Abstract Introduction The IκB kinase (IKK) complex is best known for its roles in cell survival and in the activation of canonical NFκB isoforms. The complex contains two kinase subunits (IKKα/β) and a dimeric polyubiquitin-binding adaptor subunit known as the NFκB essential modulator protein (NEMO) [IKBKG]. While oversimplified models often place the IKK complex entirely downstream of the adaptor protein Carma1, T cell receptor (TCR)-induced IKKβ phosphorylation is Carma1-independent. Thus, IKK complexes may play unconventional roles downstream of the TCR, but upstream of Carma1. Methods Here, we examine how IKK complexes participate in TCR proximal signals via dynamic imaging analyses of fluorescent NEMO chimeras expressed in Jurkat T cells and primary human T cell blasts. Results We observe that NEMO enters TCR- and ZAP-70-containing microclusters within ∼70 seconds of their formation, via processes that rely on the activities of Lck and ZAP-70, and that are independent of downstream effectors implicated in canonical NFκB activation. The mutational inactivation of either the K63 or the linear polyubiquitin-binding sites in NEMO, the pharmacological inhibition the K63-chain forming E2 ligase Ubc13, or the inhibition of the linear polyubiquitin forming LUBAC complex all prevent the recruitment of NEMO to the TCR. If NEMO is co-expressed with wild-type IKKβ, the co-clustering of NEMO with the TCR is largely eliminated. However, kinase dead IKKβ co-clusters with NEMO at the TCR, and IKKβ inhibitors shift wild-type IKK complexes into TCR microclusters. Conclusion These data suggest that the IKK complex is rapidly recruited to the TCR via multipoint interactions with ubiquitin polymers assembled within ∼1 minute of ZAP-70 recruitment, whereas the activity of the IKK complex releases NEMO from the TCR. These findings define a window within which partially activated IKK complexes may influence TCR-proximal signaling events upstream of Carma1. Funding Source NIH R01 AI103022 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Currently, most tools utilized in host-pathogen interaction studies depend on the use of human or mouse (Mus musculus) cells and tissues. While these species have led to countless breakthroughs in our understanding of infectious disease, there are undoubtedly important biological processes that are missed by limiting studies to these two vertebrate species. For instance, it is well-established that a common deermouse in North America, Peromyscus leucopus, has unique interactions with microbes, which likely shape its ability to serve as a critical reservoir for numerous zoonotic pathogens, including a Lyme disease spirochete, Borrelia burgdorferi. In this work, we expand the immunological toolkit to study P. leucopus biology by performing the first differentiation of deermouse bone marrow to macrophages using P. leucopus M-CSF producing HEK293T cells. We find that P. leucopus BMDMs generated through this method behave broadly very similarly to C57BL/6J macrophages generated with the L-929 supernatant, although RNA sequencing revealed modest differences in transcriptomic responses to B. burgdorferi and lipopolysaccharide. In particular, differences in Il-10 induction and caspase expression were observed between the species.

Signaling through classical death receptor Fas was mainly appreciated as a pro-death pathway until recent reports characterized pro-inflammatory outcomes of Fas-mediated activation in pathological contexts. How Fas signaling can switch to pro-inflammatory activation is poorly understood. Herein, we report that in macrophages and neutrophils, the Toll-like receptor (TLR) adapter CD14 determines the inflammatory output of Fas-mediated signaling. Our findings propose CD14 as a crucial chaperone of Fas receptor internalization in macrophages and neutrophils, resulting in Cd14-/- myeloid cells that are protected from FasL-induced apoptosis, activate nuclear factor κB (NF-κB), and release cytokines in response. As in TLR signaling, CD14 is also required for Fas to signal through the adaptor TRIF (TIR-domain-containing adapter-inducing interferon-β) and induce a pro-death complex. Our findings demonstrate that CD14 availability can determine the switch between Fas-mediated pro-death and pro-inflammatory outcomes by internalizing the receptor.

SUMMARYLocal immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in variousin vitroandin vivomodels of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immunefingerprintfor each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING- sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues.Ex vivoanalyses reveal that basal STING- signaling limits the susceptibility of TRMs to death-inducing stimuli and determines theirin situlocalization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging.HIGHLIGHTSAging tissues are associated with tissue-autonomousimmunefingerprints, primarily driven by interactions of tissue stromal and myeloid populations.STING shapes these immune fingerprints of aging tissues in unexpected ways.Loss of STING alters the location, numbers, and viability of tissue resident macrophages.STING signaling is critical for longer lifespans and maintenance of tissue architecture.

