The results presented here identify EBAG9 as an important player in the control of secretory lysosome release in CTLs. We not only present what we believe is a novel molecular component of the regulated secretory pathway within the immune system, but also provide evidence for a systemic immunosurveillance function of EBAG9 through genetic deletion in mice.
Apart from the genetic analysis of rare human disease-causing mutations and their corresponding murine mutant strains (3
), little is known about the proteins that mediate protein sorting into and fusion of lytic granules with the plasma membrane in CTL. In Ebag9–/–
mice, we obtained an enhanced release of granzyme A when CTLs were triggered with anti-CD3 antibodies. This observation is consistent with the effect of EBAG9 upon overexpression in PC12 cells, since loss of EBAG9 might lower an inhibitory step in Ca2+
-dependent regulated exocytosis (12
). In contrast, the capacity of EBAG9 to regulate specifically the granzyme A pathway, but not CTLA4 targeting, is in agreement with results from Ashen mice, which have a loss of function in the Rab27a
). The enhanced in vitro release of granzyme A translates into a substantially increased capacity of Ebag9–/–
CTLs to kill allogeneic target cells. Since the frequency of allogeneic CTLs might confound the interpretation of this result, we additionally conducted an antigen-specific in vivo kill assay (32
). CTL cytolytic capacity against syngeneic splenocytes that carry the dominant Tag peptide IV was assessed in primary and memory responses. In agreement with our in vitro results, Ebag9–/–
mice killed their targets more efficiently than did Ebag9+/+
Since effector cell frequencies could affect the interpretation of the cytotoxicity assays (43
), we determined numbers of antigen-specific CTLs during the priming and effector phases using Kb
:Tag peptide dimer staining. Furthermore, we addressed the priming capacity of KO and WT DCs in vitro and in vivo, but a difference between the genotypes was not seen. In conjunction with the comparable proliferation rate of EBAG9-deficient OT-I cells, we suggest that the magnitude of the cytolytic activity in EBAG9-deficient animals is due to an increased secretion of lytic granule content.
A contribution of NK cells to the elimination of peptide-pulsed splenocytes could be excluded, thus we infer that EBAG9 activity may define a discrete secretory mechanism in CTL, which is not found in NK cells. Furthermore, this finding indicates that the precise composition of the exocytosis machinery might vary between hematologic cell types (2
). While NK cells can provide secretory granules upon activation or inhibition of cell surface receptors almost immediately, CTLs require a longer priming and maturation process prior to gaining cytolytic competence (45
). Striking differences between CTLs and NK cells include cell surface and lysosomal proteins involved in antigen presentation, capturing, and internalization, which enable NK cells to act as APCs to CD4+
T cells (47
). Furthermore, differences between primary NK cells and NK cell lines with regard to protein profiles in secretory lysosomes were observed (48
). Thus, it could be envisaged that CTLs and NK cells employ individual vesicular transport pathways to supply secretory lysosomes with cytolytic effector molecules. To our knowledge, a comparative proteomic approach addressing the composition of secretory lysosomes from primary NK cells and defined maturational stages of CTLs has not been demonstrated yet.
