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.

Figure 1

Effect of FcμR on Fas-mediated apoptosis in Jurkat cells. (A) Jurkat cells transduced with the bicistronic retroviral construct containing both human FcμR and GFP cDNAs (FcμR/GFP) or only GFP cDNA (GFP) as a control were incubated with biotin-labeled, anti-FcμR/Toso mAbs (HM7 [γ2bκ], HM14 [γ1κ] or 1E4 [γ1κ; Abnoca Corp]) or isotype-matched control mAbs (γ2bκ or γ1κ) as described.2,4,5  The bound mAbs were detected by addition of phycoerythrin-labeled streptavidin (PE-SA). For IgM binding, cells were incubated with myeloma or hybridoma-derived IgM of both human and mouse origin at 10 μg/mL, washed, and then incubated with biotin-labeled, rat anti–mouse μ mAb (clone 332.12) for mouse IgM or mouse anti–human μ mAb (clone SA-DA4.4) for human IgM. Biotin-labeled, control mAbs of the same isotype (rat γ2bκ or mouse γ1κ) were also included. Stained cells were analyzed by Accuri C6 Flow Cytometer. Because all staining profiles with control mAbs were indistinguishable, only the result with biotin-labeled mouse IgG1 control mAb is shown for simplicity. Note the expression of FcμR/Toso on FcμR/GFP transductants as determined by reactivity with 3 different mAbs and by IgM-ligand binding. (Only the binding result with mouse IgMλ hybridoma [B1–8; anti-nitrophenyl antibody] is shown.) (B) Both cell lines were cultured at 37°C for 12 (top panel) and 20 hours (bottom panel) without or with agonistic mouse anti–human Fas mAb of IgMκ (CH11; 10 ng/mL; Millipore) or IgG3κ isotype (2R2; 1μg/mL; Invitrogen) or with a recombinant human FasL (superFasL; Enzo Life Sciences) at 3 different protein concentrations (1, 10 and 100 ng/mL). Cells were stained with 7-aminoactinomycin D (7-AAD) and allophycocyanin (APC)–labeled annexin V to identify early (annexin V+/7-AAD) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V/7-AAD+) cells by flow cytometry.2  Note the resistance of the FcμR/GFP transductant to Fas-mediated apoptosis by IgM mAb, but not by IgG3 mAb and FasL. These experiments were performed 3 times with essentially the same results.

Figure 1

Effect of FcμR on Fas-mediated apoptosis in Jurkat cells. (A) Jurkat cells transduced with the bicistronic retroviral construct containing both human FcμR and GFP cDNAs (FcμR/GFP) or only GFP cDNA (GFP) as a control were incubated with biotin-labeled, anti-FcμR/Toso mAbs (HM7 [γ2bκ], HM14 [γ1κ] or 1E4 [γ1κ; Abnoca Corp]) or isotype-matched control mAbs (γ2bκ or γ1κ) as described.2,4,5  The bound mAbs were detected by addition of phycoerythrin-labeled streptavidin (PE-SA). For IgM binding, cells were incubated with myeloma or hybridoma-derived IgM of both human and mouse origin at 10 μg/mL, washed, and then incubated with biotin-labeled, rat anti–mouse μ mAb (clone 332.12) for mouse IgM or mouse anti–human μ mAb (clone SA-DA4.4) for human IgM. Biotin-labeled, control mAbs of the same isotype (rat γ2bκ or mouse γ1κ) were also included. Stained cells were analyzed by Accuri C6 Flow Cytometer. Because all staining profiles with control mAbs were indistinguishable, only the result with biotin-labeled mouse IgG1 control mAb is shown for simplicity. Note the expression of FcμR/Toso on FcμR/GFP transductants as determined by reactivity with 3 different mAbs and by IgM-ligand binding. (Only the binding result with mouse IgMλ hybridoma [B1–8; anti-nitrophenyl antibody] is shown.) (B) Both cell lines were cultured at 37°C for 12 (top panel) and 20 hours (bottom panel) without or with agonistic mouse anti–human Fas mAb of IgMκ (CH11; 10 ng/mL; Millipore) or IgG3κ isotype (2R2; 1μg/mL; Invitrogen) or with a recombinant human FasL (superFasL; Enzo Life Sciences) at 3 different protein concentrations (1, 10 and 100 ng/mL). Cells were stained with 7-aminoactinomycin D (7-AAD) and allophycocyanin (APC)–labeled annexin V to identify early (annexin V+/7-AAD) and late (annexin V+/7-AAD+) apoptotic and dead (annexin V/7-AAD+) cells by flow cytometry.2  Note the resistance of the FcμR/GFP transductant to Fas-mediated apoptosis by IgM mAb, but not by IgG3 mAb and FasL. These experiments were performed 3 times with essentially the same results.

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Acknowledgments: The authors thank Drs John F. Kearney and Peter D. Burrows for their helpful comments.

This work was supported by National Institute of Allergy and Infectious Diseases/National Institutes of Health grants AI42127 and AI82249 (to H.K.).

Contribution: K.H. and Y.K. performed research; and H.K. designed research and wrote the paper.

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

Correspondence: Hiromi Kubagawa, MD, Shelby 506, 1825 University Blvd, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294; e-mail: hiromikubagawa@uab.edu.

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