Fig 1.
Expression and function of B7 family members.
Purified CLL cells were cultured 72 hr either alone (CLLMed) or with allogeneic PBMC + CD3/CD28 (CLLAct). CLL were then analysed by flow cytometry or purified and utilized in proliferation assays. A) Flow cytometric analysis of antigen expression. Data are shown as scatterplots of the MFI of each molecule on CLL cells obtained from 5–6 individual CLL patients. Differences between CLLMed and CLLAct were analysed by paired t-test with respective p values reported in each plot. B) CFSE-PBMC were cultured with CD3/CD28 and the indicated combinations of media alone, purified CLLAct and inhibitors (blocking antibodies (α), recombinant proteins (Ig) or controls) for 72 hours prior to analysis of proliferation. Data obtained from separate experiments with blocking antibodies (n = 4) and recombinant proteins (n = 3) were normalised relative to the respective cultures containing CLLAct and control only (defined as 100%) and shown as relative proliferation (mean % ± SEM).
Fig 2.
Effect of monocyte depletion and delayed CLLAct addition on suppression.
CFSE labelled responders were cultured with CD3/CD28 in the presence or absence of CLLAct prior to analysis of proliferation. All data were normalised relative to cultures containing PBMC + CLLAct added at T = 0 (defined as 100%) and shown as relative proliferation (mean ± SEM). (A) Relative proliferation observed in cultures using CFSE-PBMC responders that were either untreated or CD14 depleted prior to assay. Either nil or CLLAct were added at T = 0. Data are from 4 separate experiments (B) Relative proliferation observed following culture of CFSE-PBMC either alone or in the presence of CLLAct added at T = 0 or T = 24h. Data are from 4 separate experiments.
Fig 3.
Effect of enzyme and adenosine receptor inhibitors on suppression mediated by CLLAct.
Proliferation in the presence of inhibitors was normalised relative to cultures containing CLLAct and carrier alone (defined as100%) and pooled data shown as relative proliferation (mean % ± SEM). (A) Relative proliferation observed following culture (n = 4 experiments) in the presence of carrier only control, caffeine or a combination of endonuclease inhibitors specific for CD38, CD39 and CD72. (B) Relative proliferation observed following culture (n = 6 experiments) in the presence of either carrier only control, caffeine, the adenosine receptor antagonist (CGS-1493) or a combination of antagonists specific for all adenosine receptor subtypes (1, 2A, 2B, 3).
Fig 4.
Effect of Ryanodine Receptor agonist and PI3K inhibitors on suppression induced by CLLAct.
CFSE-PBMC were cultured with CD3/CD28 in the presence or absence of CLLAct with or without the addition of caffeine, ryanodine receptor agonist 4CMP or the PI3Kδ inhibitor Idelalisib. Flow cytometric analysis was then performed to analyse apoptosis at 48h and proliferation and cell size at 72h. All data were normalised relative to cultures containing PBMC + CLLAct and carrier only control (defined as 100%). A) Proliferation in the absence and presence of caffeine or 4CMP was analysed and normalized data from 5 experiments shown as relative proliferation (mean ± SEM) at 72 hrs. B) Proliferation in the absence and presence of caffeine or Idelalisib was analysed and normalized data from 11 experiments shown as relative proliferation (mean ± SEM) at 72 hrs. C) Bar graph of the relative T cell proliferation and relative apoptosis of the PBMC and CLL populations observed in activated co-cultures. Co-cultures were performed in the presence or absence of caffeine and Idelalisib. Normalised data from individual experiments (n = 4) are shown as mean ± SEM. D) Scatter plot of the size of the CLL cells present at the end of co-culture. Data from 6 separate experiments are shown as plots of mean forward scatter.
Fig 5.
Dose dependent effects of caffeine and Idelalisib on suppression induced by CLLAct.
CFSE+PBMC were cultured in the presence of CLLAct and increasing concentrations of caffeine or idelalisib, prior to analysis of proliferation. All data were normalised relative to cultures containing PBMC + CLLAct and carrier only control (defined as 100%) and shown as relative proliferation (mean ± SEM). A) Relative proliferation in the presence of increasing concentrations of Idelalisib (n = 3 experiments). B) Relative proliferation in the presence of increasing concentrations of Caffeine (n = 5 experiments).
Fig 6.
Effect of Caffeine and Idelalisib on pAkt (S473).
Expression of pAKT by CLL cells was analysed by flow cytometry either prior to or following stimulation (15 min) with anti-Ig. (A) CLL cells (n = 7) were analysed before (CLL) or after (CLLAct) activation of cells by 72h co-culture with activated PBMC. (B, C) CLLAct were cultured for 1 hr in the presence or absence of either (B) Idelalisib (n = 7) or (C) caffeine (n = 5) prior to stimulation with anti-Ig for 15 mins and analysis of pAkt (S473). Data from analysis of CLL cells from 7 different patients are shown as a scatter plot of specific MFI.