soluble α-cd28 37.51  (Bio X Cell)

Journal: bioRxiv

Article Title: Mutation of an L-Type Calcium Channel Gene Leads to a Novel Human Primary Cellular Immunodeficiency

doi: 10.1101/864280

Figure Lengend Snippet: PBMCs of patients (n=3) and healthy donors (n=4) were stimulated with α-CD3 and α-CD28/49d for 4.5 hours and subsequently stained with various antibodies for extracellular markers and intracellular cytokines. The frequencies of CD38+, IFNγ+, and IL-2+ lymphocytes are shown for CD4 and CD8 non-naïve T lymphocytes, which are classified as NOT CD27+ CD45RA+. This experiment was only done once.

Article Snippet: Cells were stimulated with α-CD3 (coated plates, OKT3) and α-CD28/CD49d (soluble, L293, L25, BD Biosciences) for 4.5 hours, in the presence of brefeldin A (BD Biosciences) and monensin (BD Biosciences).

Techniques: Staining

Journal: PLoS ONE

Article Title: Differential immunomodulation of T-cells by immunoglobulin replacement therapy in primary and secondary antibody deficiency

doi: 10.1371/journal.pone.0223861

Figure Lengend Snippet: Patient PBMCs were stimulated with α-CD3/CD28 and supernatant was analyzed for cytokine detection. (A) IFN-γ, (B) IL-10, and (C) IL-17α in supernatant were detected by cytometric bead array (CBA). Significant detectable quantities of IFN-γ were achieved after 6hr stimulation while IL-10 and IL-17α is detected in significant quantities after 48hr stimulation. All patients are shown and sub-stratified into primary (1°) (n = 17) and secondary (2°) (n = 14) antibody deficiency. IFN-γ expression in whole PBMC supernatant is significantly decreased in SAD patients post-IRT but not in PAD. Other cytokines reveal no significant change in expression post-IRT in both PAD and SAD patients. ns denotes not significant ( p > 0.05), * denotes p < 0.05. P -values were determined by Wilcoxon matched-pairs signed rank test.

Article Snippet: 2x10 6 thawed PBMCs in RP-10 media were then stimulated with 2μg/mL soluble α-CD28 antibodies (BD Biosciences) for 6 hours.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Differential immunomodulation of T-cells by immunoglobulin replacement therapy in primary and secondary antibody deficiency

doi: 10.1371/journal.pone.0223861

Figure Lengend Snippet: (A) Patient PBMCs were stimulated with α-CD3/CD28 for 6hr in the presence of BFA and stained for CD3, CD4, CD8, and IFN-γ. Gating strategy for IFN-γ + CD4 + and CD8 + T-cells in stimulated PBMC is shown. (B) CD4 + and CD8 + T-cell IFN-γ expression after α-CD3/CD28 stimulation post-IRT is significantly higher in only 2° antibody deficiency patients, while 1° antibody deficiency patients trend downwards non-significantly. IFN-γ expression is detected in significant quantities after 6hr stimulation, and is more highly expressed in CD8 T-cells than CD4 T-cells. Patients are stratified into 1° (n = 17) and 2° (n = 14) antibody deficiency. ns denotes not significant ( p > 0.05), * denotes p < 0.05, and ** denotes p < 0.01. P -values were determined by Wilcoxon matched-pairs signed rank test.

Article Snippet: 2x10 6 thawed PBMCs in RP-10 media were then stimulated with 2μg/mL soluble α-CD28 antibodies (BD Biosciences) for 6 hours.

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: Differential immunomodulation of T-cells by immunoglobulin replacement therapy in primary and secondary antibody deficiency

doi: 10.1371/journal.pone.0223861

Figure Lengend Snippet: (A) Patient PBMCs were stimulated with CEF peptides for 6hr in the presence of BFA and stained for CD3, CD8, IFN-γ, and TNF-α. Gating strategy for IFN-γ + TNF-α + CD8 + T-cells in CEF combo peptide stimulated PBMC is shown. (B) IFN-γ and TNF-α double-expression of memory CD8 + T-cells after CEF combo peptide stimulation does not significantly change post-IRT in either 1° or 2° patients. (B) IFN-γ and (C) TNF-α single positive expression of memory CD8 + T-cells after CEF combo peptide stimulation does not significantly change post-IRT in either 1° or 2° patients. Single positive expression of these cytokines in CEF combo peptide stimulation is weaker than from α-CD3/CD28 stimulation. Additionally, the magnitude of IFN-γ + TNF-α + CD8 T-cells is very low. Patients are stratified into 1° (n = 17) and 2° (n = 14) antibody deficiency. ns denotes not significant ( p > 0.05). P -values were determined by Wilcoxon matched-pairs signed rank test.

Article Snippet: 2x10 6 thawed PBMCs in RP-10 media were then stimulated with 2μg/mL soluble α-CD28 antibodies (BD Biosciences) for 6 hours.

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: Differential immunomodulation of T-cells by immunoglobulin replacement therapy in primary and secondary antibody deficiency

doi: 10.1371/journal.pone.0223861

Figure Lengend Snippet: (A) Patient PBMCs dyed with CellTrace were stimulated with α-CD3/CD28 for 4 days stained for CD3, CD4, and CD8. Gating strategy for proliferating CellTrace dye-diluted CD3 + , CD4 + , and CD8 + T-cells in PBMC is shown. CD3 + T-cell proliferation is decreased post-IRT in all patients (C), but primarily due as decreased CD4 + T-cell proliferation in 1° antibody deficiency patients (B) and primarily due as decreased CD8 + T-cell proliferation in 2° antibody deficiency patients (D). Patients are stratified into 1° (n = 17) and 2° (n = 14) antibody deficiency. * denotes p < 0.05, and ** denotes p < 0.01. P -values were determined by Wilcoxon matched-pairs signed rank test.

