Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Antagonism of the A 2A R enhances TCR-stimulated IL-2 mRNA increases in primary human CD4 + T cells and Jurkat T cells. (A) Box plots (top) and difference plots (bottom) show data from naïve and memory CD4 + T cells isolated from the peripheral blood of 20 healthy donors, stimulated with plate-bound anti-CD3 and soluble anti-CD28, and grown in conditions promoting TH1 or TH2 differentiation for three days in the presence or absence of ZM-241385 (ZM). IL-2 mRNA levels were determined by qPCR. In the box plots (top), the height of the box plots equals the interquartile range (IQR) and the horizontal line within the box indicates the median value. The whiskers extend to the lowest and highest data points within 1.5 X IQR and the open circles indicate the outliers, which lie above or below the whiskers. In the difference plots (bottom), open circles show pairwise differences in IL-2 mRNA for each sample when treated with ZM-241385 (ZM) or not (Con). To the right of the open circles are the median values (closed circles) and 95% confidence intervals. (B) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the absence or presence of ZM-241385 (ZM) for three days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05; *** , p < 0.001; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Isolation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: A dominant negative Gα s construct, Gα s DN3, which blocks signaling from G s -coupled receptors, enhances TCR-stimulated IL-2 mRNA increases. Jurkat cells were nucleofected with Gα s DN3 or empty vector (pcDNAI/Amp) and then stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days. IL-2 mRNA levels were determined by qPCR and normalized to the amount produced by the TCR-stimulated control. Data represent the mean ± SE from 8 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Dominant Negative Mutation, Construct, Plasmid Preparation, Produced, Control

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA and adenylyl cyclase inhibition with ddA decrease TCR-stimulated IL-2 mRNA levels. Jurkat cells were nucleofected with Gα s siRNA or NT siRNA as described in Methods (A-C) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days (A, C). Gα s siRNA significantly decreased levels of Gα s mRNA (A), Gα s protein (B), and IL-2 mRNA (C). Data for (A) and (C) represent the mean ± SE from 8 experiments. (B) Left, each immunoblot is representative of three immunoblots. Right, quantification of protein expression levels in the presence of Gα s siRNA relative to NT siRNA. Data represent mean ± SE from 3 experiments. (D) Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence or absence of ddA. Data represent the mean ± SE from 17 experiments. mRNA levels were determined by qPCR. * , p < 0.05; ** , p < 0.01; **** , p < 0.0001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Inhibition, Western Blot, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Inhibiting cAMP production decreases activity of the IL-2 promoter without affecting IL-2 mRNA stability. (A) ddA does not decrease stability of IL-2 mRNA. After 3 days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA, Jurkat cells were incubated for the indicated times with Actinomycin D to inhibit transcription, and the rate of IL-2 mRNA degradation was measured. In both cases, the rates of IL-2 mRNA degradation fit a single exponential. Data represent means ± SD from triplicate determinations from a single experiment representative of 4 experiments. (B) ddA decreases IL-2 promoter activity in a luciferase reporter assay. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence or absence of ddA for 3 days following nucleofection with the indicated plasmids. (B) Data represent means ± SD from triplicate determinations from a single assay representative of 6 assays. (C) Data represent the means ± SE of values from stimulated cells expressing IL2/pGL3 from the 6 assays. ** , p < 0.01.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Activity Assay, Incubation, Luciferase, Reporter Assay, Expressing

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Gα s siRNA, but not Gα s DN3, decreases TCR-stimulated cAMP. Jurkat cells were nucleofected with the indicated siRNA or plasmids and then assayed for cAMP accumulation as described in Methods. The TCR was stimulated with 2.5 µg/ml plate-bound anti-CD3 and 2.5 µg/ml soluble anti-CD28 (A and B), and the A 2A R was stimulated with 300 µM CGS-21680 (C). Data in (A) represent the mean ± SE from 3 experiments and data in (B and C) represent the mean ± SE from 9 experiments. * , p < 0.05.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Evidence for an inhibitory effect of cAMP on TCR-stimulated IL-2 mRNA levels after at least 2 days of TCR stimulation. (A) The potentiating effect of A 2A R antagonism was only observed after at least two days of TCR stimulation. IL-2 levels peaked within 24 hours of TCR stimulation and then decreased over the next 48 hours. Jurkat cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of ZM-241385 (ZM) and IL-2 mRNA levels were determined by qPCR at the indicated times. Data represent the means ± SD from a single experiment that is representative of three such experiments. (B) Stimulation of the TCR for three days followed by one hour of ddA treatment leads to potentiation of TCR-stimulated IL-2 mRNA levels by ddA. After three days of TCR stimulation with plate-bound anti-CD3 and soluble anti-CD28, Jurkat cells were treated with ddA for one hour before determination of IL-2 mRNA levels by qPCR. Data represent the mean ± SE from 14 experiments. *** , p < 0.001.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques:

