ) CD8 + ( Ldlr −/− vs T-Abc dko Ldlr −/− , p= 0.0003; aged wild-type vs T-Abc dko Ldlr −/− , p < 0.0001) T cells by qPCR and shown as fold change compared to young wild-type mice. n = 8 young wild-type, n = 6 aged wild-type, n = 6 Ldlr −/− , and n = 5 T-Abc dko Ldlr −/− mice. For all panels, error bars represent SEM. Biologically independent samples were included. p value was determined by unpaired two-tailed Student’s t test ( b , d – e , g , h ) or one-way ANOVA with Bonferroni post-test ( i , j ). * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Splenic CD4 + and CD8 + T cells from 4–5 months old male and female Ldlr −/− and T-Abc dko Ldlr −/− mice were isolated and labeled using Caspase3/7 Red (APC) Reagent (Sartorius).

Techniques: Isolation, Staining, Flow Cytometry, Labeling, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: T cell cholesterol efflux suppresses apoptosis and senescence and increases atherosclerosis in middle aged mice

doi: 10.1038/s41467-022-31135-4

Figure Lengend Snippet: Ldlr −/− and T-Abc dko Ldlr −/− mice were fed a chow diet for 12–13 months. Hearts were isolated and paraffin sections of the aortic root were stained for ( a ) H&E or ( b , c ) CD3. a Representative pictures of H&E staining (left) and quantification (right) of atherosclerotic lesion area ( p = 0.003). Scale bar represents 200 μm. n = 11 Ldlr −/− and n = 10 T-Abc dko Ldlr −/− mice. b Representative pictures of CD3 staining on atherosclerotic lesions (left) and quantification (right) of total CD3 + cells per section ( p = 0.008). T cells were identified as cells with brown plasma membrane CD3 staining and the hematoxylin staining still visible. CD3 + cells are depicted by arrows. Scale bar represents 80 μm. c CD3 + T cells as percentage of total cells per section ( p = 0.0107). b , c n = 11 Ldlr −/− and n = 10 T-Abc dko Ldlr −/− mice. d – i Aortas were collected. d Aortic CD3 + T cells were isolated, RNA was extracted, and Foxp3 , Il10 , Tgfb1 , tumor necrosis factor ( Tnf) , Bcl2 ( p = 0.023), and Cdkn1a ( p = 0.025) mRNA expression were measured by qPCR. n = 7 Ldlr −/− and n = 5 T-Abc dko Ldlr −/− mice. e Total aortic cells were labeled with CFSE, stimulated with αCD3/αCD28 beads, and stained for CD4 after 96 h of stimulation. CFSE dilution was measured by flow cytometry and the number of divisions were quantified for aortic CD4 + T cells. n = 8 Ldlr −/− and n = 8 T-Abc dko Ldlr −/− mice. f – i Total aortic cells were stained for CD4, CD44, CD62L, and cleaved caspase 3/7. f Representative flow cytometry plots of cleaved caspase 3/7 in CD4 + T cells gated as in Supplementary Fig. , and ( g ) quantification ( p = 0.015). n = 4 Ldlr −/− and n = 5 T-Abc dko Ldlr −/− mice. h Aortic CD4 + T mem/eff cells ( p = 0.0003) as percentage of aortic CD4 + T cells and ( i ) cleaved caspase3/7 in aortic CD4 + T mem/eff cells ( p = 0.033). n = 7 Ldlr −/− and n = 7 T-Abc dko Ldlr −/− mice. For all panels, error bars represent SEM. Biologically independent samples were included. p value was determined by unpaired two-tailed Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Splenic CD4 + and CD8 + T cells from 4–5 months old male and female Ldlr −/− and T-Abc dko Ldlr −/− mice were isolated and labeled using Caspase3/7 Red (APC) Reagent (Sartorius).

Techniques: Isolation, Staining, Membrane, Expressing, Labeling, Flow Cytometry, Two Tailed Test

Journal: Current protocols

Article Title: Engineering Ligand and Receptor Pairs with LIPSTIC to Track Cell-Cell Interactions.

doi: 10.1002/cpz1.311

Figure Lengend Snippet: Figure 4 Measuring interaction with LIPSTIC in immune cells ex vivo. B cells isolated from Cd40G5/G5 mice were co-cultured with CD4+ T cells isolated from Cd40lgSrtA/Y CD4-Cre OT-II an- imals in the presence or absence of the cognate antigen (Ag). After 6 hr, cells were treated with biotinylated SrtA substrate for 30 min and then analyzed by flow cytometry. (A) Identification of B and T cells in the co-culture based on expression of CD19 and CD4 markers. (B) LIPSTIC labeling identified by biotin cell surface staining in CD19+ cells gated as in A.

Article Snippet: Materials B and T cells carrying LIPSTIC-engineered molecules Complete RPMI (see recipe) 20 mM biotin-LPETG (see recipe) PBE buffer: PBS containing 0.5% (w/v) BSA and 2 mM EDTA FC block (e.g., BD Biosciences, cat. no. 553141) Antibodies and reagents for flow cytometry: Anti-biotin-APC (e.g., Miltenyi Biotec, cat. no. 130-113-288) Anti-CD4-BV421 (e.g., Biolegend, cat. no. 100437) Anti-CD19-PE-Cy7 (e.g., Biolegend, cat. no. 115520) 96-well U-bottom plates 37°C, 5% CO2 incubator for mammalian cells Alberti et al. 9 of 16 Current Protocols nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.311 by N at Prov Indonesia, W iley O nline L ibrary on [20/02/2024].

Techniques: Ex Vivo, Isolation, Cell Culture, Cytometry, Co-Culture Assay, Expressing, Labeling, Staining