cd4 t effector teff cells  (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