NF-κB and Extrinsic Cell Death Pathways – Entwined Do-or-Die Decisions for T cells
Authors: Sam Blanchett, Ines Boal-Carvalho, Scott Layzell, Benedict Seddon
Journal: Trends in Immunology (2020)

Kinases such as IKK serve dual functions in activating NF-κB and in repressing cell death pathways independently of NF-κB activation.Protection of mature thymocytes from TNF-induced cell death appears to be exclusively mediated by direct repression of RIPK1 by the IKK complex, and not by NF-κB activation.The development and homeostasis of conventional and regulatory T cells (Tregs) relies on signaling via distinct TNF receptor superfamily (TNFRSF) members that activate specific NF-κB dimers.Naïve T cell survival is predominantly mediated by RelA and by TNFRSF members including TNFR1 and CD27.Based on mouse studies, Tregs require c-Rel for development and RelA for functional competence. TCR, TNFR2, GITR, and OX40 receptors are key triggers of NF-κB activity in Tregs.Cell death and NF-κB signaling pathways are under developmental regulation in T cells at the level of RIPK1 and MLKL expression; this determines how and when signals are delivered during T cell ontogeny, and what modes of cell death can be triggered. NF-κB signaling is required at multiple stages of T cell development and function. The NF-κB pathway integrates signals from many receptors and involves diverse adapters and kinases. Recent advances demonstrate that kinases controlling NF-κB activation, such as the IKK complex, serve dual independent functions because they also control cell death checkpoints. Survival functions previously attributed to NF-κB are in fact mediated by these upstream kinases by novel mechanisms. This new understanding has led to a refined view of how NF-κB and cell death signaling are interlinked and how they regulate cell fate. We discuss how NF-κB activation and control of cell death signaling by common upstream triggers cooperate to regulate different aspects of T cell development and function. NF-κB signaling is required at multiple stages of T cell development and function. The NF-κB pathway integrates signals from many receptors and involves diverse adapters and kinases. Recent advances demonstrate that kinases controlling NF-κB activation, such as the IKK complex, serve dual independent functions because they also control cell death checkpoints. Survival functions previously attributed to NF-κB are in fact mediated by these upstream kinases by novel mechanisms. This new understanding has led to a refined view of how NF-κB and cell death signaling are interlinked and how they regulate cell fate. We discuss how NF-κB activation and control of cell death signaling by common upstream triggers cooperate to regulate different aspects of T cell development and function. There has been considerable interest over the years in understanding the role of NF-κB signaling in the development, function, and homeostasis of vertebrate T cells. The pathways of NF-κB activation are highly complex. There are a multitude of surface receptors that can activate NF-κB through one of two distinct pathways, and activate transcription factor dimers from a family of five different monomers. The two pathways of NF-κB activation and how surface receptors connect to them are described in detail in Figure I in Box 1. Given this complexity, it is perhaps unsurprising to find that NF-κB mediates functions that vary between different T cell subsets, and between different stages of differentiation in the thymus and periphery. Recent studies reveal that different types of NF-κB dimers have specific roles in the development of naïve, activated, and regulatory (Treg) T cells. Furthermore, other studies show that the kinases responsible for directly triggering NF-κB activity in fact have additional substrates that are involved in controlling cell death checkpoints [1.Dondelinger Y. et al.NF-kappaB-independent role of IKKalpha/IKKbeta in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar]. These recent advances have led to an understanding that not all NF-κB