Signaling cascades that convert the recognition of pathogens to efficient inflammatory responses by immune cells, specifically neutrophils, are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following stimulation by integrins, formyl peptide receptors and gram-negative bacteria Klebsiella pneumoniae and Yersinia pseudotuberculosis in vitro (Nguyen et al., 2020, Shaban et al., 2020, Boras et al., 2017). SKAP2 is also required for the host defense against K. pneumoniae and ΔyopH Y. pseudotuberculosis infection in vivo in mouse models (Shaban et al., 2020, Nguyen et al., 2020). Another class of pattern recognition receptors (PRR) is the C-type lectin receptors (CLR), such as Dectin-1, Dectin-2 and Mincle, that are critical to trigger innate immune responses. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 is crucial for maximal ROS response to purified CLR agonists and to the fungal pathogens Candida glabrata and C. albicans , as well as for robust killing of C. glabrata . Skap2-/- murine neutrophils failed to generate ROS and exhibited reduced cellular adhesion in response to trehalose-6,6’-dibehenate (TDB), furfurman, and curdlan, Mincle, Dectin-2, and Dectin-1 agonists, respectively. TDB, furfurman, and curdlan stimulation also led to SKAP2-independent integrin conformational changes, showing that inside-out signaling by these CLRs to integrin occurs in the absence of SKAP2. Pyk2 phosphorylation was significantly reduced after infection with C. glabrata in Skap2-/- neutrophils, while Syk phosphorylation was unaffected by the loss of SKAP2. These data strengthen the importance of SKAP2 in the activation of neutrophil ROS production by PRRs to include CLRs and extend the role of SKAP2 in host defense beyond antibacterial immunity to include Candida species.

The IkB kinase (IKK) complex coordinates inflammatory responses by activating canonical NFkB downstream of immune receptors. This complex consists of the kinases IKKa/β and the dimeric adaptor subunit NEMO (NFkB essential modulator protein). NEMO mutations cause immunodeficiencies and impair antigen receptor function. Canonical NFkB activation by immunoreceptors, such as the T cell receptor (TCR), requires the assembly of the Carma1/Bcl10/Malt1 (CBM) signalosome. While, functional studies and in vitro interactions, place the IKK complex downstream of the CBM signalosome, the spatial relationship between these complexes has never been observed in intact immune cells. We observed that NEMO is recruited into TCR microclusters and membrane compartments within ~70 seconds of TCR engagement. Point mutations impacting the K63-linked and linear polyubiquitin ubiquitin-binding domains of NEMO selectively impair NEMO recruitment into TCR microclusters. We verified the involvement of these polyubiquitin chains by showing that inhibitors of either the K63-specific Uev1A/Ubc13 ubiquitin E2 ligase or the linear ubiquitin activating complex (LUBAC) prevent NEMO from entering TCR microclusters. To examine whether the entire IKK complex is recruited to the TCR, we co-expressed IKKb chimeras with NEMO. We observed that wild-type (WT), but not kinase-dead, IKKb caused NEMO microclusters to disappear. Similarly, the treatment of cells expressing WT IKKb with inhibitors of IKKβ or its upstream activator, TAK1, restored the recruitment of IKKβ and NEMO into microclusters. These results suggest that the IKK complex is recruited to the TCR via linear- and K63-linked polyubiquitin chains and then dissociates from the TCR via IKKβ activity.

The NF-κB essential modulator protein (NEMO) is required for activation of canonical NF-κB by the T cell antigen receptor (TCR). However, the subcellular localization of NEMO during this process is not well understood. By dynamically imaging fluorescent NEMO chimeras in live human T cells, we demonstrate that NEMO is rapidly recruited into TCR microclusters via domains previously implicated in the recognition of linear and K63-linked polyubiquitin. The recruitment of NEMO into TCR microclusters requires the activities of the tyrosine kinases Lck and ZAP-70, but not the adaptor proteins LAT or SLP-76. Thus, our findings reveal that the pathways leading from TCR to NF-κB bifurcate downstream of ZAP-70 to independently control the recruitment and activation of NEMO.