The immunological phenotype of EBAG9-deleted mice could be explained mechanistically through the identification of γ2-adaptin as an interaction partner of EBAG9. Despite the structural similarities to the γ1 subunit of the AP1 complex, which itself functions as an adaptor complex for transport carriers between the TGN and endosome, a unique function of γ2-adaptin was identified. To our knowledge, γ2-adaptin–containing AP complexes have not been identified so far. In addition, localization to the cytoplasm and perinuclear structures of the cis-Golgi apparatus as well as localization to CD63+
granules, late endosomes, and multivesicular bodies (MVBs) differed markedly from the localization described for AP1 (7
). Evidence for a sorting function within the endosomal-lysosomal sorting pathway came from virus infection models. For HBV maturation it was shown that γ2-adaptin interacted via its ear domain with the viral L-protein, whereas the head domain recruited the core particle (11
). Thus, γ2-adaptin may organize HBV assembly platforms on endosomal membranes. Functionally, depletion of γ2-adaptin correlated with diminished HBV virion release without compromising constitutive protein secretion. Unexpectedly, it was also shown that γ2 overexpression had the same effect on HBV and retroviral Gag protein release, which was partially explained by imposing a dominant inhibitory effect on endogenous γ2-adaptin (50
). The unique function of γ2-adaptin may rely on its ubiquitin-binding activity, a common feature of monomeric adaptors (e.g., epsin and hepatocyte growth factor–regulated tyrosine kinase substrate), which control ubiquitin-dependent endocytotic sorting processes to the MVB/lysosome. Although differences between cellular physiology and virus infections could be a concern, from the HBV model it was inferred that γ2-adaptin may not act as a typical vesicle-forming adaptor, but functions in connecting HBV production and in the subsequent selection of cargo protein destined for inclusion into coated vesicles (11
In support of a role in the endosomal/MVB sorting pathway, it was shown that γ2-adaptin depletion impaired the degradation of internalized epidermal growth factor and resulted in defective MVB morphology in liver and HeLa cells (51
). More specifically, γ2-adaptin depletion induced an endosomal enlargement. Conversely, in EBAG9-deleted CTLs we obtained reduced secretory lysosome volumes, indicating that EBAG9 could act as a negative modulator on the endosomal transport route. In this scenario, EBAG9 inhibits γ2-adaptin cargo recruitment and sorting functions. As a potential consequence of this association, EBAG9 could restrain the upregulated sorting to the secretory lysosome compartment in CTLs. The absence of EBAG9 would allow enhanced activity of γ2-adaptin in these processes instead.
An interaction between EBAG9 and γ2-adaptin, as revealed by yeast 2-hybrid screening, was confirmed by GST–γ2-adaptin pulldown and coimmunoprecipitation studies. In the pulldown assay, the ear domain was sufficient to recruit EBAG9-GFP, which is consistent with the role of this domain to bind other accessory molecules (rabaptin-5, γ-synergin, enthoprotin, and NECAP1) (52
). Using truncated versions of EBAG9, we identified a sequence between aa 30 and aa 175 as an interaction domain sufficient for γ2-ear domain binding. This sequence was predicted to be largely unstructured and therefore accessible for interactions with γ2-ear domains. In support of a physical interaction, strong colocalization between EBAG9-GFP and γ2-adaptin–HA was seen in a perinuclear region of T cells. This result is in agreement with the colocalization of EBAG9 and the cis
-Golgi marker GM130 under resting conditions (12
) and with the reported colocalization of γ2-adaptin in epithelial cell lines with the cis
-Golgi/intermediate compartment marker Helix pomatia
). Since it was also shown that γ2-adaptin resides together with the HBV L-protein at the Golgi complex, collectively these data support the notion that EBAG9 and γ2-adaptin interact physically and functionally.
An alternative interpretation of the functional role of EBAG9 in endosomal-lysosomal sorting and lysosome-related organelle formation involves an interaction with the BLOC-1 subunits BLOS2 and Snapin, both of which were retrieved in the yeast 2-hybrid assay. BLOC-1 regulates specific cargo-trafficking steps through interactions with other known trafficking regulators, including AP3 and BLOC-2 (53
). Within the secretory pathway, BLOC-1 is required for cargo-specific sorting from early endosomes toward lysosome-related organelles (55
). However, a functional association between EBAG9 and the BLOC-1 complex is difficult to reconcile with our data. BLOC-1 defects, as shown in pallidin
, and sandy
mouse strains, had no functional consequences on CTL cytolytic activity (55
). Thus, it seems unlikely that CTL granules mature in the absence of those subunits while still dependent on Snapin and BLOS2. Furthermore, we failed to validate the interaction with Snapin and BLOS2 in coimmunoprecipitation experiments; in particular, we did not obtain evidence for the retrieval of those molecules within the context of an intact BLOC-1 complex.
