Expression of B Cell Differentiating Factors Is Decreased in Spleen Tissue from Children with Sickle Cell and Unswitched Memory B Cell Deficiency

The spleen is among the earliest organs to be negatively impacted by sickle cell disease (SCD). Multiple prior reports have identified histopathological changes in the spleen in SCD, most notably the variable depletion of the marginal zone from the follicle. In the peripheral blood, depletion of marginal zone B cells can be detected by a reduced number of IgD+CD27+ B cells, or the unswitched memory B (UMBC) cell subset. The mechanism for these changes in the spleen is not defined. To further explore a potential mechanism for marginal zone B cell deficiency in SCD, we performed RNASeq on human spleen tissue.

We previously identified UMBC deficiency in the peripheral blood of children with SCD. Fresh spleen tissue was collected from 14 patients scheduled to undergo surgical splenectomy. B cell subsets were defined from peripheral blood collected within one week prior to surgery and analyzed by flow cytometry. FlowJo 10.6.1 (Becton Dickinson, Ashland, OR) and Prism 8.0.0 (San Diego, California USA) were used for statistical analysis of peripheral blood B cell subsets. Following removal, spleen tissue was submerged in RNALater and stored at -20^oC until processing. RNA was isolated and sequencing performed by MedGenome Inc (Foster City ,CA). EdgeR (Bioconductor.org) was used for detection of differentially expressed genes. The false discovery rate was defined using the Q-value method, with a cutoff of 2-fold change and a false discovery rate of 5%.

Thirteen of 14 spleen tissue samples collected had available clinical and laboratory data. For the gene expression analysis, spleens were grouped according to peripheral blood results for UMBC fraction: Low (n=8) and Normal (n=5). The mean UMBC fraction for the Normal group was significantly higher than the Low group (8.3% vs 3.5%, p<0.01). From the RNA comparison, 29 genes had higher and 78 genes had lower expression in the Normal group compared to the Low group. In particular, the Normal group demonstrated greater than 2-fold higher expression of genes involved in B cell differentiation, including IL21R (2.2-fold, adjusted p = 0.03), PF4 (2.6-fold, adjusted p = 0.04), and CX3CR1 (3.1-fold, adjusted p = 0.01). Splenic expression of IL21R and PF4 correlated with peripheral blood UMBC levels (Pearson R=0.59 and R=0.74, respectively). Genes with lower expression in the Normal group compared to the Low group were related to vascular smooth muscle contraction, calcium signaling pathways, and focal adhesion.

UMBC deficiency was common, with 8 out of 13 patients with SCD about to undergo splenectomy having low UMBC levels in peripheral blood. Our analysis of splenic gene expression showed that patients with normal UMBC levels had higher expression of genes known to be involved in B cell differentiation. Disruption of these pathways may be responsible for the depletion of marginal zone B cells in patients with low UMBC levels. Additional studies are currently underway to understand how physiological changes in the spleen in SCD may impact B cell differentiation.

Tubman:Global Blood Therapeutics: Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding; Agios Pharmaceuticals: Research Funding.