cd4 cd25 t effector cells teff  (Miltenyi Biotec)

Journal: Scientific Reports

Article Title: Preclinical and clinical characterization of the RORγt inhibitor JNJ-61803534

doi: 10.1038/s41598-021-90497-9

Figure Lengend Snippet: Effects of JNJ-61803534 on human immune cells. (a) Effect of JNJ-61803534 on IL-17A, IL-17F and IL-22 production in human CD4 + T cells under Th17 differentiation, and on IFNγ production in human CD4 + T cells under Th1 conditions, respectively. Data are averages of duplicates and presented as the percentage of vehicle control group. (b) Effect of JNJ-61803534 on FOXP3 gene expression after 6 days under Treg differentiation conditions. Data are presented as fold change in gene expression over vehicle control group (mean ± SD, n = 3). (c) Effect of JNJ-61803534 on Treg suppression of IFNγ production from effector T cells. Data are presented as mean ± SD, n = 3. (d) Dose-dependent inhibition of IL-17A production in 1:1 diluted human whole blood by JNJ-61803534. Data are average of duplicates.

Article Snippet: Frozen purified human CD4 + CD25 + natural Treg cells (nTreg), monocyte-derived dendritic cells (DC) and CD4 + CD25 - T effector cells (Teff) (Allcells, LLC, Alameda, CA) were thawed and co-cultured in the presence or absence of JNJ-61803534.

Techniques: Control, Gene Expression, Inhibition

Journal: American Journal of Translational Research

Article Title: Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells

doi:

Figure Lengend Snippet: The impact of Jak2 depletion on T cell development. Jak2 deficiency was induced by tamoxifen injection. Four days after last induction, the mice were sacrificed and used for following experiments. A. PCR analysis of tail genomic DNA to check the presence of floxed null allele. B. Western blot analysis to confirm Jak2 depletion in splenic cell lysates. C. Comparison of total splenic cell numbers between Jak2-/- and control mice. Relative cell numbers were normalized by body weight. D. Flow cytometry analysis of splenic cell populations by plots on forward scatter and side scatter. Comparison was carried out for the lymphoid cluster (lower left corner) between Jak2-/- and WT controls. E. Flow cytometry analysis for the percentage of CD3+CD4+ T cells in total splenocytes. F. Flow cytometry analysis of lymphoid cells in peripheral blood. The number of lymphoid cells (lower left corner) was compared between Jak2-/- and WT controls. G. Comparison for the proportion of CD3+CD4+ T cells in peripheral blood mononuclear cells (PBMCs) between Jak2-/- and WT controls. All data were expressed as means ± SD, and four mice were included in each study group.

Article Snippet: CD4 + CD25 + Tregs and CD4 + CD25 - effector T cells (Teff) were isolated using a mouse CD4 + CD25 + regulatory T cell isolation kit (Miltenyi, San Diego, CA, USA) as instructed.

Techniques: Injection, Western Blot, Comparison, Control, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells

doi:

Figure Lengend Snippet: The impact of Jak2 deficiency on CD4+ T cell development. Loss of Jak2 did not affect the percentage of CD4+ T cells in total CD3+ splenic cells (A), total CD3+ lymph node cells (B), and total CD3+ PBMCs (C). However, the percentage for CD4+CD44highCD62Llow effector/memory T cells (TEM cells) in total CD4+ splenocytes (gated on CD4+ cells) was significantly reduced (D), and similarly, a significant reduction for the percentage of IFN-γ+ Th1 cells was noted (E). On the contrary, a significant increase for the proportion of Foxp3+ Treg cells in CD4+ splenocytes was characterized (F). All flow cytometry data were expressed as mean ± SD, and four mice were analyzed for each group.

Article Snippet: CD4 + CD25 + Tregs and CD4 + CD25 - effector T cells (Teff) were isolated using a mouse CD4 + CD25 + regulatory T cell isolation kit (Miltenyi, San Diego, CA, USA) as instructed.

Techniques: Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells

doi:

Figure Lengend Snippet: The effect of Jak2 deficiency on Th1 and Treg development. CD4+CD62LhighCD44low naïve CD4+ T cells were purified from Jak2-/- and control mice by magnetic beads as described (cell purity > 85%). A. Loss of Jak2 impaired Th1 development. Naïve CD4+ T cells were cultured under Th1 condition in the presence (lower) or absence (upper) of IFN-γ (50 ng/ml) for five days. The production of IFN-γ secreting Th1 cells were estimated by intracellular staining followed by flow cytometry analysis. B. Loss of Jak2 enhanced Treg production. Naïve CD4+ T cells were induced under Treg condition in the presence (lower) or absence (upper) of anti-CD28 (1 ug/ml) for five days. The production of Foxp3+ Tregs was estimated by flow cytometry as above. Three mice were analyzed in each study group, and the studies were conducted with three replications.

Article Snippet: CD4 + CD25 + Tregs and CD4 + CD25 - effector T cells (Teff) were isolated using a mouse CD4 + CD25 + regulatory T cell isolation kit (Miltenyi, San Diego, CA, USA) as instructed.

