The tumor-associated antigen EBAG9 negatively regulates the cytolytic capacity of mouse CD8+ T cells
Constantin Rüder, … , Bernd Dörken, Armin Rehm
Constantin Rüder, … , Bernd Dörken, Armin Rehm
Published July 20, 2009
Citation Information: J Clin Invest. 2009;119(8):2184-2203. https://doi.org/10.1172/JCI37760.
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Research Article

The tumor-associated antigen EBAG9 negatively regulates the cytolytic capacity of mouse CD8+ T cells

Abstract

CTLs eliminate virus-infected and tumorigenic cells through exocytosis of cytotoxic agents from lytic granules. While insights into the intracellular mechanisms facilitating lytic granule release have been obtained through analysis of loss-of-function mutations in humans and mice, there is a paucity of information on negative regulators of secretory lysosome release at the molecular level. By generating and analyzing estrogen receptor–binding fragment-associated antigen 9–KO (Ebag9 KO) mice, we show here that loss of EBAG9 confers CTLs with enhanced cytolytic capacity in vitro and in vivo. Although loss of EBAG9 did not affect lymphocyte development, it led to an increase in CTL secretion of granzyme A, a marker of lytic granules. This resulted in increased cytotoxicity in vitro and an enhanced cytolytic primary and memory T cell response in vivo. We further found that EBAG9 interacts with the adaptor molecule γ2-adaptin, suggesting EBAG9 is involved in endosomal-lysosomal biogenesis and membrane fusion. Indeed, granzyme B was sorted to secretory lysosomes more efficiently in EBAG9-deficient CTLs than it was in WT CTLs, a finding consistent with the observed enhanced kinetics of cathepsin D proteolytic processing. While EBAG9 deficiency did not disrupt the formation of the immunological synapse, lytic granules in Ebag9–/– CTLs were smaller than in WT CTLs. These data suggest that EBAG9 is a tunable inhibitor of CTL-mediated adaptive immune response functions.

Authors

Constantin Rüder, Uta E. Höpken, Jana Wolf, Hans-Willi Mittrücker, Boris Engels, Bettina Erdmann, Susanne Wollenzin, Wolfgang Uckert, Bernd Dörken, Armin Rehm

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Figure 4

Priming and expansion of antigen-specific CD8+ T cells.

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Priming and expansion of antigen-specific CD8+ T cells. (A) Frequenc...
(A) Frequency of antigen-specific CTLs is unaltered in Ebag9–/– mice. Mice were immunized as described in Figure 3A, and splenocytes (on day 4) were cultured in the presence of Tag peptide (1 μg/ml) for another 4 days. Cells were stained with anti-CD8a mAb and H-2Kb:Ig dimers loaded with Tag peptide, followed by flow cytometry analysis. Shown is the percentage of Tag dimer–positive CD8+ T cells. n = 7 Ebag9+/+ (filled squares) and n = 8 Ebag9–/– mice (open triangles). Data are from 2 independent experiments. (B) For intracellular IFN-γ staining, cells were stimulated as described in A and stained as described in Methods. The percentage of IFN-γ–positive CD8+ cells at day 8 is shown (n = 4 animals per group; data are from 2 independent experiments). (C) The proliferative capacity of Ebag9–/– Tag-specific CD8+ T cells was unaltered. Mice were immunized as described above, followed by in vitro culture of splenocytes (on day 4) in the presence of Tag peptide for another 4 days. For the last 24 hours, 10 μM BrdU was included in the culture medium. Splenocytes were surface stained with H-2Kb dimers and CD8a, followed by intracellular anti-BrdU staining. The percentage of proliferating cells was assessed by flow cytometry. (D) As described in C, the proliferative rate (percentage) of CD8+IFN-γ+ effector T cells was assessed by intracellular BrdU staining. Horizontal bars indicate the mean value.