Characterization of B Lymphocyte Deficiency in Pediatric Sickle Cell Disease

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PROJECT SUMMARY Infections are frequent complications in sickle cell disease (SCD), occurring in up to 45% of patients. In Africa, where up to 90% of children with SCD die by age 5 years, many of these deaths are attributed to infection. In SCD, the spleen is damaged in infancy and results in marginal zone B cell deficiency. The clinical implications of B cell deficiency in SCD are not fully understood and requires investigating both disease-intrinsic and - extrinsic modifiers of B cell subsets. In this investigation, I will quantify B cell subsets in children with and without SCD in Texas and in Tanzania, investigate mechanisms of B cell deficiency, and compare them to important clinical endpoints. Aim 1: Characterize mechanisms of MZB deficiency in SCD. Hypotheses: There is a difference in the development, survival, and function of B cells from SCD and non-SCD subjects due to B-cell intrinsic factors. Differences in B cell populations may be due to differences in kinetics of differentiation and proliferation and/or survival. To examine differentiation and proliferation, I will measure the efficiency of differentiation of transitional B cells from SCD and non-SCD donors into MZBs, and MZBs into plasma cells using an in vitro B cell co-culture system. To examine survival, I will measure the rate of apoptosis and expression of markers of apoptosis (e.g. annexin V, caspase 3/9, BCL2) in native and culture-derived MZBs in culture. Aim 2: Determine the effect of the microenvironment of the spleen in SCD on B cells. Hypothesis: Extrinsic factors such as the inflammatory environment in SCD impair B cell development, survival, and function. To examine the effect of the microenvironment on B cells in culture, I will culture non-SCD B cells in media supplemented with SCD serum or spleen extract. To investigate which components of the extract impact B cells, I will develop a cytokine profile for the extract and serum using multiplex ELISA. Aim 3: Determine whether low MZB is associated with infectious and inflammatory complications of SCD. Hypothesis: Life-threatening infections in SCD are more common among children with severe MZB deficiency. To determine whether B-cell tropic viruses have a confounding effect on MZB number, I will measure immune response to EBV and malaria using a custom multiplex ELISA panel. To determine whether infectious complications are associated with more severe MZB deficiency in SCD, I will compare MZB among children with and without complications such as acute chest, osteomyelitis, or bacteremia. Despite a shared driver mutation, the clinical phenotype in SCD is highly variable. Together, the studies in this proposal will overcome critical barriers to risk-stratifying patients for infectious and inflammatory complications of SCD. Increased insights into how SCD catalyzes B cell deficiency may help identify novel therapeutic targets or biomarkers to mitigate the impact of infections on SCD. My central hypothesis is that B cell abnormalities contribute to morbidity and mortality in children with SCD.