dimers are equal, and have identified a new and previously unappreciated function of death signaling in controlling T cell homeostasis and development – functions mistakenly attributed to NF-κB in the past. This review discusses how the parallel activities of NF-κB and death signaling control T cell development, tune the long-term survival of naïve T cells, guide the development and function of Tregs, and expose activated T cells to alternative modes of cell death.Box 1NF-κB: A Tale of Two Pathways and Many TriggersThe NF-κB family of transcription factors are hetero- or homodimers of Rel family member proteins, of which there are five – RelA, c-Rel, p50, RelB, and p52 – that are conserved between mouse and human. The genes Rela, Rel, and Relb encode RelA, c-Rel, and RelB proteins, respectively, while p50 and p52 are cleavage products of p105 and p100 proteins that are encoded by the Nfkb1 and Nfkb2 genes. A family of inhibitory proteins, the inhibitors of κB (IκB) that include p105 and p100 [84.Savinova O.V. et al.The Nfkb1 and Nfkb2 proteins p105 and p100 function as the core of high-molecular-weight heterogeneous complexes.Mol. Cell. 2009; 34: 591-602Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar], bind to Rel dimers and sequester them in the cytoplasm. Release of NF-κB dimers is regulated in much the same way in mouse and human. The IKK complex, a trimeric complex of two kinases, IKK1 (IKKα) and IKK2 (IKKβ) plus a regulatory subunit Nemo (IKKγ), phosphorylates IκB (P in Figure I), resulting in its degradation by the proteasome and permitting NF-κB dimers to enter the nucleus. This pathway regulates activation of RelA:p50 and c-Rel:p50 NF-κB dimers sequestered by IκBα or p105, and is termed the canonical NF-κB pathway (Figure I). The activity of RelB:p52 dimers is instead regulated by the so-called alternative NF-κB pathway. Dimers are sequestered specifically by p100 protein whose phosphorylation and degradation are specifically controlled by an IKK complex composed of an IKK1 homodimer. Activation of this IKK1 complex is instead triggered by NF-κB-inducing kinase (NIK).Surface receptors connect with canonical NF-κB pathways by various mechanisms. However, all have in common the recruitment of E3 ubiquitin ligases that allow the creation of a ubiquitin scaffold that is essential for the recruitment and activation of the TAB1/2/TAK1 complex, in turn activating the IKK complex. Following T cell receptor triggering, PKCθ phosphorylates Card11, allowing further recruitment of BCL10 and MALT1 molecules to form the large multimeric CBM complex (reviewed in detail in [85.Lork M. et al.Ubiquitination and phosphorylation of the CARD11–BCL10–MALT1 signalosome in T cells.Cell. Immunol. 2019; 340103877Crossref PubMed Scopus (16) Google Scholar]). Recruitment of the E3 ubiquitin ligase, tumor necrosis factor receptor (TNFR)-associated factor (TRAF)-6, results in the creation of the ubiquitin scaffold. By contrast, many TNFRSF proteins have TRAF-interacting motifs that can directly recruit TRAF proteins. In addition to TRAF6, TRAF 2, 3, and 5 also have E3 ubiquitin ligase activity [86.Yang X.-D. Sun S.-C. Targeting signaling factors for degradation, an emerging mechanism for TRAF functions.Immunol. Rev. 2015; 266: 56-71Crossref PubMed Scopus (58) Google Scholar] that creates the ubiquitin scaffold necessary to recruit TAB1/2/TAK1 and IKK complexes. Other TNFRSF members, such as TNFR1 and DR3, lack TRAF-interacting motifs and instead have death domains that allow the recruitment of adapters such as TNFR1-associated death domain protein (TRADD), that in turn can bind to and recruit TRAFs. By contrast, activation of the alternative pathway results from stabilization of NIK protein following inhibition of its degradation by TRAF2/3. In mouse T cells, expression of both Nfkb2 and Relb is dependent upon intact canonical signaling [24.Webb L.V. et al.Survival of single positive thymocytes depends upon developmental control of RIPK1 kinase signaling by the IKK complex independent of NF-κB.Immunity. 