PD Dr. Dirk Alexander Mielenz
Abteilung für Molekulare Immunologie
Medizinische Klinik III
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.
Finger, Y., Habich, M., Gerlich, S., Urbanczyk, S., van de Logt, E., Koch, J., Schu, L., Lapacz, K.J., Ali, M., Petrungaro, C., …Mielenz, D…, et al. (2020). Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import. EMBO J., e103889.
Reimer, D., Meyer-Hermann, M., Rakhymzhan, A., Steinmetz, T., Tripal, P., Thomas, J., Boettcher, M., Mougiakakos, D., Schulz, S.R., Urbanczyk, S., ..., Niesner, R*., and Mielenz, D*. (2020). B Cell Speed and B-FDC Contacts in Germinal Centers Determine Plasma Cell Output via Swiprosin-1/EFhd2. Cell Rep. 32, 108030.
Schuh, W., Mielenz, D., and Jäck, H.M. (2020). Unraveling the mysteries of plasma cells. Adv. Immunol. 146, 57-107.
Cossarizza, A., Chang, H.D., Radbruch, A., Acs, A., Adam, D., Adam-Klages, S., Agace, W.W., Aghaeepour, N., Akdis, M., Allez, M.,Urbanczyk, S, ...Mielenz, D. …, et al. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur. J. Immunol. 49, 1457-1973.
Urbanczyk, S., Stein, M., Schuh, W., Jäck, H.M., Mougiakakos, D., and Mielenz, D. (2018). Regulation of Energy Metabolism during Early B Lymphocyte Development. Int. J. Mol. Sci. 19, 2192.
Lee, A.Y.S., Reimer, D., Zehrer, A., Lu, M., Mielenz, D.*, and Körner, H*. (2017). Expression of Membrane-Bound CC Chemokine Ligand 20 on Follicular T Helper Cells in T-B-Cell Conjugates. Front. Immunol. 8, 1871.
Pracht, K., Meinzinger, J., Daum, P., Schulz, S.R., Reimer, D., Hauke, M., Roth, E., Mielenz, D., Berek, C., Corte-Real, J., et al. (2017). A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry. Eur. J. Immunol. 47, 1389-1392.
Reimer, D., Lee, A.Y., Bannan, J., Fromm, P., Kara, E.E., Comerford, I., McColl, S., Wiede, F., Mielenz, D.*, and Körner, H.* (2017). Early CCR6 expression on B cells modulates germinal centre kinetics and efficient antibody responses. Immunol. Cell Biol. 95, 33-41.
Stein, M., Dütting, S., Mougiakakos, D., Bosl, M., Fritsch, K., Reimer, D., Urbanczyk, S., Steinmetz, T., Schuh, W., Bozec, A., and Mielenz, D. (2017). A defined metabolic state in pre B cells governs B-cell development and is counterbalanced by Swiprosin-2/EFhd1. Cell Death Differ. 24, 1239-1252.
Wiesmann, V.,Reimer, D., Franz, D., Mielenz, D.,andWittenberg, T. (2015). Automated high-throughput analysisofBcell spreadingonimmobilizedantibodies withwhole slideimaging. Current Directions in Biomedical Engineering, 1:224-227
Fürnrohr, BG, Stein, M, Rhodes, B, Chana, PS, Schett, G, Vyse, TJ, Herrmann, M*, Mielenz, D*. (2015). Reduced fluorescence versus scatter ToF and increased peak versus integral fluorescence ratios indicate surface receptor clustering. J. Immunol, 195(1):377-85, *authors contributed equally