recombinant human cytokine standards  (R&D Systems)

Journal: Journal of Leukocyte Biology

Article Title: Phosphatase inhibitor PPP1R11 modulates resistance of human T cells toward Treg‐mediated suppression of cytokine expression

doi: 10.1002/JLB.2A0618-228R

Figure Lengend Snippet: siRNA‐mediated PPP1R11 silencing renders T cells resistant to Treg‐mediated suppression . T cells were treated with 2 µM of PPP1R11 siRNA pool or nontargeting siRNA as control for 4.5 days. ( A and B ) Responder T cells were then cocultured with allogenic Tregs or control T cells as effectors for 75 min at 37˚C and stimulated in respective cocultures for 3 h with cross‐linked anti‐CD3/‐CD28 (TCR) Abs. Individual mRNA levels were measured in responder T cells (Tstim and Tsup) post coculture separation by qRT‐PCR. The mRNA levels were normalized to GAPDH and are represented as fold changes compared to expression levels in unstimulated cells treated with control siRNA (set to 1). Figures on the left side are representative donors of respective cytokine expressions for 4 donors (mean ± sd of PCR duplicates). Percentage of Treg‐mediated suppression of cytokine mRNA expression in suppressed cells (Tsup, grey) after removal of Tregs is additionally shown in comparison to respective cytokine expression in control cells (Tstim, white) after removal of allogenic T cells for both control and PPP1R11 siRNA‐treated T cells. Right: Individual values depict percentage of suppression of individual mRNAs between Tstim and Tsup upon treatment with respective siRNAs ( P = 0.013 for IL2 and P = 0.029 for IFNG) represented by different colored line per donor. P ‐values were determined by paired, 2‐sided Student's t ‐test ( * P < 0.05). ( C and D ) Responder T cells after treatment with respective siRNAs were either stimulated alone, or in 1:1 cocultures with Tregs for 4.5 days at 37˚C with plate‐bound anti‐CD3 and soluble anti‐CD28 Ab stimulation. Respective cytokine concentrations in the supernatant were measured by multiplex bead‐array immunoassay and concentrations are represented by figures on the left (representative of 4 donors). Percentage of Treg‐mediated suppression of secreted cytokines in supernatants from respective Tcell:Treg coculture (grey) is additionally shown in comparison to respective cytokine concentration from T cells alone (white) for both control and PPP1R11 siRNA‐treated T cells after stimulation. Right: individual values depict percentage of suppression of individual cytokines from supernatants (between the samples with and without Tregs) after treatment with respective siRNAs ( P = 0.043 for IL‐2 and P = 0.021 for IFN‐γ) represented by different symbols per donor. P ‐values were determined by paired, 2‐sided Student's t ‐test ( * P < 0.05)

Article Snippet: All monoclonal capture Abs, biotinylated polyclonal detection Abs, and human recombinant cytokine standards were purchased from R&D systems.

Techniques: Control, Quantitative RT-PCR, Expressing, Comparison, Multiplex Assay, Concentration Assay

Journal: Journal of Leukocyte Biology

Article Title: Phosphatase inhibitor PPP1R11 modulates resistance of human T cells toward Treg‐mediated suppression of cytokine expression