2019; 50: 348-361Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. The NF-κB family of transcription factors are hetero- or homodimers of Rel family member proteins, of which there are five – RelA, c-Rel, p50, RelB, and p52 – that are conserved between mouse and human. The genes Rela, Rel, and Relb encode RelA, c-Rel, and RelB proteins, respectively, while p50 and p52 are cleavage products of p105 and p100 proteins that are encoded by the Nfkb1 and Nfkb2 genes. A family of inhibitory proteins, the inhibitors of κB (IκB) that include p105 and p100 [84.Savinova O.V. et al.The Nfkb1 and Nfkb2 proteins p105 and p100 function as the core of high-molecular-weight heterogeneous complexes.Mol. Cell. 2009; 34: 591-602Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar], bind to Rel dimers and sequester them in the cytoplasm. Release of NF-κB dimers is regulated in much the same way in mouse and human. The IKK complex, a trimeric complex of two kinases, IKK1 (IKKα) and IKK2 (IKKβ) plus a regulatory subunit Nemo (IKKγ), phosphorylates IκB (P in Figure I), resulting in its degradation by the proteasome and permitting NF-κB dimers to enter the nucleus. This pathway regulates activation of RelA:p50 and c-Rel:p50 NF-κB dimers sequestered by IκBα or p105, and is termed the canonical NF-κB pathway (Figure I). The activity of RelB:p52 dimers is instead regulated by the so-called alternative NF-κB pathway. Dimers are sequestered specifically by p100 protein whose phosphorylation and degradation are specifically controlled by an IKK complex composed of an IKK1 homodimer. Activation of this IKK1 complex is instead triggered by NF-κB-inducing kinase (NIK). Surface receptors connect with canonical NF-κB pathways by various mechanisms. However, all have in common the recruitment of E3 ubiquitin ligases that allow the creation of a ubiquitin scaffold that is essential for the recruitment and activation of the TAB1/2/TAK1 complex, in turn activating the IKK complex. Following T cell receptor triggering, PKCθ phosphorylates Card11, allowing further recruitment of BCL10 and MALT1 molecules to form the large multimeric CBM complex (reviewed in detail in [85.Lork M. et al.Ubiquitination and phosphorylation of the CARD11–BCL10–MALT1 signalosome in T cells.Cell. Immunol. 2019; 340103877Crossref PubMed Scopus (16) Google Scholar]). Recruitment of the E3 ubiquitin ligase, tumor necrosis factor receptor (TNFR)-associated factor (TRAF)-6, results in the creation of the ubiquitin scaffold. By contrast, many TNFRSF proteins have TRAF-interacting motifs that can directly recruit TRAF proteins. In addition to TRAF6, TRAF 2, 3, and 5 also have E3 ubiquitin ligase activity [86.Yang X.-D. Sun S.-C. Targeting signaling factors for degradation, an emerging mechanism for TRAF functions.Immunol. Rev. 2015; 266: 56-71Crossref PubMed Scopus (58) Google Scholar] that creates the ubiquitin scaffold necessary to recruit TAB1/2/TAK1 and IKK complexes. Other TNFRSF members, such as TNFR1 and DR3, lack TRAF-interacting motifs and instead have death domains that allow the recruitment of adapters such as TNFR1-associated death domain protein (TRADD), that in turn can bind to and recruit TRAFs. By contrast, activation of the alternative pathway results from stabilization of NIK protein following inhibition of its degradation by TRAF2/3. In mouse T cells, expression of both Nfkb2 and Relb is dependent upon intact canonical signaling [24.Webb L.V. et al.Survival of single positive thymocytes depends upon developmental control of RIPK1 kinase signaling by the IKK complex independent of NF-κB.Immunity. 2019; 50: 348-361Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. The T cell receptor (TCR) has long been appreciated as a potent activator of NF-κB during T cell activation, and TCR signaling plays a central role in guiding different stages of T cell development in the thymus that involve selection of the TCR repertoire [2.Klein L. et al.Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see).Nat. Rev. Immunol. 