Figure 1.
Treg inhibit IL-2 production via the CD39 pathway.
(A) Using CD39 cell surface expression, Treg/CD39+ and Treg/CD39− were sorted and co-cultured with CFSE labeled CD4+CD45RA+CD25low naive T cells (ratio ½). After overnight anti-CD3/CD28 (1 µg/ml) activation, CFSE naive T cells were resorted from co-culture and the impact of Treg on IL-2 production was evaluated by q-PCR. (B) A representative Figure of 3 independent experiments showing Treg/CD39+ suppression of IL-2 mRNA expression in anti-CD3/CD28 stimulated naive CD4+ cells. (C) Effect of anti-CD39 mAbs on suppressive function of Treg/CD39+ and Treg/CD39−. (D) The epigenetic changes on methylation of the unique specific CpG site of il-2 gene promoter (n = 3) were studied by bisulphite modification of a mixture of DNA of anti-CD3/CD28 (1 µg/ml) activated naive T cells alone or co-cultured with Treg/CD39+ or Treg/CD39− in 3 independent experiments. Following molecular cloning and bulk sequencing, 20–30 colonies were analyzed for each experimental condition. *P<0.05, **P<0.01.
Figure 2.
Inhibition of IL-2 production by A2AR induced signals during the activation of naive TCD4+ lymphocytes.
CD4+CD25−CD45RA+ naive T cells were pre-incubated with A2AR agonist CGS 21680 (10 µM) and/or A2AR antagonist ZM 241385 (2 µM). After 6H of anti-CD3/CD28 (2 µg/ml) activation, IL-2 production was evaluated by q-PCR. (A) % of inhibition of IL-2 mRNA expression by A2AR stimulation (pooled data of 3 experiments). (B) Inhibition of demethylation of the unique specific CpG site of il-2 gene promoter was performed by bisulphite modification of DNA following molecular cloning and bulk sequencing on anti-CD3/CD28 (2 µg/ml) activated naive CD4+ T cells pre-incubated with A2AR agonist CGS 21680 (10 µM) and/or A2A antagonist ZM 241385 (2 µM) in 3 independent experiments. 20–30 colonies were analyzed for each experimental condition. *P<0.05, **P<0.01.
Figure 3.
Activation of Adenyl cyclase inhibit IL-2 production by naive CD4+ T.
CD4+CD25−CD45RA+ naive T cells were pre-incubated with the Adenyl cyclase inhibitor 2′,5′-DiDeoxyadenosine (ddADA) (200 µM), or the adenyl cyclase activator Forskolin (2 µM) for 30 mins. After 6H of anti-CD3/CD28 (2 µg/ml) activation, IL-2 production was evaluated by q-PCR. (A) % Inhibition of IL-2 mRNA expression by adenyl cyclase activator (pooled data of 3 experiments). (B) The epigenetic changes on methylation of the unique essential CpG site of il-2 gene promoter. The cells were pre-incubated with ddADA (200 µM) or Forskolin (2 µM) for 6H before anti-CD3/CD28 mAbs activation in 3 independent experiments. 20–30 colonies were analyzed for each experimental condition. 20–30 colonies were analyzed for each experimental condition. *P<0.05, ***P = 0.0001.
Figure 4.
Inhibition of anti-CD3/CD28 mAbs stimulated naive CD4+ T cell proliferation and IL-2 production by cAMP.
(A) Representative histograms showing the anti-CD3/CD28 stimulated proliferation of purified naive CD4+ T alone or treated with cAMP. Percentages of proliferating (CFSElow) CD4+ T cells are shown for each condition (one representative experiment out of 4 performed in triplicate); (B) Inhibition of CD4+ T cell proliferation by cAMP (pooled data of 4 experiments). (C) % Inhibition of IL-2 mRNA expression by cAMP (pooled data of 4 experiments). (D) The epigenetic changes on methylation of the essential CpG site of il-2 gene promoter. The cells were pre-incubated with cAMP before anti-CD3/CD28 activation in 3 independent experiments. 20–30 colonies were analyzed for each experimental condition. *P<0.05.
Figure 5.
cAMP induced inhibition of IL-2 production by CD4+ T cell subsets.
(A) Gating strategy. (B) Inhibition of intracellular IL-2 production by cAMP in all anti-CD3/CD28 (2 µg/ml) stimulated sub-populations of CD4+ T cells (representative figure of 5 experiments). (C) % Inhibition of intra-cellular IL-2 production (pooled data of 5 experiments) by all CD4+ T cells sub-populations. *P<0.05.
Figure 6.
High levels of A2AR and endogenous cAMP in CD4+ T cells from HIV infected patients and lack of IL-2 production
. (A) CD45RA+ and CD45RA− CD4+ T cells were purified from the blood of ART-naive HIV-infected patients (n = 5) and healthy controls (n = 5). A2AR mRNA expression was assessed using qPCR. Horizontal lines correspond to the mean for each data set. (B) CD4+ T cells were purified from the blood of ART-naive HIV-infected patients (n = 6) and healthy controls (n = 8). Intra-cellular cAMP is measured using the cAMP direct enzyme immunoassay from GE healthcare Biosciences. (C) Purified naive CD4+ T cells form HIV+ART- patients and healthy controls were stimulated with high doses of anti-CD3/CD28 mAbs (5 µg/ml) during 6H and IL-2 mRNA levels were quantified by RT-PCR. (D) The epigenetic changes on methylation of the unique essential CpG site of il-2 gene promoter in naïve CD4+ T cells from HIV+ART- patients vs. healthy controls following anti-CD3/CD28 mAbs activation. To reach the technical limitation due to the low frequency of naïve CD4+ T cells in HIV+ART- patients, the Clonal analysis was performed on a pool of the extracted DNA from naïve CD4 T cells of HIV+ patients and healthy controls before and after CD3/CD28 mAbs activation. *P<0.05, **P<0.01.