Journal: Nature Communications

Article Title: PI3Kγ signaling controls trafficking of CD8 + T cells between lymphoid and non-lymphoid organs and drives hypertension in a murine model

doi: 10.1038/s41467-025-61009-4

Figure Lengend Snippet: a Schematics of the experiment: CD8 + T lymphocytes were purified from total splenocytes from PI3Kγ +/+ , PI3Kγ −/− , and PI3Kγ KD/KD mice receiving angiotensin II or vehicle, and were plated on wells coated with anti-CD3 and anti-CD28 antibodies. PIP3 production was analyzed by immunocytochemistry, and Akt phosphorylation at Thr308 and Ser473 sites by western blot. b , c Immunocytochemistry representative images ( b ) of PIP3 (green) expression on CD8 + T cells (red); DAPI (blue) was used to identify nuclei. Images were acquired by confocal microscopy at 40X magnification (scale bar = 20 µm). c Quantitative analysis of the % of PIP3 + CD8 + T cells from the spleen of n = 3 PI3Kγ +/+ Veh, n = 3 PI3Kγ +/+ AngII, n = 3 PI3Kγ −/− Veh, n = 3 PI3Kγ −/− AngII, n = 3 PI3Kγ KD/KD Veh and n = 3 PI3Kγ KD/KD AngII mice. Representative western blot images ( d , f ) and quantitative analysis ( e, g ) of Akt phosphorylation of Thr308 ( d , e ) and Ser473 ( f , g ) in CD8 + T cells isolated from the spleen of n = 6 PI3Kγ +/+ Veh, n = 6 PI3Kγ +/+ AngII, n = 5 PI3Kγ −/− Veh, n = 5 PI3Kγ −/− AngII, n = 5 PI3Kγ KD/KD Veh and n = 5 PI3Kγ KD/KD AngII mice. Quantitative analysis ( e , g ) as fold changes of pThr308 Akt ( e ) and pSer473 ( g ), normalized over β-actin levels, are shown for angiotensin II treated mice as compared to vehicle controls. All data are expressed as mean ± SEM. Statistical analysis was performed by Two-way ANOVA and Tukey’s correction for multiple comparisons in c , e , g . N represents biologically independent samples. Source data are provided as a file. Schematic in a is Created in BioRender. Perrotta, M. (2025) https://BioRender.com/1qw2qst .

Article Snippet: In the ex-vivo co-culture experiments of MRA and conditioned culture medium of CD8 + T lymphocytes, the cells were isolated from total splenocytes as described above and cultured 5 * 10 5 cells/well at a density of about 7 * 10 5 /cm 2 for 72 h at 37 °C, 5% CO 2 in TexMACS medium (Miltenyi Biotec, 130-097-196) with 10% Dialyzed FBS (Gibco 26400-044), 1% Penicillin-Streptomycin (Gibco, 15140-122), 55 μM β-mercaptoethanol (Gibco, 31350-010) in 48 well pre-coated with CD3ε antibody (pure-functional grade, Miltenyi Biotec, 130-092-973) and then stimulated with CD28 antibody (pure-functional grade, Miltenyi Biotec, 130-093-182).

Techniques: Purification, Immunocytochemistry, Phospho-proteomics, Western Blot, Expressing, Confocal Microscopy, Isolation

Journal: Nature Communications

Article Title: PI3Kγ signaling controls trafficking of CD8 + T cells between lymphoid and non-lymphoid organs and drives hypertension in a murine model

doi: 10.1038/s41467-025-61009-4

Figure Lengend Snippet: a Immunohistochemistry of CD3 + (magenta) and B220 + (green) lymphocytes to analyze the white pulp area in the spleen of PI3Kγ +/+ , PI3Kγ −/− and PI3Kγ KD/KD mice after in vivo administration of angiotensin II or vehicle as control. Nuclei are visualized in blue by DAPI. Images were acquired by confocal microscopy at 10X magnification (scale bar = 100 µm). b Quantitative analysis of the white pulp area marked by CD3 + T cells. n = 6 PI3Kγ +/+ Veh, n = 6 PI3Kγ +/+ AngII, n = 5 PI3Kγ −/− Veh, n = 7 PI3Kγ −/− AngII; n = 7 PI3Kγ KD/KD Veh and n = 5 PI3Kγ KD/KD AngII mice. c Analysis of the myogenic tone of mesenteric resistance arteries (MRA) dissected from naïve WT mice ex vivo co-cultured with CD8 + T cells purified from the spleen of PI3Kγ +/+ and PI3Kγ −/− mice stimulated in vivo for 3 days with angiotensin II or vehicle. n = 4 CD8 Veh ; n = 5 CD8 AngII-PI3Kγ+/+ and n = 5 CD8 AngII-PI3Kγ−/− . Representative plots of flow cytometry ( d ) and quantitative analysis ( e – h ) of total CD8+ T cells ( e ), naïve ( f ), effector ( g ) and resident memory ( h ) CD8+ T cells in kidneys dissected from PI3Kγ +/+ , PI3Kγ −/− and PI3Kγ KD/KD mice after in vivo administration of angiotensin II or vehicle for 28 days. n = 5 PI3Kγ +/+ Veh, n = 7 PI3Kγ +/+ AngII, n = 6 PI3Kγ −/− Veh, n = 8 PI3Kγ −/− AngII, n = 6 PI3Kγ KD/KD Veh and n = 7 PI3Kγ KD/KD AngII. All data are expressed as mean ± SEM. Statistical analysis was performed by Two-way ANOVA, followed by Tukey’s correction for multiple comparisons in b , e , f , g , h ; One-way ANOVA for repeated measures, followed by Tukey’s correction for multiple comparisons in c. N represents biologically independent samples. Source data are provided as a file. Schematics in a , c , d were created in BioRender. Perrotta M (2025) https://BioRender.com/1f77gjq , https://BioRender.com/fbmd4r3 , https://BioRender.com/g0be7ts .