2014; 14: 377-391Crossref PubMed Scopus (624) Google Scholar]. Previous studies have implied that NF-κB plays a role downstream of the pre-TCR complex (see Glossary) in CD4−CD8− double-negative thymocytes [3.Voll R.E. et al.NF-kappa B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development.Immunity. 2000; 13: 677-689Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar], and downstream of the TCR for the selection of CD8 lineage cells from CD4+CD8+ double-positive (DP) thymocytes [4.Mora A.L. et al.Lineage-specific differences among CD8+ T cells in their dependence of NF-kappa B/Rel signaling.Eur. J. Immunol. 1999; 29: 2968-2980Crossref PubMed Google Scholar, 5.Hettmann T. Leiden J.M. NF-kappa B is required for the positive selection of CD8+ thymocytes.J. Immunol. 2000; 165: 5004-5010Crossref PubMed Scopus (53) Google Scholar, 6.Jimi E. et al.Differential role of the transcription factor NF-kappaB in selection and survival of CD4+ and CD8+ thymocytes.Immunity. 2008; 29: 523-537Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. The most striking phenotype caused by interfering with NF-κB pathways occurs when upstream triggers, such as the IKK complex, TAK1, or LUBAC (Figure I in Box 2), are genetically ablated in Cd4creChukfl/flIkbkbfl/fl, Cd4creMap3k7fl/fl, or in either Cd4creHoilfl/fl or Cd4creHoipfl/fl mice, respectively, resulting in arrested thymocyte development at the CD4+ and CD8+ single-positive (SP) stages following positive selection [7.Schmidt-Supprian M. et al.Mature T cells depend on signaling through the IKK complex.Immunity. 2003; 19: 377-389Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 8.Sato S. et al.TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells.Int. Immunol. 2006; 18: 1405-1411Crossref PubMed Scopus (100) Google Scholar, 9.Wan Y.Y. et al.The kinase TAK1 integrates antigen and cytokine receptor signaling for T cell development, survival and function.Nat. Immunol. 2006; 7: 851-858Crossref PubMed Scopus (214) Google Scholar, 10.Teh C.E. et al.Linear ubiquitin chain assembly complex coordinates late thymic T-cell differentiation and regulatory T-cell homeostasis.Nat. Commun. 2016; 713353Crossref PubMed Scopus (31) Google Scholar, 11.Xing Y. et al.Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling.Nat. Immunol. 2016; 17: 565-573Crossref PubMed Scopus (79) Google Scholar]. The timing of arrest is not an artefact of the Cd4cre recombinase drivers used to delete genes in DP thymocytes because more recent studies, in which IKK1/2 genes were deleted earlier in development in double-negative progenitors in huCD2icreChukfl/flIkbkbfl/fl mice, report an identical block at the SP stage, and normal development before this stage [12.Webb L.V. et al.TNF activation of NF-κB is essential for development of single-positive thymocytes.J. Exp. Med. 2016; 213: 1399-1407Crossref PubMed Scopus (17) Google Scholar].Box 2Multi-Complex Signaling by TNFR1In mouse, ligation of TNFR1 by TNF ligands results in signaling processes that can give rise to several different signaling complexes that either result in (i) activation of NF-κB, or (ii) triggering cell death by apoptosis or by a distinct form of inflammatory cell death termed necroptosis (reviewed in [28.Vandenabeele P. et al.Molecular mechanisms of necroptosis: an ordered cellular explosion.Nat. Rev. Mol. Cell Biol. 2010; 11: 700-714Crossref PubMed Scopus (1481) Google Scholar,29.Pasparakis M. Vandenabeele P. Necroptosis and its role in inflammation.Nature. 2015; 517: 311-320Crossref PubMed Scopus (870) Google Scholar]). Initial ligation of TNFR1 allows recruitment of the E3 ligase TRAF2 via TRADD, followed by the formation of a ubiquitin scaffold. Although initiated by TRAF2, further recruitment of RIPK1 together with the activity of other ubiquitin ligases – cellular inhibitor of apoptosis proteins (cIAP) and the linear ubiquitin chain assembly complex (LUBAC) – cooperate to add ubiquitin modifications both to themselves and to RIPK1, thus generating a multiprotein complex and ubiquitin scaffold (Figure I). This results in the formation of the so-called TNFR complex I, that allows TAB1/2/TAK1 and IKK complexes to be recruited and allows IKK activation by TAK1, thereby triggering subsequent NF-κB activation. The stability of complex I may be transient in the face of deubiquitinases such as A20 and CYLD. Dissolution of complex I results in the formation of one of several cell death-inducing complexes. Recruitment of Fas-associated protein with death domain (FADD), and caspase 8 by TRADD [87.Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1773) Google Scholar,88.Wang L. et al.TNF-alpha induces two distinct caspase-8 activation pathways.Cell. 2008; 133: 693-703Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar], results in formation of complex IIa that triggers apoptosis by activating caspase 8 (CASP8), whereas additional recruitment of RIPK1 forms complex IIb that is a potent inducer of apoptosis [89.Dondelinger Y. et al.Regulation of RIPK1's cell death function by phosphorylation.Cell Cycle. 2016; 15: 5-6Crossref PubMed Scopus (9) Google Scholar, 90.Ting A.T. Bertrand M.J.M. More to life than NF-kappaB in TNFR1 signaling.Trends Immunol. 2016; 37: 535-545Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 91.Annibaldi A. Meier P. Checkpoints in TNF-induced cell death: implications in inflammation and cancer.Trends Mol. Med. 2018; 24: 49-65Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar]. In the absence of caspase 8 protease activity, RIPK3 and MLKL (mixed lineage kinase domain-like pseudokinase) are recruited to complex IIb and together trigger necroptosis, that is also RIPK1 kinase-dependent (reviewed in [28.Vandenabeele P. et al.Molecular mechanisms of necroptosis: an ordered cellular explosion.Nat. Rev. Mol. Cell Biol. 2010; 11: 700-714Crossref PubMed Scopus (1481) Google Scholar,29.Pasparakis M. Vandenabeele P. Necroptosis and its role in inflammation.Nature. 2015; 517: 311-320Crossref PubMed Scopus (870) Google Scholar]). The utilization of RIPK1 as an adapter during TNFR1 signaling is probably no accident. As a component of complex I, RIPK1 comes into close proximity with the IKK and TAB1/2/TAK1 complexes. Recent studies reveal that phosphorylation (P in Figure I) of RIPK1, by IKK and TAK1, represses RIPK1 kinase activity [1.Dondelinger Y. et al.NF-kappaB-independent role of IKKalpha/IKKbeta in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar,33.Geng J. et al.Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis.Nat. Commun. 2017; 8: 359Crossref PubMed Scopus (114) Google Scholar], preventing triggering of cell death. Indeed, blocking IKK or TAK1 activity is sufficient to sensitize many cells to TNF-induced cell death. Importantly, this protective function of RIPK1 phosphorylation can occur independently of NF-κB signaling [1.Dondelinger Y. et al.NF-kappaB-independent role of IKKalpha/IKKbeta in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar,24.Webb L.V. et al.Survival of single positive thymocytes depends upon developmental control of RIPK1 kinase signaling by the IKK complex independent of NF-κB.Immunity. 2019; 50: 348-361Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. These studies reveal the polyfunctionality of such kinases, and how they regulate cell fate decisions – not only as upstream activators of NF-κB signaling but also through targeting other substrates in the signaling pathway, such as RIPK1, to block cell death induction. In mouse, ligation of TNFR1 by TNF ligands results in signaling processes that can give rise to several different signaling complexes that either result in (i) activation of NF-κB, or (ii) triggering cell death by apoptosis or by a distinct form of inflammatory cell death termed necroptosis (reviewed in [28.Vandenabeele P. et al.Molecular mechanisms of necroptosis: an ordered cellular explosion.Nat. Rev. Mol. Cell Biol. 2010; 11: 700-714Crossref PubMed Scopus (1481) Google Scholar,29.Pasparakis M. Vandenabeele P. Necroptosis and its role in inflammation.Nature. 