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: BMC Genomics

Article Title: Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

doi: 10.1186/1471-2164-9-171

Figure Lengend Snippet: PARP-1 dependent genes in T cells a

Article Snippet: The cells were activated with plate-bound hamster anti-mouse CD3 (clone 145-2C11) (5 μg/ml) in the absence or the presence of hamster anti-mouse CD28 (clone 37.51) (5 μg/ml) both from BD PharMingen (San Diego, CA) and cultured in RPMI 1640 medium (BioWhittaker) supplemented with 10% FCS, 2 mM L-glutamine, 5 × 10 -5 M 2-mercaptoethanol, 2.5 μg/ml fungizone, 100 IU/ml penicillin, and 10 μg/ml streptomycin, all from Sigma (St. Louis, MO).

Techniques:

Journal: BMC Genomics

Article Title: Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

doi: 10.1186/1471-2164-9-171

Figure Lengend Snippet: Gene expression profile analysis of Parp-1 +/+ and Parp-1 -/- activated T cells . (A) Venn diagram showing the numbers of PARP-1-dependent genes in T cells upon stimulation with anti-CD3 mAb alone or in combination with anti-CD28 mAb. (B) Expression maps including the genes dependent on PARP-1 in response to both anti-CD3 and anti-CD3 + anti-CD28 stimulation. Red indicates higher expression in Parp-1 +/+ T cells compared to Parp-1 -/- T cells (positive regulation by PARP-1) while green indicates higher expression in Parp-1 -/- T cells compared to Parp-1 +/+ T cells (negative regulation by PARP-1). Numbers on the left column indicate the fold-change expression in Parp-1 +/+ T cells compared to Parp-1 -/- T cells. (C) Real-time PCR analysis of representative genes. Samples were normalized according to Gapdh expression levels. The y-axis represents fold-change of activated versus resting cells for both Parp-1 +/+ (white bars) and Parp-1 -/- (black bars) T cells. Results represent the mean value ± SD of two independent experiments. The ratio of gene expression in Parp-1 +/+ over Parp-1 -/- cells (positive number) or the ratio of gene expression in Parp-1 -/- over Parp-1 +/+ cells (negative number) is indicated above each pair of bars.

Article Snippet: The cells were activated with plate-bound hamster anti-mouse CD3 (clone 145-2C11) (5 μg/ml) in the absence or the presence of hamster anti-mouse CD28 (clone 37.51) (5 μg/ml) both from BD PharMingen (San Diego, CA) and cultured in RPMI 1640 medium (BioWhittaker) supplemented with 10% FCS, 2 mM L-glutamine, 5 × 10 -5 M 2-mercaptoethanol, 2.5 μg/ml fungizone, 100 IU/ml penicillin, and 10 μg/ml streptomycin, all from Sigma (St. Louis, MO).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

doi: 10.1186/1471-2164-9-171

Figure Lengend Snippet: Gene Ontology term enrichment analysis of PARP-1 dependent genes upon T cell activation . More genes than expected by chance were involved in certain processes or functions in response to anti-CD3 stimulation alone (black bar) or in response to anti-CD3 + anti-CD28 stimulation (grey bar). The length of the bar indicates the ratio between numbers of observed PARP-1 dependent genes in each processes or functions and the number expected by chance.

Article Snippet: The cells were activated with plate-bound hamster anti-mouse CD3 (clone 145-2C11) (5 μg/ml) in the absence or the presence of hamster anti-mouse CD28 (clone 37.51) (5 μg/ml) both from BD PharMingen (San Diego, CA) and cultured in RPMI 1640 medium (BioWhittaker) supplemented with 10% FCS, 2 mM L-glutamine, 5 × 10 -5 M 2-mercaptoethanol, 2.5 μg/ml fungizone, 100 IU/ml penicillin, and 10 μg/ml streptomycin, all from Sigma (St. Louis, MO).

