Is Toso an antiapoptotic protein or an Fc receptor for IgM?

To the editor:

We have read with great interest the publication by Nguyen et al entitled “Toso regulates the balance between apoptotic and nonapoptotic death receptor signaling by facilitating RIP1 ubiquitination.”¹  Because there are important discrepancies between their results¹  and ours²  with regard to the ligand specificity of the cell-surface Toso protein, we wish to provide comments and some new findings related to this issue. Toso, named after a Japanese liquor drunk on New Year's day to celebrate long life and eternal youth, was originally designated as an inhibitor of Fas/CD95-induced apoptosis and was also called the Fas apoptotic inhibitory molecule 3.³  In this originally described apoptosis assay, a mouse monoclonal antibody (mAb) of the IgM isotype (CH11) was used for ligation of Fas. Independently, we cloned a cDNA encoding an IgM Fc receptor (FcμR) based on an IgM binding strategy from cDNA libraries generated from human B-lineage cells including chronic lymphocytic leukemia (CLL) cells.²  The nucleotide sequence of this FcμR was identical to that of Toso. To determine whether the FcμR inhibits Fas-mediated apoptosis, the apoptosis-prone Jurkat human T-cell line was transduced with bicistronic retroviral constructs containing both FcμR and green fluorescent protein (GFP) cDNAs or the GFP cDNA alone as a control. Cells expressing comparable levels of GFP were enriched from each transductant by FACS and were used in apoptosis assays. The recombinant Fas-ligand (FasL) used by Nguyen et al¹  as well as 2 agonistic Fas-specific mAbs (CH11 [μκ] and 2R2 [γ3κ]) were then used to induce apoptosis of the transductants. The results are shown in Figure 1 and are clearly quite distinct from those reported by Nguyen et al. First, FcμR⁺/GFP⁺, but not GFP⁺, cells display IgM binding and are reactive with 3 different mAbs (our HM7 [γ2bκ] and HM14 [γ1κ] anti-FcμR mAbs²  and 1E4 [γ1κ] anti-Toso mAb⁴ ; Figure 1A), confirming our previous results.^2,5  Furthermore, Vire et al⁶  have also recently described a high level of Toso expression on CLL-B cells together with IgM binding and subsequent internalization; results also consistent with our recent findings.⁵  Thus, the lack of IgM binding to Toso reported by the Nguyen group apparently resulted from their usage of transient Toso-transductants, which may not have expressed sufficient levels of Toso to detect IgM binding (see supplemental Figure 12 in Nguyen et al¹ ). (By contrast, their functional studies were conducted with stable Toso-transductants.) Second, antiapoptotic activity of Toso/FcμR is observed only when the Fas receptor is ligated by the IgM mAb (CH11), but not when ligated by an IgG3 mAb (2R2) or the native FasL, indicating that Toso/FcμR per se has no inhibitory activity in Fas-mediated apoptosis (Figure 1B). Our results are thus quite different from those of Nguyen, et al who showed that Toso expression on Jurkat cells inhibits FasL-induced apoptosis (see their supplemental Figure 4). The basis for these discrepancies remains unclear and there is a possibility that Toso/FcμR may interact with an additional protein/ligand. An exchange of reagents including Toso/FcμR-transductants would facilitate the resolution of these conflicting data.