Klebsiella pneumoniae is a respiratory, blood, liver, and bladder pathogen of significant clinical concern. We show that the adaptor protein, SKAP2, is required for protection against K. pneumoniae (ATCC 43816) pulmonary infections. Skap2-/- mice had 100-fold higher bacterial burden when compared to wild-type and burden was controlled by SKAP2 expression in innate immune cells. Skap2-/- neutrophils and monocytes were present in infected lungs, and the neutrophils degranulated normally in response to K. pneumoniae infection in mice; however, K. pneumoniae-stimulated reactive oxygen species (ROS) production in vitro was abolished. K. pneumoniae-induced neutrophil ROS response required the activity of SFKs, Syk, Btk, PLCγ2, and PKC. The loss of SKAP2 significantly hindered the K. pneumoniae-induced phosphorylation of SFKs, Syk, and Pyk2 implicating SKAP2 as proximal to their activation in pathogen-signaling pathways. In conclusion, SKAP2-dependent signaling in neutrophils is essential for K. pneumoniae-activated ROS production and for promoting bacterial clearance during infection.

Vav family guanine nucleotide exchange factors (GEFs) are essential regulators of immune function. Despite their structural similarity, Vav1 promotes and Vav2 opposes T cell receptor (TCR)-induced Ca2+ entry. By using a Vav1-deficient Jurkat T cell line, we find that Vav1 facilitates Ca2+ entry via non-catalytic scaffolding functions that are encoded by the catalytic core of Vav1 and flanking linker regions. We implicate, in this scaffolding function, a previously undescribed polybasic motif that is strictly conserved in Vav1 and absent from Vav2 in tetrapods. Conversely, the catalytic activity of Vav2 contributes to the suppression of TCR-mediated Ca2+ entry. By performing an in vivo 'GEF trapping' assay in intact cells, we demonstrate that Cdc42 interacts with the catalytic surface of Vav2 but not Vav1, and that Vav1 discriminates Cdc42 from Rac1 via F56 (W56 in Rac1). Finally, the Cdc42-specific inhibitor ZCL278 and the shRNA-mediated suppression of Cdc42 each prevent the inhibition of TCR-induced Ca2+ entry by Vav2. These findings define stark differences in the functions of Vav1 and Vav2, and provide an explanation for the differential usage of these Vav isoforms by immune subpopulations.

Schistosomiasis is a major helminthic disease in which damage to the affected organs is orchestrated by a pathogenic host CD4 T helper (Th) cell-mediated immune response against parasite eggs. In the case of the species Schistosoma mansoni, the resulting granulomatous inflammation and fibrosis takes place in the liver and intestines. The magnitude of disease varies greatly from individual to individual but in a minority of patients, there is severe disease and death. S. mansoni infection in a murine model similarly results in marked strain variation of immunopathology. In the most commonly examined mouse strain, C57BL/6 (BL/6), there is relatively mild hepatic pathology arising in a Th2-dominated cytokine environment. In contrast, CBA mice develop decisively more severe lesions largely driven by proinflammatory IL-17-producing Th17 cells. Dendritic cells (DCs) from CBA mice differ sharply with those from BL/6 mice in that they vastly over-express the C-type lectin receptor (CLR) CD209a (SIGNR5), a homolog of human DC-SIGN, which senses glycans such as those produced by schistosome eggs. Silencing of CD209a, and recent studies with CD209a KO CBA mice have shown that this receptor is crucial to induce the pathogenic Th17 cell response; indeed, CD209a KO mice display markedly reduced immunopathology akin to that seen in BL/6 mice. Mechanistically, CD209a synergizes with the related CLRs Dectin-2 and Mincle to stimulate increased DC production of IL-1β and IL-23, necessary for pathogenic Th17 cell development. These findings denote key molecular underpinnings of disease variability based on selection and function of contrasting Th cell subsets.

