The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.

The tetraspanin CD53 has been implicated in B cell development and function. CD53 is a transcriptional target of EBF1, a critical transcription factor for early B cell development. Further, human deficiency of CD53 results in recurrent infections and reduced serum Igs. Although prior studies have indicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. In this study, we show that CD53 expression, which is minimal on hematopoietic stem and progenitor cells, increases throughout bone marrow B cell maturation, and mice lacking CD53 have significantly decreased bone marrow, splenic, lymphatic, and peripheral B cells. Mixed bone marrow chimeras show that CD53 functions cell autonomously to promote B lymphopoiesis. Cd53−/− mice have reduced surface expression of IL-7Rα and diminished phosphatidylinositol 3 kinase and JAK/STAT signaling in prepro- and pro-B cells. Signaling through these pathways via IL-7R is essential for early B cell survival and transition from the pro-B to pre-B cell developmental stage. Indeed, we find increased apoptosis in developing B cells and an associated reduction in pre-B and immature B cell populations in the absence of CD53. Coimmunoprecipitation and proximity ligation studies demonstrate physical interaction between CD53 and IL-7R. Together, these data, to our knowledge, suggest a novel role for CD53 during IL-7 signaling to promote early B cell differentiation.

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, most commonly originating from the transformation of progenitor cells of the B cell lineage (B cell precursor-ALL; BCP-ALL). Treatment of patients with high-risk or relapsed disease is difficult and prognosis remains poor in pediatric patients, with an even worse survival rate for adult BCP-ALL. Previous studies have shown an association of enhanced CD53 expression with many B cell malignancies, suggesting upregulation of CD53 may be implicated in carcinogenesis or maintenance of malignant cells. CD53 is a member of the tetraspanin family of transmembrane proteins, classically involved in cell adhesion, proliferation, and survival, and expressed exclusively on hematopoietic cells. While several studies have implicated a role for CD53 in regulating mature B cell proliferation, its role in early B cell development is not yet known. To elucidate the contribution of CD53 to normal and malignant B cell development, we have generated a CD53 knockout mouse. In our CD53-/- mouse, we observe no differences in total white blood cell counts, yet the fraction of peripheral blood B cells is significantly reduced by 31% compared to wild-type (WT) controls (28.3% vs. 19.5%; p<0.005). During homeostatic B lymphopoiesis, CD53 increases through development, beginning at the pre-pro-B cell stage and reaching highest expression on mature B cells. Further investigation into the loss of B cells revealed that immature pre-B cells in the bone marrow and mature B cells in the spleen and lymph nodes are significantly diminished upon loss of CD53, resulting from increased apoptosis in CD53-/- mice. B cell differentiation of CD53-/- hematopoietic stem cells (HSCs) in vitro corroborates the dependence on CD53 for normal differentiation, as CD53-/- cultures have 26% fewer B cells than controls (p=0.033). Investigation into the signaling differences between WT and CD53-/- B cell progenitors by mass cytometry (CyTOF) suggests that decreased PI3K/Akt and MAPK signaling could be driving this loss. With the observed loss of both B cell progenitors and mature B cells in CD53-deficient mice, CD53-/- mice were recently crossed to Eμ-Myc transgenic mice, a model of B-lineage leukemia/lymphoma, to generate WT, CD53-/-, Eμ-Myc+;CD53+/+, and Eμ-Myc+;CD53-/- groups to assess whether loss of CD53 alters the pathology or survival of these mice. As observed in human patients, moribund Eμ-Myc+ mice significantly upregulate CD53 on malignant cells, suggesting a potential role for CD53 during pathogenesis. Ongoing experiments are aimed at elucidating the mechanism by which CD53 promotes homeostatic B cell development and determining the potential of CD53 as a therapeutic target for B lineage malignancies. Disclosures No relevant conflicts of interest to declare.