Circulating Invariant Natural Killer T Cell Subsets Are Functionally Preserved in Splenectomized Children with Sickle Cell Disease Compared to Patients with Large Spleen

Recent data suggests that invariant natural killer T (iNKT) cells, a unique subset of T lymphocytes that express markers characteristic of both T cells and natural killer cells, play a pivotal role in the pathogenesis of sickle cell disease (SCD). Indeed, these cells are being considered as a potential target to prevent or treat acute crises of SCD in adult patients. However, the phenotypic and functional characteristics of circulating iNKT cell subsets in children with SCD remain unknown. Herein, we functionally evaluated iNKT cell subsets in relation to myeloid dendritic (mDC) and plasmacytoid dendritic (pDC) cells and markers of SCD severity in well-defined groups of children with SCD. The classification criteria for SCD was based on the history of patients, clinical examination, hematological and radiological findings. Blood iNKT cell subsets were prospectively studied in 68 children with SCD and normal controls using peripheral blood mononuclear cells, 10-color flow cytometry and culture assays. The iNKT cell subsets were identified by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells. The mDC and pDC cells were phenotypically characterized by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells. SCD patients were stratified into three groups including surgically splenectomized (n=23), large spleen (n=21) and steady state (n=24). Comparisons among study groups were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. Compared to normal individuals, splenectomized and steady state subjects, large spleen patients exhibited significantly higher levels of CD3iNKT, CD4iNKT and CD8iNKT cells (P=0.04). There were no differences in the levels of iNKT cell subsets between splenectomized and steady state subjects (P=0.56). The levels of mDC and pDC cells were also similar among the study groups (P=0.70). Interestingly, the large spleen patients tend to have higher CD4, but not CD8, Th1-like, Th2-like and Th17-like iNKT cells compared to splenectomized and steady state subjects (p=0.08). The expression levels of CD1d on both mDC and pDC were equivalent among study groups. The levels of CD3iNKT cells were negatively associated with baseline haemoglobin F levels (r=0.45, P=0.04) but not with age, time since splenectomy, spleen size or total hemoglobin levels at phlebotomy. Collectively, these results further advance the functional characterization of circulating iNKT cell subsets in children with SCD and reveal that surgically splenectomized patients have preserved function of iNKT cell subsets that are to be considered for clinical purposes.