Techniques: Purification, Control, Magnetic Beads, Cell Culture, Staining, Flow Cytometry

Journal: American Journal of Translational Research

Article Title: Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells

doi:

Figure Lengend Snippet: Analysis of the intrinsic proliferative capability of CD4+ T cells after Jak2 depletion. CD4+ T cells were prepared from Jak2-/- and control mice and labeled with CFSE to serve as responder cells, while T cell depleted control splenocytes were treated with mitomycin C to serve as accessory cells. A. Results for proliferation of responder cells stimulated with PMA (10 ng/ml) and Ionomycin (250 ng/ml). B. Proliferation results for responder cells stimulated by anti-CD3 (0.5 ug/ml) and anti-CD28 (0.5 ug/ml). C. Flow cytometry analysis of allogenic BMDCs prepared from BALB/c mice. D. Proliferation results for responder cells stimulated by BALB/c-derived allogenic BMDCs. Mitomycin C treated BMDCs were co-cultured with CFSE labeled splenic cells originated from Jak2-/- or control mice for 72 h. Cell proliferation was estimated as above based on the halving of CFSE fluorescence intensity. Similarly, four mice were included for each group and the studies were carried out with three replications.

Article Snippet: CD4 + CD25 + Tregs and CD4 + CD25 - effector T cells (Teff) were isolated using a mouse CD4 + CD25 + regulatory T cell isolation kit (Miltenyi, San Diego, CA, USA) as instructed.

Techniques: Control, Labeling, Flow Cytometry, Derivative Assay, Cell Culture, Fluorescence

Journal: American Journal of Translational Research

Article Title: Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells

doi:

Figure Lengend Snippet: Loss of Jak2 selectively repressed signals essential for Th1 development. A. IFN-γ and IL-12 were potent to stimulate Jak2 phosphorylation (p-Jak2) in CD4+ T cells, while p-Jak2 was undetectable in IL-2 stimulated CD4+ T cells. B. Loss of Jak2 in CD4+ T cells impaired IL-12 induced STAT4 activation (p-STAT4). C. Jak2-/- CD4+ T cells manifested impaired IFN-γ/STAT1 signaling. D. Jak2 deficiency did not impact IL-2/STAT5 signaling in CD4+ T cells. E. Western blot results for analysis of transcription factors relevant to Th1 development. Naïve CD4+ T cells originated from Jak2-/- and control mice were polarized under Th1 condition for five days, followed by analysis of T-bet, Hlx, Runx3 and IL-12Rβ2 expression levels by Western blotting. GAPDH was served as loading controls, and data shown here were a representative of three independent experiments.

Article Snippet: CD4 + CD25 + Tregs and CD4 + CD25 - effector T cells (Teff) were isolated using a mouse CD4 + CD25 + regulatory T cell isolation kit (Miltenyi, San Diego, CA, USA) as instructed.

Techniques: Phospho-proteomics, Activation Assay, Western Blot, Control, Expressing

Journal: Frontiers in Immunology

Article Title: Regulatory T Cells Suppress Effector T Cell Proliferation by Limiting Division Destiny

doi: 10.3389/fimmu.2018.02461

Figure Lengend Snippet: Quantitative analysis of the effect of Tregs on Teff stimulation. CD4 + CD25 − CD62L + effector T cells (Teffs) labeled with cell division dyes were co-cultured with varying ratios of Tregs and the response measured. (A) Example timecourse of cell division progression in cultures without Tregs (top panel) and with Tregs (lower panel). (B) Number of undivided Teff cells in culture over time. Data shown are mean +/– SEM of triplicate samples. One representative data set from three independent experiments is shown. (C) The effect of Tregs on the total live cell number of Teffs over time for varying ratios of suppression is shown. For each graph, the Treg ratio (closed circles) is overlaid with the control culture with no Tregs added (open circles). (D) The cohort number over time of Teff cells is shown for varying ratios of Tregs. For each graph, the Treg ratio (closed circles) is overlaid with the control culture with no Tregs added (open circles).

Article Snippet: CD4 + CD25 − CD62L + effector T cells (Teff) were isolated from pooled mouse lymph nodes (inguinal, axillary, brachial, superficial cervical, and lumbar) and spleens by negative and positive selection using the mouse naïve CD4+ T cell isolation kit (Miltenyi).

Techniques: Labeling, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: Regulatory T Cells Suppress Effector T Cell Proliferation by Limiting Division Destiny

doi: 10.3389/fimmu.2018.02461

Figure Lengend Snippet: Log-dampener model of suppression of CD4 + effector T cell proliferation by Tregs. (A) In the absence of Tregs, signals from antigen (signal 1—gray), co-stimulation (signal 2—blue) and cytokines (signal 3—red) sum linearly according to the number of divisions contributed to division destiny by each signal, resulting in an exponential increase in the magnitude of the T cell response (note the log scale on the y-axis). “a” and “b” refer to different types of co-stimulatory and cytokine signals contributing positively to the T cell response. (B) Tregs regulate division destiny by reducing positive signals (1, 2a and 3a) and by adding negative signals (2c and 3c). (C) Illustration of the effect of Tregs on the size of the effector T cell response (note the linear y-axis). The blue dots represent the peak of the T cell response in the absence of Tregs (dark blue), after subtracting of positive signals (medium blue) and after addition of negative signals (light blue). (D) Schematic showing different types of signals being integrated within the CD4 + effector T cell according to the rules of the “cellular calculus”.

