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: Journal for Immunotherapy of Cancer

Article Title: Expression of VISTA regulated via IFN-γ governs endogenous T-cell function and exhibits correlation with the efficacy of CD19 CAR-T cell treated B-malignant mice

doi: 10.1136/jitc-2023-008364

Figure Lengend Snippet: CAR-T cell-induced tumor lysis was not sufficient to reduce VISTA expression on syngeneic activated T cell. (A) Gating strategy of flow cytometry. (B) Percentages of B-ALL cell death in vitro. B-ALL cell alone or co-cultured with purified T-cell stimulated with anti-CD3 and anti-CD28 (T activated ) in vitro were used as the control and T-cell groups, respectively. (C) The production of cytokines was evaluated in the co-culture cellular system for 6 hours. Naïve T cells were included as a control group without antibody activation (T unactivated ). (D) The representative flow cytometry histograms of VISTA levels. The T cells lacking CAR chimerism in the CAR-T groups were designated as GFP − T cells. Three independent experiments were repeated. B-ALL, B acute lymphoblastic leukemia; CAR, chimeric antigen receptor; FMO, fluorescence minus one; GFP, green fluorescent protein; VISTA, V-domain Ig suppressor of T-cell activation.

Article Snippet: Prior to flow cytometry analysis, staining was performed using the following monoclonal antibodies: CD3 FITC (fluorescein isothiocyanate) (clone17A2; BioLegend), CD3 BV650 (clone17A2; BioLegend), CD3ε PE/Cy5 (clone145-2C11; Elabscience), CD4 PE/Cyanine7 (cloneGK1.5; BioLegend), CD4 Alex Flour700 (cloneRM4-5; BioLegend); CD8a APC (allophycocyanin) (clone53-6.7; BioLegend), CD8 APC/Cyanine7 (clone53-6.7; BioLegend); CD11b PB (Pacific Blue) (cloneM1/70; BioLegend), F4/80 BV605 (cloneBM8; BioLegend), MHC-II AF700 (cloneM5/114.15.2; BioLegend), CD19 PE-cf594 (clone6D5; BioLegend), CD19 BV510 (clone6D5; BioLegend), CD11c PE-cf594 (cloneN418; BioLegend), IFN-γ PE/Cyanine7 (cloneXMG1.2; BioLegend), TNF-α APC (cloneMP6-XT22; BioLegend); Granzyme B eFlour450 (cloneM5/114.15.2; Thermo Fisher Scientific); CD62L PB (cloneMEL-1; BD Biosciences), CD44 APC/eFlour780 (cloneIM7; BD Biosciences); PD-1 PE/Cyanine7 (clone RMP1-30; BioLegend), TIGIT (Vstm3) PE (clone1G9; BioLegend); NGFR (CD271) APC (cloneME204; BioLegend), VISTA (PD-1H) PE (cloneMIH63; BioLegend), CD69 BV510 (cloneH1.2F3; BioLegend), and CD20 APC/Cyanine7 (clone SA275A11; BioLegend).

Techniques: Lysis, Expressing, Flow Cytometry, In Vitro, Cell Culture, Purification, Control, Co-Culture Assay, Activation Assay, Fluorescence