doi: 10.1002/JLB.2A0618-228R

Figure Lengend Snippet: PPP1R11 silencing augments TCR‐induced cytokine expression in T cells . T cells were treated with 2 µM of PPP1R11 siRNA pool or nontargeting siRNA for 4.5 days and then activated for (A) 3 h (mRNA studies) with cross‐linked anti‐CD3/‐CD28 (TCR) Ab stimulation or (B and C) 4.5 days with plate‐bound anti‐CD3 and soluble anti‐CD28 Abs at 37˚C. ( A ) Respective cytokine mRNA levels measured by qRT‐PCR were normalized to GAPDH and represented as fold changes compared to expression levels in unstimulated cells (set to 1) treated with respective siRNAs. (Left) Representative figure for IL2 mRNA (mean ± sd of PCR triplicates) expression upon treatment with control siRNA (white bars) and PPP1R11 siRNA (grey bars). (Middle and right) Averaged log 2 value for respective mRNAs (mean ± sem of 8 donors) ( P = 0.005 for IL2 and P = 0.003 for IFNG ) are expressed as fold change with respect to control siRNA (log 2 set to 0). P ‐values were determined by unpaired, one sample, 2‐sided t ‐test ( * P < 0.05). ( B and C ) T cells were labeled with CFSE prior to siRNA‐treatment. ( B ) Respective cytokine concentrations in the supernatants of T cells treated with respective siRNAs were measured by multiplex bead‐array immunoassay and represented by figures on the left. (Middle and right) Averaged figures for respective cytokine concentrations (mean ± sem of 10 donors; P = 0.0004 for IL‐2 and P = 0.0001 for IFN‐γ) are expressed as fold change with respect to control siRNA‐treated cells (log 2 set to 0). P ‐values were determined by unpaired, one sample, 2‐sided t ‐test ( * P < 0.05). ( C ) Left panel: Proliferation was assessed by flow cytometry by gating on live (viability dye‐negative), singlet, CD3 + CD4 + CFSE + T cells. Unstimulated T cells (dotted lines) were used as control. Overlaid CFSE histograms are shown for a representative donor (for control siRNA‐treated cells in black and PPP1R11 siRNA‐treated cells in grey, and for 2 different starting cell densities each as indicated by thick or thin lines, respectively). The right panel represents averaged values for percentage of proliferating cells (gate as indicated in the left panel) from 10 donors in 3 separate experiments (mean ± sem ). Each symbol represents an individual donor. Percentage of proliferating cells was calculated as percentage of CFSE‐diluting cells of total CFSE‐positive cells. P ‐values were determined by paired, 2‐sided Student's t ‐test

Article Snippet: All monoclonal capture Abs, biotinylated polyclonal detection Abs, and human recombinant cytokine standards were purchased from R&D systems.

Techniques: Expressing, Quantitative RT-PCR, Control, Labeling, Multiplex Assay, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Phosphatase inhibitor PPP1R11 modulates resistance of human T cells toward Treg‐mediated suppression of cytokine expression

doi: 10.1002/JLB.2A0618-228R

Figure Lengend Snippet: Chemical inhibition of the PPP1R11 target PP1 dampens T cell activation‐associated cytokines . T cells were treated with indicated concentrations of PP1 inhibitor tautomycetin for 5 h at 37˚C and activated for ( A ) 3 h with soluble cross‐linked anti‐CD3/‐CD28 Abs for mRNA studies or ( B ) 5.5 days with plate‐bound anti‐CD3 and soluble anti‐CD28 Abs for cytokine studies in the supernatant at 37˚C. DMSO‐treated and PBS‐treated cells were used as vehicle control and negative control, respectively, while EGTA‐treated cells were used as positive control for mRNA studies. DMSO‐treated cells were used as vehicle control for cytokine studies in the supernatant. Both unstimulated and stimulated cells were measured for mRNA studies by qRT‐PCR while only stimulated cells were measured for cytokine protein studies by multiplex bead‐array immunoassay. (A) Respective cytokine mRNA levels were normalized to GAPDH and are represented as fold changes compared to expression levels in unstimulated DMSO‐treated cells (set to 1). Representative results for IL2 mRNA (left) and IFNG mRNA (right) from 2 donors are shown (mean ± sd of PCR triplicates). (B) Respective cytokine concentrations for varying doses of tautomycetin are represented as fold change compared to cytokine concentration for DMSO‐treated cells (set to 1). Averaged result for IL‐2 (left), IFN‐γ (middle), and TNF‐α (right) is shown (mean ± sem of 4 donors). P ‐values were determined by paired, one‐sample, 2‐sided t ‐test ( * P < 0.05 and ** P < 0.005). Individual symbols represent individual donors

Article Snippet: All monoclonal capture Abs, biotinylated polyclonal detection Abs, and human recombinant cytokine standards were purchased from R&D systems.

Techniques: Inhibition, Activation Assay, Control, Negative Control, Positive Control, Quantitative RT-PCR, Multiplex Assay, Expressing, Concentration Assay