Article Snippet: In the ex-vivo co-culture experiments of MRA and conditioned culture medium of CD8 + T lymphocytes, the cells were isolated from total splenocytes as described above and cultured 5 * 10 5 cells/well at a density of about 7 * 10 5 /cm 2 for 72 h at 37 °C, 5% CO 2 in TexMACS medium (Miltenyi Biotec, 130-097-196) with 10% Dialyzed FBS (Gibco 26400-044), 1% Penicillin-Streptomycin (Gibco, 15140-122), 55 μM β-mercaptoethanol (Gibco, 31350-010) in 48 well pre-coated with CD3ε antibody (pure-functional grade, Miltenyi Biotec, 130-092-973) and then stimulated with CD28 antibody (pure-functional grade, Miltenyi Biotec, 130-093-182).

Techniques: Immunohistochemistry, In Vivo, Control, Confocal Microscopy, Ex Vivo, Cell Culture, Purification, Flow Cytometry

Journal: Nature Communications

Article Title: PI3Kγ signaling controls trafficking of CD8 + T cells between lymphoid and non-lymphoid organs and drives hypertension in a murine model

doi: 10.1038/s41467-025-61009-4

Figure Lengend Snippet: a , b Immunocytochemistry representation ( b ) of PIP3 + (green) expression in CD8 + (red) T cells isolated from the spleen of PI3Kγ +/+ and PI3Kγ CX/CX mice and plated onto anti-CD3 and anti-CD28 coated wells. Nuclei are stained by DAPI and visualized in blue. b Quantitative analysis of the % of PIP3 + CD8 + T cells was performed in n = 3 PI3Kγ +/+ and n = 4 PI3Kγ CX/CX mice. Images were acquired by confocal microscopy at 40X magnification (scale bar = 20 µm). c – f Western blot analysis of Akt phosphorylation in Thr308 (c) and Ser473 ( e ) in CD8 + T cells isolated from the spleens of PI3Kγ +/+ and PI3Kγ CX/CX mice. Representative blot ( c , e ) and quantitative analysis of phosphorylated Akt at Thr308 ( d ) and Ser473 ( f ) as fold changes over β-actin levels, in n = 6 PI3Kγ +/+ and PI3Kγ CX/CX mice in d , and n = 8 PI3Kγ +/+ and PI3Kγ CX/CX mice in ( f ). Metabolic-flux analysis by the Seahorse XF Analyzer was used to measure the mitochondrial respiration and the aerobic glycolysis by the oxygen-consumption rate (OCR) ( g – j ) and extracellular acidification rate (ECAR) ( k – m ) under basal conditions and after drug-induced mitochondrial inhibition in CD8 + T cells purified from the spleen of PI3Kγ CX/CX and PI3Kγ +/+ mice. For g – m , n = 9 PI3Kγ +/+ and n = 8 PI3Kγ CX/CX animals were analyzed and measured in triplicates. All data are expressed as mean ± SEM. Statistical analysis was performed by unpaired Mann–Whitney test in b ; two-sided unpaired T -test in d , f , h , i , j , l , m . N represents biologically independent samples. Source data are provided as a file.

Article Snippet: In the ex-vivo co-culture experiments of MRA and conditioned culture medium of CD8 + T lymphocytes, the cells were isolated from total splenocytes as described above and cultured 5 * 10 5 cells/well at a density of about 7 * 10 5 /cm 2 for 72 h at 37 °C, 5% CO 2 in TexMACS medium (Miltenyi Biotec, 130-097-196) with 10% Dialyzed FBS (Gibco 26400-044), 1% Penicillin-Streptomycin (Gibco, 15140-122), 55 μM β-mercaptoethanol (Gibco, 31350-010) in 48 well pre-coated with CD3ε antibody (pure-functional grade, Miltenyi Biotec, 130-092-973) and then stimulated with CD28 antibody (pure-functional grade, Miltenyi Biotec, 130-093-182).

Techniques: Immunocytochemistry, Expressing, Isolation, Staining, Confocal Microscopy, Western Blot, Phospho-proteomics, Inhibition, Purification, MANN-WHITNEY

Journal: iScience

Article Title: Targeted deletion of Interleukin-3 results in asthma exacerbations

doi: 10.1016/j.isci.2022.104440

Figure Lengend Snippet:

Article Snippet: V450 Hamster anti-Mouse CD3e , BD Bioscience, Heidelberg, Germany , Cat# 560801, RRID: AB_2034005.

Techniques: Purification, Recombinant, Lysis, Enzyme-linked Immunosorbent Assay, Cell Isolation, Isolation, Software

Journal: Cell reports

Article Title: Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection

doi: 10.1016/j.celrep.2022.110560

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD3e (152 Sm) , Fluidigm , Cat# 3152004B; RRID: AB_2687836.

Techniques: Recombinant, Western Blot, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Cell Culture, IP Phosphatase Assay, Software