Journal: bioRxiv

Article Title: Limited protection against early-life cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1101/2024.07.10.602923

Figure Lengend Snippet: Effector T cells primed in adults protect from early-life MCMV lung infection (A) Experimental setup for (B-J): CD3 + CD44 + effector T cells were purified from adult lungs 7 dpi and 8x10 6 cells per animal were transferred into MCMV infected neonatal mice which were then analysed 8 dpi, controls did not receive T cells. (B and C) (B) Representative flow cytometry plots and (C) quantitative analysis of CD45 + cells obtained from neonatal lungs at 8 dpi. (D and E) (D) Representative flow cytometry histograms and (E) quantitative analysis of CD44 expression in isolated lung T cells. (F) Immunohistology of lungs in general view (left panels) and high resolution (right panels) illustrating inflammation and presence of NIFs with MCMV-infected cells. Dotted squares indicate the zoomed areas shown in the right panels. (G-H) Quantitative analysis of immunohistology with (G) number of NIFs per lung slice, (H) NIF area and (I) number of MCMV-infected cells per NIFs. (J) Quantitative analysis of lung viral loads as indicated. Data acquired from four independent experiments. Statistical differences were calculated in C, E and G-J with Mann-Whitney U test and the p values are provided above the graphs.

Article Snippet: CD44 + T cells were isolated from lungs of MCMV-infected Wt adult mice 7 dpi by positive selection of CD45 + cells with CD45 Microbeads (Miltenyi Biotec) followed by FACS cell sorting for CD3 + CD44 + T cells.

Techniques: Infection, Purification, Flow Cytometry, Expressing, Isolation, MANN-WHITNEY

Journal: Nature immunology

Article Title: Protein phosphatase 2A is requisite for the function of regulatory T cells

doi: 10.1038/ni.3390

Figure Lengend Snippet: ( a, b ) Naïve CD4 + T cells (a) or T conv and T reg cells (b) were stimulated with CD3 plus CD28 antibodies for the indicated time periods. Immunoblotting for p-PP2A C (Y307) was then performed. Data are from one experiment representative of two independent experiments with similar results (n=3 mice per experiment). ( c, f ) Splenocytes isolated from Foxp3 IRES-GFP mice were stimulated with CD3 plus CD28 antibodies for 24 hours or left unstimulated. Intracellular staining was then performed for PP2A C (c) or SET (f). Data are from one experiment representative of three independent experiments with similar results (n=3 mice per experiment). ( d ) Naïve CD4 + T cells were stimulated with CD3 plus CD28 for 0, 0.5, 1, 3, 6, 18 and 24 hours. Intracellular staining was then performed for SET and p-PP2A C (Y307). A representative histogram (left) and quantification of the results (right) is shown from one of two independent experiments with similar results (n=3 per group). ( e ) Intracellular staining for p-PP2A C (Y307) of naïve CD4 + T cells activated with CD3 plus CD28 antibodies for 24 hours and then spin-infected with an empty or a Set -expressing mCherry lentiviral vector. The analysis was done on mCherry + T cells (n=5 per group, one of two experiments with similar results is shown). Mean ± s.e.m. is shown, MFI: Mean fluorescent intensity, * P <0.01 (unpaired, two-tailed t-test).

Article Snippet: CD45.1 + CD4 + CD62L + CD25 – CD44 – T cells (1 × 10 5 , T eff ) were stained with CFSE (Life Technologies) and cultured for 4 days with PP2A wt or PP2A flox T reg cells in serially decreasing ratios (1:1, 1:2, 1:4, 1:8, only T eff cells) in the presence of 1 × 10 5 irradiated splenocytes and plate-bound anti-CD3 (2 μg/ml, clone 145-2C11; BioXcell).

Techniques: Western Blot, Isolation, Staining, Infection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature immunology

Article Title: Protein phosphatase 2A is requisite for the function of regulatory T cells

doi: 10.1038/ni.3390

Figure Lengend Snippet: ( a, b ) Naïve CD4 + T cells were stimulated with anti-CD3 plus anti-CD28 for 24h and then treated with sphingomyelinase (SMase, 0.5 units/mL) or vehicle (50% glycerol in PBS) for 1h. The cells were then stained for ceramide (a) or p-PP2A C (Y307) (b). A representative histogram (left) and quantification of the results (right) is shown (n=3, one of two experiments with similar results is shown). ( c ) T conv and T reg cells were stimulated for 24h and then subjected to ESI-MS/MS for the quantification of ceramide species. ( d ) Ceramide content of CD3 + CD4 + Foxp3 − and CD3 + CD4 + Foxp3 + was quantified using flow cytometry (n=3 mice). Data are from one of three experiments with similar results. ( e ) T conv and T reg cells were subjected to chromatin immunoprecipitation using a Foxp3-specific antibody or an IgG control antibody. Shown is binding enrichment at the mouse Sgms1 gene, normalized to the input (n=3 per group, one representative of two independent experiments with similar results is shown). ( f ) Naïve CD4 + T cells were stimulated for 24h and then spin-infected with an mCherry-expressing lentivirus that harbored or not the murine Sgms1 coding sequence. CD4 + mCherry + cells were analyzed for ceramide content using flow cytometry. A representative histogram (left) and quantification of the results (right) is shown. Data are from one experiment representative of two independent experiments with similar results. Mean ± s.e.m. is shown, MFI: Mean fluorescent intensity, * P <0.05, ** P <0.01, *** P <0.001 (unpaired, two-tailed t-test).