Article Snippet: CD4 + CD25 − CD62L + effector T cells (Teff) were isolated from pooled mouse lymph nodes (inguinal, axillary, brachial, superficial cervical, and lumbar) and spleens by negative and positive selection using the mouse naïve CD4+ T cell isolation kit (Miltenyi).

Techniques:

Journal: Brain

Article Title: Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke

doi: 10.1093/brain/awx111

Figure Lengend Snippet: Thrombolytic treatment inhibits the repopulation of circulating Tregs in stroke patients. (A) Representative gating strategy for the FACS analysis of CD3+CD4+CD25+CD127− Tregs in human blood cells. (B–D) The number of Tregs (per µl blood) decreased after stroke onset (upon admission) in both male (C) and female (D) patients (n = 84 for stroke patients; n = 115 for healthy controls). (E–G) The percentages of Tregs (in CD3+CD4+ T cells) decreased after stroke onset (upon admission) in both male (F) and female (G) patients. (H–J) tPA treatment inhibited the repopulation of Tregs at Day 1 and Day 7 after stroke. (H) The number of Tregs at Day 0, Day 1, and Day 7 after stroke in tPA-treated and non-tPA-treated patients. (I–J) Changes in Treg numbers at Day 1 (I) and Day 7 (J) after stroke with or without tPA-treatment (n = 42 for non-tPA-treated; n = 23 for tPA-treated). Data are mean ± SE. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Regulatory T cell suppression assay CD4 + CD25 − T effector cells (Teffs) were plated at 2 × 10 5 per well in a U bottom 96-well plate in the presence of anti-CD3/CD28 activation beads (Miltenyi) to stimulate their proliferation.

Techniques:

Journal: Brain

Article Title: Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke

doi: 10.1093/brain/awx111

Figure Lengend Snippet: Administration of Tregs dramatically reduces tPA-induced intracerebral haemorrhage in a murine suture model of stroke. (A) Scheme for experimental design in a suture model of MCAO. tPA (10 mg/kg) was continuously infused into the femoral vein over 20 min at 2 h after MCAO. Tregs were isolated from pooled spleens and lymph nodes of donors and transferred (2 × 106 cells/animal) to ischaemic recipients immediately after tPA delivery through the femoral vein. The timeline for outcome measurements is also illustrated. (B) The percentages of Tregs in CD3+CD4+ T cells in the blood collected from sham, MCAO, and MCAO+tPA groups. (C) Representative flow cytometry plots of CD25+Foxp3+ cells in CD4+CD25+ Tregs and CD4+CD25− T effective cells (Teff) after double selection. (D) T cell suppression test. Graded numbers of Tregs were added to the Teff cells in the presence of anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml) stimulation. The proliferation of Teff was measured 24 h later by BrdU incorporation test. (E and F) Regional cerebral blood flow (CBF) was monitored using 2D laser speckle imaging. (E) Representative images of cerebral blood flow before MCAO, during MCAO, and at 10 min after reperfusion for each group. (F) Quantification of cerebral blood flow. Results are expressed as percent change from baseline (pre-MCAO). n = 4–5 per group. (G) Representative images of the dorsal (top) and ventral (middle) surfaces of the brain and a coronal section (bottom) showing the location of the intracerebral haemorrhage 1 day after stroke in mice treated with PBS, PBS+Treg, tPA+PBS, or tPA+Treg. (H) Quantification of cerebral haemorrhage after different doses of Treg treatment by spectrophotometric haemoglobin assay 1 day after stroke. (I) Exogenous Tregs reduced tPA-induced brain haemorrhage in the absence of endogenous Tregs. Mice were injected intraperitoneally with either PBS (control) or 300 mg of CD25-specific antibody (CD25 Ab) 2 days prior to MCAO. tPA (10 mg/kg) was given at 2 h after MCAO. Brain haemorrhage was measured 1 day after MCAO. (n = 4–6/group). (J) Tregs did not directly inhibit tPA activity. 20 µg tPA was incubated with or without 1.6 × 105 Tregs in 160 µl media for 30 min or 1 h. The activity of tPA was determined by zymography (top) and tPA chromogenic activity assay (bottom). Data are mean ± SE. *P ≤ 0.05; **P ≤ 0.01.

Article Snippet: Regulatory T cell suppression assay CD4 + CD25 − T effector cells (Teffs) were plated at 2 × 10 5 per well in a U bottom 96-well plate in the presence of anti-CD3/CD28 activation beads (Miltenyi) to stimulate their proliferation.

Techniques: Isolation, Flow Cytometry, Selection, BrdU Incorporation Assay, Imaging, Injection, Control, Activity Assay, Incubation, Zymography