The functions of EFhd1 and EFhd2 during checkpoints of B cell differentiation

The pre-B and germinal center (GC) B cell checkpoints (Fig. 1) control specific humoral immunity but they are not yet fully understood. Both checkpoints have in common the principle of an expansion of B cell clones expressing an appropriate Ig(m) heavy chain. In other words, both checkpoints are involved in the selection of the B cell receptor repertoire, that is, the entity of different BCRs expressed in the whole body. The pre BCR checkpoint shapes the primary repertoire whereas the GC checkpoint shapes the secondary repertoire. Metabolic changes, cell migration and signaling mediated by the second messenger Ca²⁺ play important roles during these processes, implicating a coordinated expression of signaling molecules. Therefore, understanding the function of signaling proteins enables understanding of complex processes such as the pre-BCR or GC checkpoints..

Pre B cell checkpoint / EFhd1:

In the previous funding period, we have been working on these checkpoints using transgenic and knock-out mouse models of two homologous Ca²⁺ binding adaptor proteins, Swiprosin-1/EFhd2 (EFhd2) and Swiprosin-2/EFhd1 (EFhd1). We have shown that EFhd1 is expressed in pro B cells, but becomes down-regulated by surface expression of the pre BCR (Fig. 1). While the abundance of EFhd1 in pro B cells limits their glycolysis (shown for the pro-B cell line 38B9), its down-regulation by the surface pre-BCR enables proper mitochondrial function in primary pre-B cells (Stein et al., 2017), suggesting that EFhd1 acts as a brake for aerobic glycolysis driving large pre-B cell expansion. In line, we have shown that down-regulation of EFhd1 by the pre-BCR fosters early B cell development at the pre-B cell checkpoint. The exact function of EFhd1 in pro-B cells, at the pre-B cell checkpoint and in later stages, is not clear. EFhd1 resides in the inner mitochondrial membrane and transmits Ca²⁺ currents into mitochondrial flashes (“mitoPHlashes”; Rosselin et al, EMBO Rep., 2017; Hou et al., Cell Calcium, 2016). We will therefore focus on the function of EFhd1 in B cell development and differentiation through analysis of B cell specific EFhd1-deficient mice with an emphasis on mitochondrial and glycolytic pathways. These experiments will help to understand metabolic requirements of pro- and pre-B cells.

 

GC checkpoint / EFhd2:

We have furthermore shown that knock-out of the homologues protein EFhd2 increases GC reactions and type 2 immunity in response to infection with the helminth Nippostrongylus brasiliensis in a B cell intrinsic manner (Brachs et al., 2014). EFhd2 is a cytoskeletal protein and controls activity of the actin remodeling protein cofilin after Ca²⁺ binding and dimerization (Mielenz & Gunn-Moore, Biochem J., 2016). We therefore hypothesize that the regulation of type 2 immunity by EFhd2 is linked to the GC checkpoint as well as to cytoskeleton associated processes. To better understand this connection, we will characterize the function of EFhd2 in the B cell cytoskeleton and finally in GC dynamics, especially with regard to B cell migration within germinal centers. EFhd2 is a STAT6 target gene (Mokada-Gopal et al., J. Immunol., 2017) and obviously a negative regulator of STAT6 dependent Th2 type GC reactions (Brachs et al., 2014). We envision that up-regulation of EFhd2 by STAT6 constitutes a negative feedback loop controlling GC reactions through cytoskeletal regulation. In accordance, EFhd2 expression is up-regulated in GC B cells (Fig. 1). Understanding this pathway will especially help understanding STAT6-dependent immunity like allergy and immune reactions towards parasites.