The IκB kinase (IKK) complex mediates the activation of canonical NFκB isoforms following T cell receptor (TCR) ligation. This complex consists of the kinases IKKα and IKKβ and an essential adaptor subunit, the NFκB essential modulator protein (NEMO). Most models suggest that the IKK complex is activated within oligomeric Carma1/Bcl10/Malt1 (CBM) signalosomes. However, we observed that NEMO enters TCR microclusters before CBM complexes are assembled, within ~70 seconds of TCR engagement. NEMO also entered mobile vesicles and in larger membrane-bounded structures (hereafter ‘macroclusters’). The recruitment of NEMO into TCR microclusters is prevented by Src kinase inhibitors and by the catalytic inactivation of ZAP-70, but occurs in the absence of either SLP-76 or Carma1. Further, NEMO fails to co-localize with TCR-induced CBM polymers. Thus, the recruitment of NEMO to the TCR occurs via a CBM-independent mechanism. The deletion the zinc-finger (ZnF) domain of NEMO disables NFκB signaling and eliminates NEMO from microclusters, while preserving NEMO macroclusters. Since the ZnF domain interacts with polyubiquitin chains, we generated point mutations impacting the ubiquitin-binding site in the ZnF domain and two independent sites within the NEMO ubiquitin-binding ‘NUB’ domain. These mutations impair the ability of NEMO to capture K63-linked and/or linear polymers, hinder NFκB signaling, and eliminate NEMO from microclusters without disrupting NEMO macroclusters. These findings suggest that NEMO is rapidly recruited to polyubiquitin chains associated with the TCR, rather than the CBM complex, and that the CBM complex augments IKK-dependent NFκB signaling via a distinct, recruitment-independent mechanism.

The immunopathology caused by schistosome helminths varies greatly in humans and among mouse strains. A severe form of parasite egg-induced hepatic granulomatous inflammation, seen in CBA mice, is driven by Th17 cells stimulated by IL-1β and IL-23 produced by dendritic cells that express CD209a (SIGNR5), a C-type lectin receptor (CLR) related to human DC-SIGN. Here, we show that CD209a-deficient CBA mice display decreased Th17 responses and are protected from severe immunopathology. In vitro, CD209a augments the egg-induced IL-1β and IL-23 production initiated by the related CLRs Dectin-2 and Mincle. While Dectin-2 and Mincle trigger an FcRγ-dependent signaling cascade that involves the tyrosine kinase Syk and the trimolecular Card9-Bcl10-Malt1 complex, CD209a promotes the sustained activation of Raf-1. Our findings demonstrate that CD209a drives severe Th17 cell-mediated immunopathology in a helminthic disease based on synergy between DC-SIGN- and Dectin-2-related CLRs.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

Cryptosporidium spp. are the causative agents of diarrheal disease worldwide, but effective treatments are lacking. Cryptosporidium employs mucin-like glycoproteins with O-glycans to attach to and infect host intestinal epithelial cells. The Tn antigen (GalNAcα1-Ser/Thr) is an O-glycan essential for these processes, as Tn-specific lectins and a Tn-specific monoclonal antibody block attachment to and infection of host cells in vitro. The enzymes in Cryptosporidium catalyzing their synthesis, however, have not been studied. Previously, we identified four genes encoding putative UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) in the genomes of three Cryptosporidium spp. Here we report the in silico analysis, cloning, expression, purification, and characterization of one of the four enzymes Cryptosporidium parvum (Cp)-ppGalNAc-T4. This enzyme contains the characteristic domains and motifs conserved in ppGalNAc-Ts and is expressed at multiple time points during in vitro infection. Recombinant soluble Cp-ppGalNAc-T4 was enzymatically active against an unmodified EA2 peptide suggesting that it may function as an "initiating" ppGalNAc-T. Cp-ppGalNAc-T4 also exhibited a strong preference for UDP-GalNAc over other nucleotide sugar donors and was active against unmodified and O-glycosylated versions of the C. parvum gp40-derived peptide, with a preference for the former, suggesting it may play a role in modifying this glycoprotein in vivo. Given the importance of mucin-type O-glycosylation in Cryptosporidium spp., the enzymes that catalyze their synthesis may serve as potential therapeutic targets.

Significance Here we demonstrate that Yersinia YopJ-induced murine macrophage death involves caspase-8–induced cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME). The ensuing cell death is rapid, morphologically is similar to pyroptosis, and induces IL-1 release. Recently, both GSDMD and GSDME were reported to be critical effectors of caspase-1/11–driven pyroptosis and caspase-3–dependent secondary necrosis, which prompted the redefinition of pyroptosis as cell death-mediated by gasdermin activation. Our work extends these studies and shows that activation of caspase-8 in the context of TAK1 inhibition results in cleavage of both GSDMD and GSDME, leading to pyroptotic-like cell death. Further study will be needed to determine whether caspase-8 cleaves GSDMD directly or via intermediate substrates.

ABSTRACT Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3β1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi. We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin β1 play a role in the activation of the Src kinase pathway. However, only integrin β1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.

Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo.

Abstract The magnitude of immunopathology and pro-inflammatory cytokine production in murine Schistosoma mansoni infection is strain-dependent. Severe hepatic egg-induced granulomatous inflammation in CBA mice is associated with Th1 and Th17 cytokine responses, whereas BL/6 mice develop milder lesions in a Th2-polarized cytokine environment. Pathogenic Th17 cell responses in CBA mice are dependent on the production of IL-1β and IL-23 by egg-stimulated dendritic cells (DC); by comparison, such Th17 cells fail to develop in BL/6 mice. The reasons for strain-dependent differences in DC reactivity to eggs remain unclear. Genome-wide gene profiling revealed significant differences between CBA vs. BL/6 DCs in C-type lectin receptors (CLRs), a family of pattern recognition receptors that binds glycans such as those produced by schistosome eggs. Expression of the CLR CD209a, a murine homologue of human DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), was strikingly higher in several APC populations from CBA mice; however, only CBA DC, but not macrophages, B cells, or granulocytes elicited Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DC, and over-expression in BL/6 DC, demonstrated CD209a to be necessary for egg-induced DC production of ERK1/2 map kinase-dependent IL-1β and IL-23 as well as subsequent Th17 cell development. These findings reveal a novel mechanism controlling the development of Th17 cell-mediated immunopathology in helminthic disease.

The hallmark of an adaptive immune response is the activation and clonal expansion of antigen-specific T and B lymphocytes. The recruitment of a T cell into an immune response requires the coordinated delivery of three distinct signals. Signal 1 is delivered when the T cell receptor (TCR) engages cognate antigenic peptides presented by major histocompatibility complexes (MHC) displayed on the surface of an antigen-presenting cell (APC). Signal 2 is delivered upon the recognition of costimulatory ligands presented by APCs that have been exposed to proinflammatory stimuli. Lastly, signal 3 is delivered by combinations of cytokines that support the survival, proliferation, effector function, and differentiation of T cells that have received signals 1 and 2. In particular, cytokines that signal through the common gamma chain, such as interleukin-2 (IL-2), contribute to signal 3 by promoting the survival and expansion of T cells. This system is exquisitely selective, and for the most part ensures that nonspecific ‘‘bystander’’ lymphocytes do not participate in immune responses. The effectiveness of this safety mechanism is remarkable, given that the vast majority of the T cells present in secondary lymphoid organs are not specific for antigens derived from the offending pathogen and that many of these cells are capable of recognizing self-antigens. These potentially hazardous cells are exposed to the same milieu of costimulatory ligands and cytokines available to neighboring antigen-specific T cells. Consequently, the mechanisms that prevent the inappropriate expansion of bystander T cells might contribute to the avoidance of autoimmune disorders. In this issue of Immunity, Watanabe et al. (2014) provide insight into this process by demonstrating that the tumor suppressor p53 (TP53 in human; Trp53 in mouse) imposes the checkpoint that prevents the proliferation of bystander T cells exposed to signal 3, in the form of IL-2, and that this checkpoint is lifted by signals delivered through the TCR. A tremendous amount of study has been devoted to the roles of p53 in cancer because most human cancers involve either inactivating mutations in p53 itself or in components of the p53 pathway (Green and Kroemer, 2009). Given the importance of p53 in cancer biology, it is somewhat surprising that so little is known about its functions in T cells. The best characterized role of p53 is that of a transcription factor that either halts the cell cycle or induces apoptotic cell death in response to DNA damage. Consequently, several earlier studies have addressed the roles of p53 in T cell development, during the recombination of genomic TCR loci. Consistent with its roles in cancer biology, p53 contributes to the suppression of thymocyte proliferation during the checkpoint associated with the recombination of the TCRb locus and the subsequent formation of a functional pre-TCR. This process requires the introduction of double-strand DNA breaks and is expected to activate p53. Remarkably, the loss of p53 is sufficient to permit thymocytes that are incapable of generating a functional pre-TCR to progress from the coreceptor ‘‘doublenegative’’ (DN) stage to the CD4 CD8 ‘‘double-positive’’ (DP) stage (Guidos et al. 1996; Haks et al. 1999). B cell development is similarly restored in recombinase-deficient mice lacking p53. The ability of activated B cells to undergo cycles of expansion and somatic hypermutation is also influenced by p53. In particular, BCL6, a transcriptional