Pediatric Sickle Cell Disease Humoral Immunophenotype Alterations

Sickle cell disease (SCD) is a red blood cell disorder associated with hemolysis and inflammation affecting hematopoietic system homeostasis. While both innate and T cell driven adaptive immune responses are involved in driving SCD-linked inflammation, the involvement of the B cell compartment in the pathophysiology of SCD, especially in pediatric patients, is much less understood. To determine the range of immune changes, we assessed the frequency of twelve immune populations via flow cytometry and levels of B cell related cytokines of the peripheral blood (PB) of pediatric (n=13) and adult (n=12) patients with SCD on hydroxyurea as compared to pediatric (n=5) and adult (n=10) race-matched controls. We also evaluated bulk RNA sequencing of the PB of pediatric SCD patients and controls. The majority of SCD patients carried the hemoglobin SS genotype (92.3% pediatrics and 66.7% adults). Mean Hydroxyurea dose was 23.4 mg/kg and 20.0 mg/kg for pediatric SCD (PSCD) and adult SCD (ASCD), respectively. Laboratory data revealed anemia with mean hemoglobin 9.4 g/dl in PSCD and 9.7 g/dl in ASCD with elevated hemolysis in pediatric and adult SCD with mean reticulocyte count of 7.1% in PSCD and 8.8% in ASCD with mean absolute reticulocyte count (ARC) 203 ± 94 for pediatric SCD and 214 ± 169 for adult SCD. Flow cytometry data revealed 1.9-fold increase (p value 0.012) of CD3+CD4+ cells in pediatric SCD versus pediatric controls; this change was not seen in adult SCD versus adult controls (decrease 17%, p value 0.302). Pediatric SCD patients had 3.5-fold increase (p=0.005) in CD19+ B cells without further increase in differentiated B cells, and this change was not seen in adult SCD versus adult controls (increase 7%, p value 0.815). We performed bulk RNA sequencing on the PB of pediatric patients with SCD as compared to the pediatric controls. Obtained transcriptomic data indicated significant changes in transcripts linked to humoral immunity. Over Representation Analysis (ORA) of cell type revealed upregulation of transcripts linked to pro-B cells (27 genes, top 3:E2F2,RAD51,ASPM) and plasma cells (37 genes, top 3:IGF1,TNFRSF17,DERL3). Analysis of B cell related cytokines revealed significant increase in IL2 (p value 0.013), IL4 (p value 0.026), TNFβ (p value 0.039), and IL13 (p value 0.034) inpediatric SCD versus pediatric controls withno significance for these cytokines in adult SCD versus adult controls. BAFF was elevated in both pediatric and adult SCD versus age-matched controls (p value0.041 and p value 0.005, respectively). In sum, our data indicate significant changes in CD4+ and CD19+ cells andsuggest significant alteration of the B cell maturation in pediatric SCD samples. Our findings point to previously unreported effect of SCD on the humoral immune system in children with SCD treated with hydroxyurea, warranting further investigation.