Influence of Genetic Modifiers on Levels of Invariant Natural Killer T-Cells in Patients with Sickle Cell Anemia

Although patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been associated with five different beta S-globin gene cluster haplotypes (βS-haplotypes) that show different clinical expression. Because genetic modifiers can modulate treatment response, we hypothesized that βS-haplotypes can affect levels of invariant natural killer T (iNKT) cells, which are known to play a key role in the pathogenesis of SCD, and are being considered as a potential target to treat acute crises in SCD patients. Herein we evaluated the impact of βS-haplotypes and HbF concentration on iNKT cell and dendritic cell (DC) subsets in a well-defined group of patients with SCD in a steady state. Sickle cell anemia patients were selected based on the history of patients, clinical examination and hematological findings. The βS-haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism analysis for seven restriction sites. The iNKT cell subsets were characterized 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 using flow cytometry and cell culture. The myeloid DC (mDC) and plasmacytoid DC (pDC) cells were identified by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells by flow cytometry. Comparisons among βS-haplotypes were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. Among the 125 sickle cell anemia patients studied, Benin haplotype (40%) was the most common followed by Bantu (21%), Arab/Indian (18%), Atypical (12%) and Benin/Bantu or Benin/Arab-Indian or Bantu/Arab-Indian (9%). The majority of subjects with Benin/Benin βS-haplotype had a severe to moderate clinical profile similar to Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.23). A trend toward increased levels of CD3iNKT, CD4iNKT and CD8iNKT cell subsets was observed in the subjects with the Benin/Benin βS-haplotype, when compared to other βS-haplotype groups (P=0.06). Interestingly, subjects with the Benin/Benin βS-haplotype exhibited slightly higher levels of Th1-like cells, but not Th2-like and Th17-like cells, when compared to subjects with the Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.047). Comparisons between the levels of mDC and pDC cell subsets, as well as the expression of CD1d on these DC cells, showed no statistically significant differences among the βS-haplotype groups (P=0.42). Similarly, levels of iNKT cell subsets, Th1-like, Th2-like, and Th17-like cells as well as DC subsets were similar among patients who received or not hydroxyurea therapy, independently of βS-haplotype groups (P= 0.09). Likewise, coexistence of high HbF and βS-haplotypes did not show any significant association with iNKT cell subsets. Collectively, our findings suggest that neither the βS-haplotypes or HbF levels nor the hydroxyurea therapy or clinical severity appeared to be associated with iNKT cell or DC cell subsets in patients with SCD. The effects of other genetic modifiers such as alpha/beta-thalassemia and G6PD deficiency on iNKT cell subsets need to be evaluated in future studies.