Techniques: Activation Assay

Journal: BMC Genomics

Article Title: Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

doi: 10.1186/1471-2164-9-171

Figure Lengend Snippet: PARP-1 dependent genes in T cells involved in the immune response . (A) Expression maps including all the genes belonging to GO terms "immune response". Red indicates higher expression in Parp-1 +/+ T cells compared to Parp-1 -/- T cells (positive regulation by PARP-1) while green indicates higher expression in Parp-1 -/- T cells compared to Parp-1 +/+ T cells (negative regulation by PARP-1). Numbers in the left column indicate the fold-change expression in Parp-1 +/+ T cells compared to Parp-1 -/- T cells. (B) Real-time PCR analysis of representative genes in response to anti-CD3 stimulation or (C) in response to anti-CD3 + anti-CD28 stimulation. Samples were normalized according to Gapdh expression levels. The y-axis represents fold-change of activated versus resting cells for both Parp-1 +/+ (white bars) and Parp-1 -/- (black bars) T cells. Results represent the mean value ± SD of two independent experiments. The ratio of gene expression in Parp-1 +/+ over Parp-1 -/- cells (positive number) or the ratio of gene expression in Parp-1 -/- over Parp-1 +/+ (negative number) cells is indicated above each pair of bars.

Article Snippet: The cells were activated with plate-bound hamster anti-mouse CD3 (clone 145-2C11) (5 μg/ml) in the absence or the presence of hamster anti-mouse CD28 (clone 37.51) (5 μg/ml) both from BD PharMingen (San Diego, CA) and cultured in RPMI 1640 medium (BioWhittaker) supplemented with 10% FCS, 2 mM L-glutamine, 5 × 10 -5 M 2-mercaptoethanol, 2.5 μg/ml fungizone, 100 IU/ml penicillin, and 10 μg/ml streptomycin, all from Sigma (St. Louis, MO).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Transcriptional regulation by Poly(ADP-ribose) polymerase-1 during T cell activation

doi: 10.1186/1471-2164-9-171

Figure Lengend Snippet: T cell proliferation and apoptosis in Parp-1 +/+ and Parp-1 -/- T cells . (A) Splenic T cells from Parp-1 +/+ (white bars) and Parp-1 -/- (black bars) were stimulated with plate-bound anti-CD3 mAb alone or in combination with anti-CD28 mAb for 24 h, 48 h and 72 h. Proliferation was measured by 3 H-thymidine uptake during the last 12 h of culture. Data are representative of 3 separate experiments carried on in triplicate. Results represent the mean value ± SD. *P < 0.05 in t-test. (B) Apoptosis of T cells from Parp-1 +/+ (white bars) and Parp-1 -/- (black bars) was detected by FITC-Annexin-V and Propidium iodide staining and flow cytometric analysis at 24 hours after stimulation. Results represent the mean value ± SD of two independent experiments.

Article Snippet: The cells were activated with plate-bound hamster anti-mouse CD3 (clone 145-2C11) (5 μg/ml) in the absence or the presence of hamster anti-mouse CD28 (clone 37.51) (5 μg/ml) both from BD PharMingen (San Diego, CA) and cultured in RPMI 1640 medium (BioWhittaker) supplemented with 10% FCS, 2 mM L-glutamine, 5 × 10 -5 M 2-mercaptoethanol, 2.5 μg/ml fungizone, 100 IU/ml penicillin, and 10 μg/ml streptomycin, all from Sigma (St. Louis, MO).