Journal: Journal of Molecular Signaling

Article Title: Inhibition of Gα s /cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4 + T Helper Cells

doi: 10.5334/1750-2187-10-2

Figure Lengend Snippet: Model of how the source and context of activated Gα s and cAMP may determine whether they enhance or inhibit TCR-stimulated IL-2 transcription. Interactions between the TCR and peptide-major histocompatibility complex (MHC) lead to recruitment of CD4 and its associated kinase, p56-Lck, which phosphorylates tyrosine residues in the cytoplasmic tails of the TCR subunits, leading to recruitment and phosphorylation of the tyrosine kinase, ZAP-70. CD28 co-stimulation provides an additional signal that is needed for complete T cell activation and regulation of IL-2 production . ZAP-70 and p56-Lck then phosphorylate and activate numerous downstream target proteins, including PLC-γ, leading to Ca 2+ increases and activation of a variety of downstream pathways including translocation of NFAT to the nucleus and activation of IL-2 transcription (black and white pathway). Gα s stimulated by a mechanism that does not involve G s PCRs, but which could potentially involve the TCR, enhances TCR-stimulated IL-2 transcription by a mechanism that may involve binding of pCREB to the CRE site of the IL-2 promoter [ ] during the initial stages of TCR stimulation (green pathway, Stimulatory Step 1). In contrast G s PCRs decrease TCR-stimulated IL-2 transcription, potentially by utilizing both Gα s and Gβγ signaling in cells that have been exposed to at least two days of TCR stimulation (red pathway, Inhibitory Step 2). The inhibitory G s PCR/Gα s /cAMP pathway may involve binding of CREM, which gradually replaces pCREB, to the CRE site of the IL-2 promoter or the formation of NFAT/ICER complexes on NFAT/AP-1 composite sites in the IL-2 promoter , leading to repression of transcription (Inhibitory Step 2). Previous studies suggest that cAMP increases stimulated by the TCR are smaller and more transient than those stimulated by G s PCRs, as depicted by the relative sizes of the cAMP symbols, and this may contribute to the opposite effects on IL-2 transcription. Simultaneously, Gβγ may inhibit TCR-stimulated IL-2 transcription by decreasing TCR-stimulated Ca 2+ increases through Ca v 1 channels (Inhibitory Step 2), which are activated by the TCR by an unknown mechanism . Ca 2+ -calmodulin-activated calcineurin dephosphorylates NFAT, exposing a nuclear localization sequence (NLS) and leading to nuclear translocation.

Article Snippet: Cells were plated at a density of 2–9 × 10 6 cells/ml (depending on yield) in 24-well dishes coated with 2.5 μg/ml anti-CD3 antibody (Miltenyi) in RPMI containing 10% fetal bovine serum, 2.5 μg/ml anti-CD28 antibody (Miltenyi) and IL-2 (2 ng/ml) (R&D Systems).

Techniques: Immunopeptidomics, Phospho-proteomics, Activation Assay, Translocation Assay, Binding Assay, Sequencing

Journal: Molecular Medicine Reports

Article Title: Cytotoxic effect of CLL-1 CAR-T cell immunotherapy with PD-1 silencing on relapsed/refractory acute myeloid leukemia