2015; 517: 311-320Crossref PubMed Scopus (870) Google Scholar]). Initial ligation of TNFR1 allows recruitment of the E3 ligase TRAF2 via TRADD, followed by the formation of a ubiquitin scaffold. Although initiated by TRAF2, further recruitment of RIPK1 together with the activity of other ubiquitin ligases – cellular inhibitor of apoptosis proteins (cIAP) and the linear ubiquitin chain assembly complex (LUBAC) – cooperate to add ubiquitin modifications both to themselves and to RIPK1, thus generating a multiprotein complex and ubiquitin scaffold (Figure I). This results in the formation of the so-called TNFR complex I, that allows TAB1/2/TAK1 and IKK complexes to be recruited and allows IKK activation by TAK1, thereby triggering subsequent NF-κB activation. The stability of complex I may be transient in the face of deubiquitinases such as A20 and CYLD. Dissolution of complex I results in the formation of one of several cell death-inducing complexes. Recruitment of Fas-associated protein with death domain (FADD), and caspase 8 by TRADD [87.Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1773) Google Scholar,88.Wang L. et al.TNF-alpha induces two distinct caspase-8 activation pathways.Cell. 2008; 133: 693-703Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar], results in formation of complex IIa that triggers apoptosis by activating caspase 8 (CASP8), whereas additional recruitment of RIPK1 forms complex IIb that is a potent inducer of apoptosis [89.Dondelinger Y. et al.Regulation of RIPK1's cell death function by phosphorylation.Cell Cycle. 2016; 15: 5-6Crossref PubMed Scopus (9) Google Scholar, 90.Ting A.T. Bertrand M.J.M. More to life than NF-kappaB in TNFR1 signaling.Trends Immunol. 2016; 37: 535-545Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 91.Annibaldi A. Meier P. Checkpoints in TNF-induced cell death: implications in inflammation and cancer.Trends Mol. Med. 2018; 24: 49-65Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar]. In the absence of caspase 8 protease activity, RIPK3 and MLKL (mixed lineage kinase domain-like pseudokinase) are recruited to complex IIb and together trigger necroptosis, that is also RIPK1 kinase-dependent (reviewed in [28.Vandenabeele P. et al.Molecular mechanisms of necroptosis: an ordered cellular explosion.Nat. Rev. Mol. Cell Biol. 2010; 11: 700-714Crossref PubMed Scopus (1481) Google Scholar,29.Pasparakis M. Vandenabeele P. Necroptosis and its role in inflammation.Nature. 2015; 517: 311-320Crossref PubMed Scopus (870) Google Scholar]). The utilization of RIPK1 as an adapter during TNFR1 signaling is probably no accident. As a component of complex I, RIPK1 comes into close proximity with the IKK and TAB1/2/TAK1 complexes. Recent studies reveal that phosphorylation (P in Figure I) of RIPK1, by IKK and TAK1, represses RIPK1 kinase activity [1.Dondelinger Y. et al.NF-kappaB-independent role of IKKalpha/IKKbeta in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar,33.Geng J. et al.Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis.Nat. Commun. 2017; 8: 359Crossref PubMed Scopus (114) Google Scholar], preventing triggering of cell death. Indeed, blocking IKK or TAK1 activity is sufficient to sensitize many cells to TNF-induced cell death. Importantly, this protective function of RIPK1 phosphorylation can occur independently of NF-κB signaling [1.Dondelinger Y. et al.NF-kappaB-independent role of IKKalpha/IKKbeta in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling.Mol. Cell. 2015; 60: 63-76Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar,24.Webb L.V. et al.Survival of single positive thymocytes depends upon developmental control of RIPK1 kinase signaling by the IKK complex independent of NF-κB.Immunity. 2019; 50: 348-361Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. These studies reveal the polyfunctionality of such kinases, and how they regulate cell fate decisions – not only as upstream activators of NF-κB signaling but also through targeting other substrates in the signaling pathway, such as RIPK1, to block cell death induction. SP thymocytes undergo a phase of maturation lasting many days [13.McCaughtry T.M. et al.Thymic emigration revisited.J. Exp. Med. 2007; 204: 2513-2520Crossref PubMed Scopus (191) Google Scholar,14.Sinclair C. et al.Asymmetric thymocyte death underlies the CD4:CD8 T-cell ratio in the adaptive immune system.