Article Snippet: CD45.1 + CD4 + CD62L + CD25 – CD44 – T cells (1 × 10 5 , T eff ) were stained with CFSE (Life Technologies) and cultured for 4 days with PP2A wt or PP2A flox T reg cells in serially decreasing ratios (1:1, 1:2, 1:4, 1:8, only T eff cells) in the presence of 1 × 10 5 irradiated splenocytes and plate-bound anti-CD3 (2 μg/ml, clone 145-2C11; BioXcell).

Techniques: Staining, Tandem Mass Spectroscopy, Flow Cytometry, Chromatin Immunoprecipitation, Binding Assay, Infection, Expressing, Sequencing, Two Tailed Test

Journal: Nature immunology

Article Title: Protein phosphatase 2A is requisite for the function of regulatory T cells

doi: 10.1038/ni.3390

Figure Lengend Snippet: (a ) Jurkat T cells were treated with SMase (0.5 units/mL) or vehicle (50% glycerol in PBS) for 1 hour and then stained for p-AKT T308 , p-AKT S473 and p-S6. Data are from one experiment representative of two independent experiments with similar results. ( b ) Naïve CD4 + T cells were stimulated with CD3 plus CD28 antibodies (2 μg/mL) for 24h and then incubated with okadaic acid or DMSO for 3 hours. During the last hour the cells were treated with SMase or vehicle and then stained for p-AKT T308 , p-AKT S473 and p-S6 (n=3 per treatment group). Data are from one experiment representative of two independent experiments with similar results. ( c ) Foxp3 + T reg cells were isolated from PP2A wt and PP2A flox mice and stained for p-S6 (n=3 mice per group). A representative histogram (left) and quantification of the results (right) is shown. Data are from one experiment representative of three independent experiments with similar results. Mean ± s.e.m., MFI: Mean fluorescent intensity, ns P >0.05, * P <0.05, ** P <0.01, *** P <0.001 (unpaired, two-tailed t-test).

Article Snippet: CD45.1 + CD4 + CD62L + CD25 – CD44 – T cells (1 × 10 5 , T eff ) were stained with CFSE (Life Technologies) and cultured for 4 days with PP2A wt or PP2A flox T reg cells in serially decreasing ratios (1:1, 1:2, 1:4, 1:8, only T eff cells) in the presence of 1 × 10 5 irradiated splenocytes and plate-bound anti-CD3 (2 μg/ml, clone 145-2C11; BioXcell).

Techniques: Staining, Incubation, Isolation, Two Tailed Test

Journal: Nature immunology

Article Title: Protein phosphatase 2A is requisite for the function of regulatory T cells

doi: 10.1038/ni.3390

Figure Lengend Snippet: ( a, b ) FACS-sorted PP2A wt and PP2A flox T reg cells were stimulated with CD3 plus CD28 and then expanded for 4 days in the presence of 100 IU/mL of IL-2. During the final 24 hours, they were treated with 100nM of Rapamycin or DMSO. (a) The T reg cells (gated on FoxP3-YFP + CD4 + T cells) were re-stimulated with PMA/Ionomycin for 6 hours and stained for IL-17 and IL-2. (b) The ECAR and OCR of PP2A wt and PP2A flox T reg cells (sorted again as Foxp3-YFP + CD4 + T cells at the end of the culture) were measured (n=3 mice per group, unpaired, two-tailed t-test). Data are from one experiment representative of two independent experiments with similar results. ( c ) Left : H&E staining of the lungs and the salivary glands of PP2A wt and PP2A flox mice treated or not with rapamycin (scale bar represents 100 μm). Images are from one experiment representative of two independent experiments with similar results. Right : combined clinical score of inflammation in the liver, skin, stomach, salivary glands, lungs and pancreas of PP2A wt and PP2A flox mice treated or not with rapamycin (one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test). ( d, e ) Ex vivo amounts of p-S6 (d) and CD98 expression (e) in T regs (FoxP3-YFP + ) from mice in (c). (f) In vitro suppression assay using effector CD45.1 + CD4 + (T eff ) T cells and T reg cells from PP2A wt or PP2A flox rapamycin-treated and vehicle-treated mice. Representative histograms showing CFSE dilution and percentage of divided cells (ratio T reg /T eff : 1/1). Data are from one experiment representative of two independent experiments with similar results. Mean ± s.e.m., * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: CD45.1 + CD4 + CD62L + CD25 – CD44 – T cells (1 × 10 5 , T eff ) were stained with CFSE (Life Technologies) and cultured for 4 days with PP2A wt or PP2A flox T reg cells in serially decreasing ratios (1:1, 1:2, 1:4, 1:8, only T eff cells) in the presence of 1 × 10 5 irradiated splenocytes and plate-bound anti-CD3 (2 μg/ml, clone 145-2C11; BioXcell).

Techniques: Staining, Two Tailed Test, Ex Vivo, Expressing, In Vitro, Suppression Assay