Background: Sickle cell disease (SCD) is a chronic degenerative disease punctuated by periodic painful vaso-occlusive crises (pVOC). When sickle hemoglobin polymerizes, it causes erythrocytes to become rigid and sticky and produces multi-cellular aggregates that can occlude venules. We have shown that invariant natural killer T (iNKT) cells are pivotal in causing inflammation and injury in sickle cell disease (Wallace KL, Marshall MA, et al. Blood. 2009; 114(3):667-76). Activation of iNKT cells causes an induction of anti-inflammatory adenosine A2A receptors (A2ARs) that have a negative feedback role to limit inflammation. We found that an A2AR activation inhibits iNKT cell activation and protects SCD mice from tissue injury (Wallace KL, Linden J. Blood 2010; 116(23):5010-20). We have also seen in adult SCD patients that the expression of the A2AR is significantly higher in iNKT cells during pVOC than at steady state (Field JJ, Lin G, et al. Blood 2013; 121(17):3329-42). In addition, CD39 and CD73, ectonucleotidases that generate adenosine from ATP, reduce inflammation by activating the A2AR (Crikis S, Lu B, et al. Am J Transplant 2010; 10(12):2586-95).

Hypothesis and Aims: We hypothesized that pediatric SCD patients will have elevated A2AR and CD39 expression on iNKT cells during pVOC compared to steady state. We also hypothesized that the elevated A2AR immunoreactivity on activated iNKT cells is associated with enhanced A2AR signaling and reduced iNKT cell activation and inflammation. Our aims were: 1) to measure A2ARs and CD39 on iNKT cells of pediatric SCD patients at steady state and during pVOC, and also on cultured human iNKT cells in vitro with and without activation; and 2) to measure immunoreactive A2AR protein on activated cultured iNKT cells and correlate it with functional A2AR signaling.

Methods: We collected blood from pediatric SCD patients at steady state and during pVOC. We used fluorescence-activated cell sorting (FACS) assays to evaluate the level of A2AR and CD39 immunofluorescence. For cultured human iNKT cell studies, human iNKT cells were prepared from healthy donor whole blood and expanded in culture. iNKT cells were activated through engagement of TCRs (with anti-CD3 and anti-CD28 Abs), cytokines (IL-12 and IL-18), or a combination of TCR engagement and cytokines. We treated the activated iNKT cells with vehicle or the A2AR agonist, regadenoson. iNKT cells were analyzed at 48 hours by FACS for A2AR and CD39. To determine if induced A2ARs are functional we utilized cAMP assays. A2AR is a Gs coupled receptor, which when activated, activates adenylyl cyclase to generate cAMP.

Results: iNKT cells from pediatric sickle cell patients have higher A2AR and CD39 immunofluorescence during pVOC than at steady state. In cultured iNKT cells we found both A2AR and CD39 immunofluorescence are higher in activated iNKT cells than in non-activated cells. We discovered that iNKT cell activation through TCRs and cytokines is significantly stronger than either activation method alone. We also found both in vivo and in vitro that high A2AR expression on iNKT cells is associated with high CD39 expression. The A2ARs induced on activated iNKT cells are functional by showing significantly higher cAMP from activated iNKT cells exposed to Regadenoson.

Conclusions: Our findings indicate that CD39 and the A2AR function together to provide a mechanism to gradually turn off activated iNKT cells and modulate the inflammatory response. Understanding this negative feedback system will help us to better understand the control of inflammation in SCD during vaso-occlusive crises and to identify novel therapies to treat and prevent the complications of SCD. Further studies may include determining if A2AR activation decreases iNKT cell activation, and if blockade of CD39 enhances iNKT cell activation.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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