EBAG9 localization within cytotoxic T cells is dynamic, as shown by the TCR-induced relocalization toward the immunological synapse. A similar activation-dependent redistribution toward the plasma membrane and the immunological synapse has been shown recently for the Wiskott-Aldrich syndrome protein-interacting protein (56
), which turned out to be essential for lytic granule release in NK cells. We have previously shown that EBAG9, upon nerve growth factor–induced differentiation in neuroendocrine cells, redistributed toward secretory vesicles in the vicinity of the plasma membrane. Since EBAG9 exhibits a palmitoylation anchor and is subject to phosphorylation, we suggest that these modifications contribute to EBAG9 relocalization in T cells as well (12
). The relocalization would also be consistent with the colocalization between EBAG9 and GM130 and the known polarization of the microtubule-organizing center and associated organelles toward the signaling platform at the T cell–target cell contact site. Under activated conditions, such trafficking would position EBAG9-containing structures in proximity to the endosomal-lysosomal compartment. More recently, it could be shown that lytic granule maturation is a stepwise process that involves the hMunc13-4–dependent fusion of Rab11-containing recycling endosomes with Rab27a+
late endosomal vesicles (57
). This step is followed by a separate redistribution of these endosomal exocytic vesicles and lysosomal cytotoxic granules toward the immunological synapse. Colocalization between cytotoxic granules, containing perforin, and the assembled Rab11+
endosomal structures occurred only when the granules were polarized, suggesting that a late step of granule maturation may proceed immediately prior to secretion. Notably, the efficiency of the latter process was strongly dependent on the expression levels of hMunc13-4, with a moderate efficiency in the presence of endogenous hMunc13-4 and a strong correlation when hMunc13-4 was overexpressed. Since EBAG9 in WT CTLs equipped with endogenous hMunc13-4 was found to reside mainly in a synapse compartment separate from polarized cytotoxic granules, these data suggest that the molecule could restrain sorting to the lytic granules from an upstream position.
Polarization of lytic granules toward the immunological synapse was unaltered in Ebag9–/–
mice, and hence vesicle docking and tethering were apparently not influenced. Instead, in support of a pivotal function of EBAG9 in endosomal-lysosomal protein sorting, we found that loss of EBAG9 imposed a change in the steady-state distribution of the target SNARE syntaxin 7, which is required for both homotypic late endosome fusion and heterotypic fusion with lysosomes (58
). In contrast to syntaxin 7, in our subcellular fractionation experiments we observed no relocalization of the plasma membrane SNARE syntaxin 4 or the recycling endosome marker syntaxin 13.
Confocal microscopy analysis revealed a distinct colocalization pattern of lysosomal marker molecules Lamp1, granzyme B, and cathepsin D in EBAG9-deleted and WT CTLs. This suggested that granzyme B is prone to a more efficient trafficking to lysosomes in EBAG9-deficient CTLs. An important distinction between these markers includes their sorting pathways (6
). As an integral lysosomal membrane protein, Lamp1 does not require sorting receptors, since its cytosolic tail interacts directly with multimeric AP proteins, foremost AP3 for lysosomal targeting. In contrast, the acid hydrolases cathepsin D and granzyme B associate with the M6PR for proper delivery from the TGN to endosomes. Of course, the finding of a decreased targeting of Lamp1 to cathepsin D–containing lysosomes in EBAG9-deficient CTLs could be equally interpreted as a decreased targeting of cathepsin D to Lamp1-containing lysosomes. We consider this view to be unlikely, since loss of EBAG9 causes enhanced sorting of the soluble precursor form of cathepsin D to the lysosomal compartment, as shown by pulse-chase and subcellular fractionation analysis. A role for EBAG9 in the proper delivery of secretory lysosome content and lytic granule maturation is further substantiated by an altered vesicle size distribution with smaller granule volumes in EBAG9-deficient CTLs. In agreement with the involvement of EBAG9 in the regulated secretory pathway in CTLs, transit dynamics of secretory proteins upon TCR engagement was found to be enhanced. Taken together, we conclude that EBAG9 tightly cooperates with γ2-adaptin in the sorting of cytolytic effector molecules to secretory lysosomes. Immunologically, this modulation of endosomal-lysosomal trafficking is intimately linked with the cytolytic capacity of activated CTLs.
Finally, estrogen induces gene activation of EBAG9 (22
). Since estrogen receptor expression on CD4+
T cells has been recorded, EBAG9 would be in a position to modulate the cytolytic T cell responsiveness toward antigenic target cells, depending on the prevalent estrogen levels (61
). In view of the suggested immunosurveillance function of CTLs in cancer, the potential therapeutic benefit of estrogen activity inhibition warrants further study.