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The RIP3-RIP1-NF-κB signaling axis is dispensable for necroptotic cells to elicit cross-priming of CD8+ T cells

Cellular & Molecular Immunology volume 14, pages 639–642 (2017)Cite this article

Apoptosis and necroptosis are two types of programmed cell death with distinct morphological features. Necroptosis has been assumed to be more inflammatory than apoptosis, but a recent paper1 concluded that necroptotic cells cannot elicit cross-priming of CD8+ T cells and that NF-κB activation in necroptotic cells is required for maximal CD8+ T-cell cross-priming. In contrast, our data have shown that necroptotic cells are sufficient to elicit robust CD8+ T-cell cross-priming and that NF-κB activation is not required for necroptotic cell-mediated CD8+ T-cell cross-priming. We have concluded that compared with apoptosis, necroptosis is a much more potent promoter of CD8+ T-cell cross-priming.

Necroptosis is a form of programmed cell death with a different morphology and regulatory mechanism from those of the best-characterized form of programmed cell death, apoptosis. Apoptosis is caspase dependent, and its typical morphological features include nuclear condensation, shrinkage and fragmentation of cells into membrane-bound apoptotic bodies. Unlike apoptosis, necroptosis, also called necrosis, is a lytic cell death program. Both apoptosis and necroptosis can be induced by tumor necrosis factor-α (TNF). Upon TNF treatment, TNF receptor 1 trimerizes and recruits several effector proteins, including receptor-interacting protein (RIP) 1, which interacts with caspase-8 via FADD and initiates caspase cascade-mediated apoptosis. However, when cells have a high concentration of RIP3, RIP1 can interact with RIP3 and trigger a necrosis pathway, now termed necroptosis. The RIP1–RIP3 complex, also known as the necrosome (ripoptosome), triggers phosphorylation and activation of an effector pseudokinase, mixed lineage kinase domain-like (MLKL). Upon activation, MLKL multimerizes and translocates to the cell membrane, where it induces cation influx, leading to cell swelling and rupture.2 Oligomerization is a common mechanism to activate cell death mediator proteins. Accordingly, inducible dimerization systems based on FK506 binding domain F36V mutant (Fv) or estrogen-induced homodimerization of hormone-binding domain G521R mutant (HBD*) have been successfully used to initiate apoptosis and necroptosis by inducing caspase-8 or RIP3 oligomerization, respectively.3, 4 MCA205 cells stably transfected with Fv-caspase-8, Fv-RIP3, or Fv-RIP3 RHIM-AAA (QIG449-451AAA mutant) expression vectors (Supplementary Figure S1a) underwent cell death upon AP20187 treatment. Regarding the forms of cell death, caspase-8 oligomerization-induced apoptosis or RIP3 oligomerization-induced necroptosis can be distinguished by Annexin V staining and membrane permeabilization of propidium iodide (PI) (Supplementary Figure S1b), as well as the cleavage of caspase-3/PARP and phosphorylation of MLKL (Supplementary Figure S1c). Apoptosis does not induce inflammation because the cell contents are restricted within the intact plasma membrane during this process. Conversely, necroptosis is lytic cell death and is assumed to stimulate immune responses. Indeed, compared with apoptotic cells, necroptotic MCA205 cells released more damage-associated molecular patterns (DAMPs), such as ATP and chromatin-binding protein high-mobility group B1 (HMGB-1) (Supplementary Figure S1d and e), and promoted more DC maturation (Supplementary Figure S1f) and cross-presentation to CD8+ T cells (Supplementary Figure S1g and h), which results in T-cell-mediated cytotoxicity (Supplementary Figure S1i and j). However, necroptosis may not be more immunogenic than apoptosis in all aspects because unlike apoptosis, there is no surface exposure of calreticulin in necroptotic cells (Supplementary Figure S1k). While our study was under way, a report stated that necroptotic cells were not sufficient for CD8+ T-cell cross-priming.1 Using an approach similar to that used in our study to induce oligomerization of RIP3 and caspase-8, the authors showed that reverse signaling from RIP3 to RIP1 via RHIM domain interaction initiated RIP1-dependent NF-κB activation, which is required for dying cells to elicit maximal CD8+ T-cell cross-priming. The major evidence for this conclusion is that necroptotic cells induced by full-length RIP3 oligomerization but not by a RIP3 C-terminal RHIM domain deleted mutant can elicit cross-priming. We used a RIP3 RHIM-AAA mutant but, intriguingly, did not observe any difference between the mutant and wild-type RIP3 in the induction of ATP and HMGB-1 release, DC maturation, CD8+ T-cell cross-priming and T-cell-mediated cytotoxicity (Supplementary Figure S1d–j). We further analyzed NF-κB activation and IL-6 secretion but did not find any difference between RIP3 and RIP3 RHIM-AAA mutant-mediated necroptotic cells (Supplementary Figure S2a and b).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (91029304, 31420103910, 31330047 and 81630042), the National Basic Research Program of China (973 Program; 2015CB553800, 2013CB944903 and 2014CB541804), the 111 Project (B12001), and the National Science Foundation of China for Fostering Talents in Basic Research (J1310027).

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  1. State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China

    Junming Ren, Xian Jia, Yihao Zhao, Wenke Shi, Jiongcong Lu, Yingying Zhang, Jianfeng Wu, Bo Liang, Rui Wu, Guo Fu & Jiahuai Han

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Ren, J., Jia, X., Zhao, Y. et al. The RIP3-RIP1-NF-κB signaling axis is dispensable for necroptotic cells to elicit cross-priming of CD8+ T cells. Cell Mol Immunol 14, 639–642 (2017). https://doi.org/10.1038/cmi.2017.31

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