doi: 10.3892/mmr.2021.11847

Figure Lengend Snippet: Construction of CLL-1 CAR, shRNA PD-1/CLL-1 CAR and CLL-1-overexpressing K562 cells. (A) CLL-1 CAR vector. The third-generation CAR was composed of the variable-light domain, followed and variable-heavy domain of the CLL-1 scFv, the CD8 hinge and TM region, CD28, the OX40 co-stimulatory domains and the CD3 + signaling domain. (B) shRNA PD-1/CLL-1 CAR vector. The third-generation shRNA PD-1/CLL-1 CAR was composed of the variable-light domain, followed and variable-heavy domain of the CLL-1 scFv, the CD8 hinge and TM region, CD28, the OX40 co-stimulatory domains, the CD3 + signaling domain and shRNA PD-1. (C) PSE2970 vector. The PSE2970 lentiviral vector, was constructed to express the CLL-1 antigen in the K562 cell line (stK562). (D) Flow cytometry plots exhibiting the phenotype of CAR-T cell subsets. The frequency of CD4 + and CD8 + T cells was assessed in primary T cells from patients with AML or healthy donors transduced with CLL-1 CAR or shRNA PD-1/CLL-1 CAR. (E) Bar graph of flow cytometry data illustrating the immune phenotype of CLL-1 and shRNA PD-1/CLL-1 CAR-T cells. NS, the proportion of CD4 + vs. CD8 + CAR-T cells from patients; *P<0.05, the proportion of CD4 + vs. CD8 + CAR-T cells from healthy donors. (F) Representative flow cytometry dot plots demonstrating transduction efficiency. (G) Transfection efficiency was evaluated in CLL-1 and shRNA PD-1/CLL-1 CAR-T cells. (H) Representative flow cytometry dot plots for PD-1 expression on the CLL-1 CAR-T cells and shRNA PD-1/CLL-1 CAR-T cells. (I) PD-1 expression was evaluated in CLL-1 CAR-T and shRNA PD-1/CLL-1 CAR-T cells using flow cytometry. *P<0.05. (J) PD-1 mRNA expression was measured in CLL-1 and shRNA PD-1/CLL-1 CAR-T cells using reverse transcription-quantitative polymerase chain reaction. *P<0.05. AML, acute myeloid leukemia; CLL-1, C-type lectin-like molecule-1; scFv, single-chain variable fragment; TM, transmembrane; PD-1, programmed cell death-1; CAR, chimeric antigen receptor; APC, allophycocyanin; PerCP, peridinin chlorophyll; NS, not significant.

Article Snippet: The T cells were cultured in AIM-V T cell medium (Gibco; Thermo Fisher Scientific, Inc.) containing 100 IU/ml recombinant human IL-2 (cat. no. AF-200-02; PeproTech, Inc.), 5 ng/ml recombinant human IL-7 (cat. no. AF-200-07; PeproTech, Inc.) and 5 ng/ml recombinant human IL-15 (cat. no. AF-200-15; PeproTech, Inc.), and co-stimulated with anti-CD3 (cat. no. 170-076-116; Miltenyi Biotec, Inc.) and -CD28 antibodies (cat. no. 170-076-117; Miltenyi Biotec, Inc.) for 18–24 h in a 5% CO 2 atmosphere at 37°C.

Techniques: shRNA, Plasmid Preparation, Construct, Flow Cytometry, Transduction, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation

doi: 10.1016/j.xcrm.2024.101441

Figure Lengend Snippet:

Article Snippet: CD3/CD28 activation beads , Miltenyi Biotec , #130-095-925.

Techniques: Blocking Assay, Control, Virus, Recombinant, Activation Assay, Adjuvant, Selection, Isolation, Staining, Software

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with a mixture of 3 different allogeneic B cell lines. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 6 different experiments having similar results in each are shown. (B) Cumulative data of proliferating memory CD28+ vs. CD28− memory CD8 T cells from the 6 different experiments is shown with mean percent proliferation of each T cell population ± SD. *p < 0.01

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Isolation, Labeling, Cell Culture

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with or without a mixture of 3 different allogeneic B cell lines in the absence or presence of the indicated amounts of recombinant IL-15. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 4 different experiments having similar results in each are shown.

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Isolation, Labeling, Cell Culture, Recombinant

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with a mixture of 3 different allogeneic B cell lines in the absence or presence of 10 ng/ml of recombinant IL-15. At the indicated time after culture initiation, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 4 different experiments having similar results in each are shown.

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Isolation, Labeling, Cell Culture, Recombinant

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with or without a mixture of 3 different allogeneic B cell lines in the absence or presence of 10 ng/ml of recombinant IL-15, IL-2 or IL-7. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed in a histogram as an indication of proliferation. Representative results from a single experiment of 4 different experiments having similar results in each are shown. (B) Cumulative data of proliferating memory CD28+ vs. CD28− memory CD8 T cells from the 4 different experiments is shown with mean percent proliferation of each T cell population ± SD. *p < 0.001 for CD28− cells with allogeneic B cells plus IL-15 vs. all other groups and **p < 0.01 for CD28+ cells with allogeneic B cells plus IL-15 vs. T cells cultured with B cells only. (C) CD28− RO+ CD8 T cells were isolated from peripheral blood mononuclear cells, labeled with CFSE, and aliquots cultured with or without a mixture of 3 different allogeneic B cell lines in the absence or presence of the indicated concentrations of recombinant IL-15, IL-2 or IL-7. After 96 hours, the cells were collected, washed and the dilution of CFSE by the CD8 T cells was analyzed and the proliferation observed reported as in Figures 2 and ​and3.3. Representative results from a single experiment of 3 different experiments having similar results in each are shown.