Article Snippet: 2x10 6 thawed PBMCs in RP-10 media were then stimulated with 2μg/mL soluble α-CD28 antibodies (BD Biosciences) for 6 hours.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: CD49b defines functionally mature Treg cells that survey skin and vascular tissues

doi: 10.1084/jem.20181442

Figure Lengend Snippet: Treg cells stably express CD49b in a maturation-dependent manner. (A) Percentage of CD49b + cells among Treg cells stimulated in culture with α-CD3/CD28 and IL-2 for 4 or 8 d. (B) Percentage of CD62L + or CD49b + cells among aTreg cells in tissues of 5-d-old or 8-wk-old mice. (C) Expression of Rag2 GFP in Treg subsets from 4-wk-old Rag2 GFP mice. (D) Quantification of C. **, P ≤ 0.01 by paired t test; ns, not significant. (E) The indicated Foxp3 GFP Treg subsets were separately transferred into sublethally irradiated congenic Foxp3 GFP mice. Shown is the expression of CD49b versus dilution of CTV by transferred Treg cells recovered from spleen after 6 d. (F) Quantification of CD49b expression by transferred Treg cells. Data are representative of two independent experiments. Bars depict means ± SD.

Article Snippet: Single-cell suspensions were restimulated in U-bottom plates with 50 ng/ml PMA and 500 ng/ml ionomycin or 5 µg/ml soluble α-CD3 (clone 145-2C11; BioXCell) and 5 µg/ml soluble α-CD28 (clone 37.51; BioXCell), both in the presence of 1 µg/ml brefeldin A.

Techniques: Stable Transfection, Expressing, Irradiation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lineage-specific metabolic properties and vulnerabilities of T cells in the demyelinating central nervous system

doi: 10.4049/jimmunol.1600825

Figure Lengend Snippet: (A) Dose dependent inhibition of GAPDH and hexokinase activities in a lymph nodes single cell suspension by 3-BrPa. (B) Effect of 3-BrPa on T cell proliferation. Lymph node cells were stimulated with α-CD3 (1 μg/mL) and α-CD28 (10 μg/mL). At 24 hours, a time when glycolysis is engaged, but no cell divisions have occurred, 3-BrPa was added to a final concentration of 10 μM, and subsequent divisions were assessed after 48 additional hours. Proliferation of CD8+ T cells is shown. Gated on live, singlet CD8+ cells. (C) Effect of increasing concentrations of 3-BrPa on TNFα and IL-2 production in T cells stimulated with α-CD3 (1 μg/mL) and α-CD28 (2 μg/mL). Gated on live, CD11b− CD19− TCRβ+ CD4+ or CD8+. (D) Effect of 3-BrPa on CD69 induction on T cells after 6 hours of activation with α-CD3 (1 μg/mL) α-CD28 (2 μg/mL). Gated on live, singlet, CD11b− B220− CD4+ (E) Effect of 3-BrPa on phosphorylation of ERK in TCRβ+ CD4+ T cells stimulated with α-CD3 (1 μg/mL) and α-CD28 (2 μg/mL) for 60min. Gated on singlet, TCRβ+ CD4+. The same results were observed in CD8+ T cells

Article Snippet: To assess the effects of 3-BrPa on T cell proliferation, T cells were isolated from spleens and lymph nodes of C57BL/6 mice using the R&D CD3 + T cell enrichment Column (MTCC-10), stained with 20 μM Cell Proliferation Dye eFluor450 (eBioscience #65-0842-85), and stimulated to divide in complete RPMI with platebound α-CD3 (BioXcell #BE0001-1) (1 μg/mL) and soluble α-CD28 (BioXcell #BE0015-5) (10 μg/mL) antibodies for 72hrs.

Techniques: Inhibition, Suspension, Concentration Assay, Activation Assay, Phospho-proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lineage-specific metabolic properties and vulnerabilities of T cells in the demyelinating central nervous system

doi: 10.4049/jimmunol.1600825

Figure Lengend Snippet: ELISA (A) and qRT-PCR (B) for IFN-γ and IL-17A production by in vitro derived Th1 and Th17 cells stimulated with α-CD3 (2 μg/mL) and α-CD28 (2 μg/mL) in the presence of 3-BrPa (10 μM) for 20 hours; (C) flow cytometry for IFN-γ and IL-17A production by in vitro derived Th1 and Th17 cells stimulated as above for 6 hours in the presence of Brefeldin A. Gated on live, singlet, TCRβ+, CD4+ (A–C) are N=1 experiment with n=1–2 mice per group

Article Snippet: To assess the effects of 3-BrPa on T cell proliferation, T cells were isolated from spleens and lymph nodes of C57BL/6 mice using the R&D CD3 + T cell enrichment Column (MTCC-10), stained with 20 μM Cell Proliferation Dye eFluor450 (eBioscience #65-0842-85), and stimulated to divide in complete RPMI with platebound α-CD3 (BioXcell #BE0001-1) (1 μg/mL) and soluble α-CD28 (BioXcell #BE0015-5) (10 μg/mL) antibodies for 72hrs.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, In Vitro, Derivative Assay, Flow Cytometry