Bergmann, Hannes2016-12-05b40393525http://hdl.handle.net/1885/111074Unknown mechanisms that cause failures in B cell development can lead to humoral immunodeficiencies, autoimmune disorders or B cell malignancies. This thesis demonstrates that the endosomal intramembrane cleaving protease signal-peptide-peptidase-like 2A (SPPL2A) is critical for survival of developing B cells and reveals SPPL2A-deficiency as a previously unknown mechanism of B cell loss. Mice with an ENU-mutagenesis-induced inactivating mutation in Sppl2a exhibited profound humoral immunodeficiency and specifically lacked mature B cells, phenotypically mirroring BAFF-deficient mice. Surviving B cells were characterised by abnormal endosomal membrane expansions and low surface BAFFR and BCR expression. Mature B cells, but not BAFFR and BCR surface levels, could be rescued by over-expression of the pro-survival protein BCL2, indicating that SPPL2A-deficient B cells failed to mature due to a survival defect rather than developmental arrest. On a molecular level SPPL2A-deficiency blocked the ultimate intramembrane proteolytic processing step of CD74 (MHCII invariant chain), causing a dramatic build-up of residual 8kD CD74 (p8-CD74) membrane stubs leading to endosomal enlargement. The accumulation of p8-CD74 was responsible for the loss of mature B cells, as inhibition of p8-CD74 build-up by deletion of CD74 in SPPL2A-deficient animals reconstituted mature B cell numbers to levels seen in mice solely deficient in CD74, and restored surface BCR and BAFFR expression on B cells. Interestingly, endosomal enlargement has been similarly observed in B cells from Cathepsin S-deficient mice that also accumulate a CD74 degradation product, p10-CD74. Despite more severe endosomal expansion in these B cells, they did not exhibit survival abnormalities. This finding illustrates the distinct capabilities of p10-CD74 and p8-CD74 to regulate endosomal biology and B cell survival. Despite its role in CD74 metabolism, SPPL2A appeared disconnected from the classical sequence of endosomal CD74 degradation steps, as p8-CD74 production required neither penultimate processing by Cathepsin S nor MHCII-chaperoning. Additionally, it was found that p8-CD74 already accumulated in immature B cells from the bone marrow of SPPL2A-deficient mice, which contrasted with Cathepsin S-deficient mice where CD74 accumulation first occurred in transitional B cells. This indicates that Cathepsin S processes CD74 later than SPPL2A during B cell development. p8-CD74 processing by SPPL2A was logically expected to play a role in other CD74-expressing immune cells. However, only CD8- dendritic cell (DC) numbers were reduced in SPPL2A-deficient mice, whilst CD8+ DC and pDC subsets, as well as macrophages and monocytes, appeared to survive. CD8- DCs showed elevated surface MHCII levels, whereas CD8+ DCs were characterised by an MHCII-chaperoning-dependent 10-fold increase in CD74 surface expression, highlighting differences in the wiring of MHCII and CD74 trafficking responses in these DC subsets. Functionally, SPPL2A-inactivation interfered with the ability of spleen DCs to present antigen on MHCII to CD4+ T cells, but not with MHCI associated cross-presentation. In summary, these findings reveal that SPPL2A mediated CD74 intramembrane proteolysis is a previously unknown mechanism required for humoral immunity, and survival of developing B cells and CD8- DCs. Targeting SPPL2A or CD74 may provide new therapeutic avenues for the control of endosome and B cell related diseases in the future.enB cell developmentdendritic cell developmentSPPL2ACD74invariant chainMHCIICathepsin Santigen presentationMIICendosomeintramembrane proteolysisRIPi-CLiPsmembrane protein degradationstorage diseasemultilamellar bodies201610.25911/5d7634cebddd6