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Isolation, Labeling, Cell Culture, Recombinant

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were labeled with CFSE and aliquots cultured with allogeneic B cells plus 10 ng/ml of recombinant IL-15. After 96 hours, the cultured cells were stained with fluorochrome labeled antibodies to detect expression of CD25, CD127, CD215 and ICOS on gated T cells that were (blue line) or were not (red line) proliferating as assessed by CFSE dilution. Representative results from a single experiment of 5 different experiments having similar results in each are shown. (B) CD28− memory CD8 T cells were isolated, labeled with CFSE, and aliquots cultured with pooled allogeneic B cells in culture media with 10 ng/ml IL-15. After 24 hours, the IL-15 supernatant was removed and replaced with media alone or media with 10 ng/ml IL-2. IL-2 was also added without removal of IL-15. All cells were analyzed for proliferation at 96 hours after culture initiation. Representative results from a single experiment of 4 different experiments having similar results in each are shown. (C) CD28+ and CD28− RO+ CD8 T cells were isolated, labeled with CFSE, and aliquots cultured with or without allogeneic B cell lines in the presence of 10 ng/ml IL-15. After 48 or 72 hours, the cells were collected and whole cell RNA isolated for PCR analysis of IL-15 receptor α chain. The results indicate the mean relative quantitation (RQ) of IL-15Rα in the memory CD8 T cells cultured alone or with allogeneic B cells ± SD for 4 individual samples. *p < 0.01 for expression of CD28− vs. CD28+ memory T cells.

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Labeling, Cell Culture, Recombinant, Staining, Expressing, Isolation, Quantitation Assay

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) CD28+ and CD28− RO+ CD8 T cells were labeled with CFSE and aliquots cultured with allogeneic B cell lines with 10 ng/ml of recombinant IL-15. After 96 hours, the cultured cells were stained with fluorochrome labeled antibodies to detect expression of CD107a expression. The CFSE dilution patterns were used to gate the T cells into non-proliferating (gate A), early proliferating (gate B) and late proliferating (gate C) cells and the expression levels of CD107a on T cells in each gate were determined. Representative results from a single experiment of 4 different experiments having similar results in each are shown. (B) CD107a expression was compared by overlaying histograms of late proliferating CD28− (red) and CD28+ (green) memory CD8 T cells following 96 hours of culture with allogeneic B cells plus IL-15. (C) After 96 hours, supernatants were removed from cultures of CD28+ and CD28− RO+ CD8 T cells with allogeneic B cells plus 10 ng/ml IL-15. Production of IFN-γ and TNF-α was tested by ELISA and expressed as mean concentration ± SD for 4 samples per group. *p < 0.01 for cultures with IL-15 vs. those without IL-15.

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Labeling, Cell Culture, Recombinant, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: IL-15 Induces Alloreactive CD28 − Memory CD8 T Cell Proliferation and CTLA4-Ig Resistant Memory CD8 T Cell Activation

doi: 10.1111/ajt.12719

Figure Lengend Snippet: (A) Peripheral blood mononuclear cells (PBMC) were labeled with CFSE cultured with irradiated pooled allogeneic B cells +/− 10 ng/ml IL-15 in the presence of absence of 100 ug/ml CTLA-4Ig. Proliferation of the (CD3+) T cells was analyzed by flow cytometry 96 after initiation of the cultures. (B) Proliferation of CD28+ versus CD28− memory CD8 T cells alone or in response to allogeneic B cells plus 10 ng/ml IL-15 in the presence or absence of 100 ug/ml CTLA-4Ig. (C) Cumulative data from 4 separate experiments indicating the ability of IL-15 to confer resistance of alloantigen-reactive memory CD8 T cell or peripheral blood T cell proliferation to CTLA-4Ig. *p < 0.01 compared to all cultures with CTLA-4Ig but without IL-15. (D) Isolated CD28+ memory CD8 T cells were labeled with CFSE and cultured with pooled allogeneic B cells in the presence or absence of 10 ng/ml IL-15. After 96 hours, the cultured cells were washed and stained with anti-CD28 mAb and the expression of CD28 on non-proliferating and proliferating CD8 T cells was determined by flow cytometry. In the third (far right)panel, the expression of CD28 was compared on proliferating CD28+ memory CD8 T cells following 96 hours of cultures with the allogeneic B cells +/− IL-15 by overlaying the histograms from each of the other two panels.

Article Snippet: The unbound CD8 memory T cells were then processed using the Human CD28 Microbead Kit (MACS, Miltenyi Biotec).

Techniques: Labeling, Cell Culture, Irradiation, Flow Cytometry, Isolation, Staining, Expressing