Alterations in the Humoral Immunophenotype of Children with Sickle Cell Disease on Hydroxyurea

Background: Sickle Cell Disease (SCD) is a red blood cell disorder associated with hemolysis and inflammation affecting homeostasis of the blood system. Both innate and T-cell driven adaptive immune systems are involved in SCD-linked inflammation. The involvement of B-cell compartment in etiology of SCD is much less studied. While complex interactions of ischemia-reperfusion, inflammation, and hemolysis affecting the bone marrow and immune system have been studied in adults with SCD, much less is known how their interplay affects SCD etiology in children.

Objective: To determine the range of immune changes we assessed the frequency of 12 immune populations via flow cytometry, evaluated B cell related cytokines, and performed bulk RNA sequencing of the peripheral blood (PB) of pediatric patients with SCD as compared to pediatric race-matched controls as well adult patients with SCD as compared to adult race-matched controls.

Method: Adult control PB was obtained from Rhode Island Blood Center. Pediatric control and pediatric and adult SCD PB were obtained from Rhode Island Hospital Hematology and Primary Care clinics. All informed consents/assents were obtained. PB samples were stained using markers identifying 12 immune populations including CD8, CD4, T Regulatory, B cells, Plasmablasts, Monocytes, Dendritic, Granulocytes, Neutrophils, Basophils, Eosinophils, and Stem Cells. Flow cytometry was performed on BD^TM LSRII and analyzed using FlowJo. RNA was isolated from pediatric SCD or control PB and RNA-seq was performed by Novogene. Plasma from all patients (SCD and controls) was processed for cytokine analysis using Biolegend's LEGENDplex Human B Cell Panel. Demographics, clinical, and laboratory data were obtained from chart review.

Results: Thirteen pediatric patients (ages 1-16, mean 8.7 years) and 12 adult patients (ages 8-15, mean 32.2 years) with SCD on Hydroxyurea were enrolled. HbSS genotype was found in 92.3% pediatrics and 66.7% adults. Mean Hydroxyurea dose was 23.4 mg/kg or 20.0 mg/kg for pediatric or adult SCD, respectively. Five pediatric controls (ages 9-14, mean 11.4 years) and 10 adult controls (ages 18-53, mean 31.0 years) were enrolled.

Laboratory data indicated elevated hemolysis in pediatric and adult SCD with mean Absolute Reticulocyte Count (ARC) 203 ± 94.2 for pediatric SCD and 214 ± 169 for adult SCD. Flow cytometry data revealed 1.9-fold increase (p= 0.012) of CD3+CD4+ cells in Pediatric SCD versus pediatric controls, this change was not seen in adult SCD versus adult controls (Table 1). Pediatric SCD patients had 3.5-fold increase (p=0.005) in CD19+ B cells without further increase in differentiated B cells, this change was not seen in adult SCD versus adult controls (Table 1). Subsequent analysis of B cell related cytokines revealed significant increase in IL2 (p =0.013), IL4 (p=0.026), TNFβ (p= 0.039), and IL13 (p=0.034) in pediatric SCD versus pediatric controls; there was no significance for these cytokines in adult SCD versus adult controls. BAFF was elevated in both pediatric and adult SCD versus age-matched controls (p=0.041 and p=0.005, respectively). Finally, bulk RNA sequencing of pediatric SCD or control PB 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 (29 genes), plasma cells (37 genes) and CD34 Positive Pre-B cells (10 genes), (Figure 1). These results suggested significant alteration of the B cell maturation in pediatric SCD samples.

Conclusions: Our data indicate significant changes in CD4+ and CD19+ cells in pediatric SCD patients versus controls. Increased CD4+ cell frequencies in children with SCD were previously reported (PMID 29926339). Our new finding of altered humoral immunity and differentiation of B cells noted by flow cytometry were consistent with elevated B-cell related cytokine levels in pediatric SCD versus pediatric controls. Elevated levels of pro-B cells, plasma cells, and CD34+ pre-B cell transcripts detected by RNA-seq in pediatric SCD patients versus controls further confirmed humoral alterations 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.

No relevant conflicts of interest to declare.