Evidence for Quantitative and Functional Immune Deviation in Pediatric Patients with Sickle Cell Disease
Authors: Kean Leslie , Sen Sharon , Felder Martha Ann , Tangpricha VIN , Adisa Olufolake , JAMES-Herry Angelica , Buchanan Iris , Ziegler Thomas , Alvarez Jessica , Beus Jonathan , Worthington-White Diana , Robertson Jennifer , George Jackie , Cetron Joshua , Ofori-Acquah Solomon Fiifi , Osunkwo Ifeyinwa 
Published: October 2019

Abstract
Abstract 1054
While hematopoietic stem cell transplantation (HSCT) is a curative therapy for individuals with sickle cell disease (SCD), these patients are at increased risk of graft rejection, especially after non-myeloablative HSCT. This suggests that SCD patients may exhibit immune activation at baseline. However, a rigorous analysis of the extent and character of this immune activation, using newly available multiplexed flow cytometric techniques, has not been previously reported. The objective of this study was to describe the extent of immune deviation in a cohort of pediatric subjects with SCD during steady state, given that this age group represents the majority of SCD patients undergoing HSCT.

Methods:
An IRB-approved prospective cross-sectional study design was used to compare patients, aged 10 –16y with SCD (homozygous SS or Sb0-thalassemia) during steady state (at least 21 days from an acute SCD exacerbation or other illness including infections requiring antibiotics, and, at least 8 weeks from any RBC transfusion) with an ethnic and age-matched control group of healthy individuals without SCD. Patients were recruited from the Aflac Comprehensive Sickle Cell Clinic at Children's Healthcare of Atlanta. Controls were recruited using fliers from general pediatric clinics and other community centers in the metro Atlanta area. Inclusion criteria included a confirmed documented diagnosis of homozygous SS or sickle b0 thalassemia for cases and the absence of any sickle hemoglobinopathy for controls. Subjects could only participate once in this study, were excluded if they had renal disease, any significant illness that might be associated with an immune defect such as SLE, were on oral/parenteral corticosteroids, had liver disease (AST/ALT >3ULN), or were unable to give informed consent or complete all study procedures.
We have currently enrolled 23/40 SCD patients and 18/30 controls. All patients and controls were African American. Other baseline demographic characteristics of both groups were similar with regards to gender and age (p=0.775 and 0.8314 respectively). Following informed consent, each subject had blood drawn for quantitative immune analysis: total WBC, ANC, ALC, total T cell count (CD4/CD8 T cell subsets), total B cell and NK cell count, and enumeration of CD4 and CD8 T cells for their memory subpopulations. Functional immune assessment was done on a subset of samples (18 patients, 6 controls) using multiplexed enumeration of 25 serum cytokines using the Invitrogen 25-plex human cytokine panel. Quantitative and qualitative flow cytometric and cytokine analysis was performed using FloJo software and the Prism software statistical package.

Results:
Our results provide evidence for quantitative and functional immune deviation in SCD patients compared to controls. In addition to the well-documented increases in total WBC and ANC (1.9-fold and 1.7-fold compared to controls, respectively, p <0.05), SCD patients also demonstrated significantly higher total lymphocytes, monocytes and both cytotoxic and cytokine-secreting NK cells (2.9, 2.4, and 2.2-fold compared to controls, p <0.01 for all). Circulating B cells were significantly higher in SCD patients (3.3 fold, p= 0.0005). Circulating T cells were also increased in SCD patients, but this effect was specific to CD4+ T cells (1.7-fold, p = 0.002). There was no difference in CD8+ T cells (p= 0.4). The increased CD4+ T cell count predominantly involved memory CD4+ T subpopulations, with both central and effector memory CD4+ T cells significantly increased compared to controls (1.9-fold and 2.5-fold, respectively, p< 0.01). SCD patients also showed evidence of functional immune activation at steady state; plasma cytokine analysis revealed higher circulating CXCL10, CCL4 and IL-15 (2.4, 2.1, and >10 fold compared to controls, p <0.05 for all). These cytokines, which are secreted by activated endothelial cells and monocytes, are implicated in promoting the proliferation (IL-15) and the activation (CXCL10 and CCL4) of both T cells and NK cells.

Implications:
Our results confirm that even during steady state, pediatric patients with SCD exhibit evidence of significant quantitative and functional immune deviation. This observation suggests that targeting immune pathways impacting NK cell, B cell, and CD4 T cell function may be required for successful engraftment of SCD patients during non-myeloablative HSCT.

Disclosures:
No relevant conflicts of interest to declare.