Techniques: Staining

Journal: eLife

Article Title: The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells

doi: 10.7554/elife.70555

Figure Lengend Snippet: Figure 4. Reduced in vivo but not in vitro suppressive capacity of PHD2-deficient Treg. (a) Treg function was assayed following adoptive co-transfer of CD45.2 Foxp3-expressing cells with naive, CFSE-labeled congenic CD45.1 CD4+ lymphocytes (Treg: Tconv ratio 1:3) into syngeneic lymphopenic male mice (Rag2-/-). Recipient mice were euthanized at day 6 post-transfer, and their spleen and mesenteric lymph node (mLN) cells analyzed by flow cytometry. (b) Representative flow cytometry expression profiles of CFSE-labeled cells (CD45.1 gate in the spleen) with or without co-transferred Foxp3+ cells from Foxp3cre or PHD2ΔTreg male mice. (c) Percentage of suppression established from CFSE staining profiles. (d) Frequency of activated (CD4+ CD45.1+ CD44hi CD62Llo) cells in the indicated lymphoid organs. (e) Frequency of Treg cells in the indicated organs 6 days post-transfer. (f, g) CFSE- labeled, naive conventional CD4+ T cells from CD54.1 mice were co-cultured with ex vivo-purified Treg cells from Foxp3cre or PHD2ΔTreg mice at the indicated ratios in the presence of anti-CD3 antibodies (0.5 μg/ml) and splenic feeder cells. (f) Representative flow cytometry profiles of CSFE staining. (g) Percent of suppression of proliferation as compared to cultures in which Treg cells were omitted. Data are representative of three independent experiments with n = 15 (b–e) or n = 4 (f, g) per group. Values are presented as the mean ± SD and were compared by two-way ANOVA with Tukey’s multiple comparisons test (c–e) or two-tailed unpaired Student’s t-test (g). Only significant differences are indicated as follows: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70555 19 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- mouse CD4- A700 (RM4- 5, mouse monoclonal) BD Biosciences 557956; RRID:AB_396956 (3:500) Antibody Anti- mouse CD8- A700 (53- 6.7, mouse monoclonal) BD Biosciences 557959; RRID:AB_396959 (3:500) Antibody Anti- mouse CD4- PB (RM4- 5, mouse monoclonal) BD Biosciences 558107; RRID:AB_397030 (1:100) Antibody Anti- mouse CD62L- A700 (MEL14, mouse monoclonal) BD Biosciences 560517; RRID:AB_1645210 (1:100) Antibody Anti- mouse GATA3- PE (L50823, mouse monoclonal) BD Biosciences 560074; RRID:AB_1645330 (1:10) Antibody Anti- mouse RORγt- PECF594 (Q31- 378, mouse monoclonal) BD Biosciences 562684; RRID:AB_2651150 (1:200) Antibody Anti- mouse STAT1 (pY701)A488(4a, mouse monoclonal) BD Biosciences 612596; RRID:AB_399879 (1:10) Antibody Anti- mouse IFNγ-PE (XMG1.2, mouse monoclonal) BD Biosciences 554412; RRID:AB_395376 (1:100) Antibody Anti- mouse IL- 10- APC (JES516E3, mouse monoclonal) BD Biosciences 554468; RRID:AB_398558 (1:100) Antibody Anti- mouse IL- 17- PerCP- Cy5.5 (N49- 653, mouse monoclonal) BD Biosciences 560799; RRID:AB_2033981 (1:100) Antibody Anti- CD3 antibody (2c11, mouse monoclonal) BioXCell 145- 2c11 20 μg/mouse peptide, recombinant protein Streptavidin- PECy7.

Techniques: In Vivo, In Vitro, Expressing, Labeling, Flow Cytometry, Staining, Cell Culture, Ex Vivo, Purification, Two Tailed Test

Journal: bioRxiv

Article Title: CD8 + T cell response promotes viral clearance and reduces chances of severe testicular damage in mouse models of long-term Zika virus infection of the testes

doi: 10.1101/2024.01.22.575592

Figure Lengend Snippet: A. Histology of A129 mice testes. B. Percentage of CD3 events in blood samples of A129 mice excluding red blood cells at 2 or 18 dpi. C. Percentage of seminiferous tubules (ST) with spermatogenesis in each A129 mouse testicular sample. D-F show histology, CD3 levels, and percentage of ST for the C57BL/6J mice. Statistical significance was assessed with one-way ANOVA Sidak’s multiple comparison correction. Bars show mean and error bars represent standard deviation. Lines represent the mean. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., no significant difference.

Article Snippet: For experiment shown in , 0.1 mg/mouse of anti-mouse CD3ε F(ab’)2 fragments (clone 145-2C11, BioXCell) or control f(ab’)2 fragments of polyclonal hamster IgG (BioXCell) were injected intraperitoneally on days -3, -1, 2, 7, and 14 relative to infection at day 0.

Techniques: Comparison, Standard Deviation