NF-κB Activation Mediates Induction Of Anti-Inflammatory Adenosine A2A Receptors In iNKT Cells Of Sickle Cell Patients During Vaso-Occlusive Episodes and Upon Activation Of Cultured Human iNKT Cells

iNKT cells are activated in mice and people with vaso-occlusive sickle cell disease (SCD) and play a pivotal role in initiating an inflammatory cascade. In mice, iNKT cell activation requires NF-κB signaling [Stanic, AK et al. J Immunol. 172:4667, 2004]. In mice with SCD, lung inflammation is associated with induction in iNKT cells of IFN-γ and anti-inflammatory adenosine A_2A receptor (A_2AR) mRNAs [Wallace, KL et al. Blood 116:5010, 2010]. The A_2AR is one of four G protein coupled adenosine receptors (A₁, A_2A, A_2B and A₃) and it is the most abundant adenosine receptor expressed in T cells. In patients with SCD, painful vaso-occlusive crisis (pVOC) is associated with NF-κB activation and increased expression of A_2AR immunoreactivity detected with the monoclonal Ab, 7F6-G5-A2, in circulating iNKT cells. Infusion of a moderate dose (6 nM) of the A_2AR agonist, regadenoson during pVOC reduced iNKT cell activation over time [Field, J. et al. Blood 121:3329, 2013]. Since A_2AR activation inhibits iNKT cell activation, we reasoned that iNKT cell activation during pVOC might occur preferentially in iNKT cells with low A_2AR expression. However, in the current study we show that activated human Vα24+ CD4+ iNKT cells concordantly (i.e. on the same cells) expressed Ser-536 phosphorylated/activated NF-κB (p-NFκB), cytokines and A_2ARs. These findings suggest that NF-κB signaling increases A_2AR expression as a counter-regulatory mechanism to render activated iNKT cells highly sensitive to inhibition by A_2AR activation. Spare receptor theory predicts that enhanced receptor expression will increase the functional potency of agonists. To test this hypothesis we infused into stable SCD patients at steady state a low dose of regadenoson (2 nM) well below its receptor binding K_D that was measured by competition for radioligand binding to be 12 nM. Low-dose regadenoson infusion reduced the percentage of p-NF-κB-high/activated iNKT cells by an average of 69% over 12 hours (P = 0.005). These findings suggest that A_2AR induction is a counter-regulatory mechanism that limits iNKT cell activation by increasing the sensitivity of activated iNKT cells to inhibition by adenosine. In order to further test the hypothesis that NF-κB activation stimulates A_2AR transcription, we examined cultured human iNKT cells. Blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of cytokine and A_2AR mRNA and protein upon iNKT activation by plate-bound anti-CD3 antibodies. In conclusion, NF-κB-mediated induction of A_2ARs in iNKT cells serves as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. We conclude that A_2AR agonists have therapeutic potential for treating inflammatory diseases associated with iNKT cell activation, including SCD.