We appreciate the interest in our work. In their comment Honjo et al argue that Toso per se would not have antiapoptotic activity on CD95-induced apoptosis,1  as, in contrast to our findings,2  inhibitory activity would only be observed upon on CD95-ligation by the IgM mAb CH11. Our conclusion that Toso exhibits antiapoptotic effects on death receptor signaling is based on multiple lines of experimental evidence. Knockdown of Toso renders cells more susceptible to CD95L-induced apoptosis, while overexpression of Toso results in decreased apoptosis in response to stimulation with CD95L, the natural ligand for CD95. Toso also provides relative protection from TNFα-induced apoptosis and necrosis and TRAIL-induced cell death, suggesting that Toso serves as a general regulator of death receptor signaling. Furthermore, the inhibitory function of Toso in death receptor-induced apoptosis was not only restricted to the human system, but was also observed in experiments using cells from Toso knockout mice. Thus, while Honjo et al draw their conclusion solely from an overexpression study in a single cell type,1  we have confirmed our findings in several different cell types and different species, including Jurkat cells, BJAB cells and human and mouse primary T cells.2  Importantly, the observation of an antiapoptotic function of Toso is further supported by independent studies that also explored apoptosis induced by physiologic death receptor ligands. Song and Jacob demonstrated that overexpression of Toso protects Jurkat cells from CD95L- and TNFα- induced apoptosis and that primary T cells from Toso transgenic mice are relatively resistant to CD95L-induced apoptosis.3  Also, consistent with our results, Richter et al reported that knock-down of Toso increases CD95L-mediated apoptosis in primary human T cells.4  As an additional note, while the specific Jurkat cell line used by Honjo et al1  seems to be unusually sensitive to CD95L-induced apoptosis (≥ 95% apoptotic cells after only 6 hours of stimulation with 10 ng/mL CD95L), careful analysis of their data presented in Figure 1B still reveals that in response to CD95L-stimulation Toso expressing cells are relatively protected from late apoptosis (there are considerably less late apoptotic cells [ = annexinV+7-AAD+ cells] in CD95L-stimulated Toso overexpressing cells compared with control cells). Finally, there is not only ample experimental evidence for an antiapoptotic function of Toso in death-receptor biology, but, by the identification of the Toso-RIP1 interaction and the dependence on RIP1 ubiquitination, we also provide a mechanistic basis for the observed antiapoptotic phenotype. In their letter, Honjo et al also provide additional data to support their earlier conclusion5  that Toso serves as a receptor for IgM. A potential binding of IgM to Toso is of high interest to us, however, despite of using several different experimental models with human and mouse Toso, so far we have not been able to experimentally confirm such an interaction (see supplemental Figures 11-12 in Nguyen et al2 ). The reason for these divergent results is currently unclear, but we agree with Honjo et al that additional ligands for Toso may exist. The identification and characterization of novel physiologic Toso ligands would certainly help to better understand Toso biology.

Authorship

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Kyeong-Hee Lee, Research Center Borstel, Leibriz Center for Medicine and Biosciences, Division of Molecular Immunology, Parkallee 22, 23845 Borstel, Germany; e-mail: klee@lz-borstel.de.

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