NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated and relocated to the cutoplasm by a network of several kinases including CK-1, GSK-3 and DYRK. Although identified originally as a major transcriptional regulator in T cells, it is now clear that NFAT transcription factors also possess important roles in other cells of the hematopoietic system including dendritic cells, mast cells, megakaryocytes and B cells.
Here we have analyzed the role of NFAT2 in B cell development. Analysis of the role of this family member in hematopoiesis has been complicated by the fact that deletion of this gene is embryonic lethal around ED 13 because of defects in heart valve development. To circumvent this problem we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice, in which the Cre recombinase is expressed under the control of the B cell-specific cd19 promoter.
B cells from these mice were isolated using CD19-labeled magnetic beads and subjected to analysis by flow cytometry. While CD19+ splenocytes from conditional NFAT2 knock-out mice occurred in normal numbers, these cells showed significantly reduced expression of CD38 and ZAP70 upon stimulation with anti-IgM antibody as compared to CD19+ splenocytes from wild-type controls. The reduction of these proteins could also be detected in B cells isolated from the peripheral blood and from bone marrow and was confirmed by western blotting and quantitative RT-PCR. CD38 and ZAP70 are well characterized prognostic factors in chronic lymphocytic leukemia (CLL) and their increased expression has been shown to correlate with poor patient survival. Our data indicate that the expression of these markers is at least in part regulated by Ca2+/NFAT signaling and that deregulation of this pathway can contribute to their overexpression in disease.
Next we analyzed bone marrow and peritoneal lavages from conditional NFAT knock-out mice by flow cytometry. While we found no significant differences in the abundance of B cell subpopulations in bone marrow, we detected an almost complete absence of CD5+CD43+ B1a cells in the peritoneal cavity, clearly demonstrating the requirement of NFAT2 in the development of this subclass. B1a cells are a phenotypically and functionally distinct population of B cells which are long-lived and typically express CD5, CD43 and high levels of surface IgM together with low surface IgD and CD45 (B220). A human B cell equivalent of the murine B1a cell has been suggested as the leukemic precursor cell in chronic lymphocytic leukemia (CLL). To further delineate the role of NFAT2 in the development of B1a cells we determined the abundance of B1 progenitor cells (B1P) in bone marrow and spleen by FACS analysis. In NFAT2 knock-out mice we observed a significant reduction of the frequency of B220- CD19+ CD93+ B1P cells in bone marrow (0.8% vs. 4.7%) and spleen (0.16% vs. 0.82%) demonstrating that NFAT2 is essential for normal development of this precursor cell population.
In summary, our data provide strong evidence that NFAT2 is critical for the expression of CD38 and ZAP70 in B cells and substantially controls B1a cell homeostasis implicating Ca2+/NFAT signaling as a potential target for the treatment of CLL.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.