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E2905-E2914Crossref PubMed Scopus (56) Google Scholar], during which time they acquire receptors that are necessary for normal recirculation, such as L-selectin, CCR7, and S1P1, as well as the ability to proliferate in response to TCR triggering [11.Xing Y. et al.Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling.Nat. Immunol. 2016; 17: 565-573Crossref PubMed Scopus (79) Google Scholar]. Transcriptional analysis of SP mouse thymocytes reveals a prominent NF-κB gene signature specifically in late-stage SP thymocytes, supporting the idea that maturation is dependent upon NF-κB (Figure 1) [11.Xing Y. et al.Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling.Nat. Immunol. 2016; 17: 565-573Crossref PubMed Scopus (79) Google Scholar,12.Webb L.V. et al.TNF activation of NF-κB is essential for development of single-positive thymocytes.J. Exp. Med. 2016; 213: 1399-1407Crossref PubMed Scopus (17) Google Scholar]. Although the TCR has been considered to be the key upstream trigger of NF-κB activity in thymocytes, NF-κB-induced genes are most evident among late-stage mouse SPs [11.Xing Y. et al.Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling.Nat. Immunol. 2016; 17: 565-573Crossref PubMed Scopus (79) Google Scholar,12.Webb L.V. et al.TNF activation of NF-κB is essential for development of single-positive thymocytes.J. Exp. Med. 2016; 213: 1399-1407Crossref PubMed Scopus (17) Google Scholar] (Figure 1), at a point when thymic development is no longer under the influence of TCR signaling [15.Sinclair C. Seddon B. Overlapping and asymmetric functions of TCR signaling during thymic selection of CD4 and CD8 lineages.J. Immunol. 2014; 192: 5151-5159Crossref PubMed Scopus (15) Google Scholar]. Furthermore, it is notable that the CARD11/BCL10/MALT1 (CBM) complex, that links TCR signaling to NF-κB activation (see Figure I in Box 1), is redundant for T cell selection [16.Ruland J. et al.Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure.Cell. 2001; 104: 33-42Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 17.Egawa T. et al.Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.Curr. Biol. 2003; 13: 1252-1258Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 18.Hara H. et al.The MAGUK family protein CARD11 is essential for lymphocyte activation.Immunity. 2003; 18: 763-775Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 19.Ruefli-Brasse A.A. et al.Regulation of NF-kappaB-dependent lymphocyte activation and development by paracaspase.Science. 2003; 302: 1581-1584Crossref PubMed Scopus (286) Google Scholar, 20.Ruland J. et al.Differential requirement for Malt1 in T and B cell antigen receptor signaling.Immunity. 2003; 19: 749-758Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 21.Schmidt-Supprian M. et al.Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-κB activation.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4566-4571Crossref PubMed Scopus (202) Google Scholar, 22.Jost P.J. et al.Bcl10/Malt1 signaling is essential for TCR-induced NF-kappaB activation in thymocytes but dispensable for positive or negative selection.J. Immunol. 2007; 178: 953-960Crossref PubMed Scopus (19) Google Scholar], further arguing against a role for TCR-triggered NF-κB in thymic development. Recent studies have shed light on this puzzle by revealing that the block in SP development is in fact the result of cell death triggered by TNF signaling. Specifically, the apparent developmental block in IKK1/2- or TAK1-deficient T cells, in huCD2icreChukfl/flIkbkbfl/fl and Cd4creMap3k7fl/fl mice, is overcome in vivo by genetic ablation of TNF or TNFR1, or by TNF-blocking monoclonal antibodies (mAbs) [11.Xing Y. et al.Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling.Nat. Immunol. 2016; 17: 565-573Crossref PubMed Scopus (79) Google Scholar,12.Webb L.V. et al.TNF activation