Signaling from the T-cell receptor (TCR) conditions T-cell differentiation and activation, requiring exquisite sensitivity and discrimination. Using mass cytometry, a high-dimensional technique that can probe multiple signaling nodes at the single-cell level, we interrogate TCR signaling dynamics in control C57BL/6 and autoimmunity-prone nonobese diabetic (NOD) mice, which show ineffective ERK activation after TCR triggering. By quantitating signals at multiple steps along the signaling cascade and parsing the phosphorylation level of each node as a function of its predecessors, we show that a small impairment in initial pCD3? activation resonates farther down the signaling cascade and results in larger defects in activation of the ERK1/2-S6 and I?B? modules. This nonlinear property of TCR signaling networks, which magnifies small initial differences during signal propagation, also applies in cells from B6 mice activated at different levels of intensity. Impairment in pCD3? and pSLP76 is not a feedback consequence of a primary deficiency in ERK activation because no proximal signaling defect was observed in Erk2 KO T cells. These defects, which were manifest at all stages of T-cell differentiation from early thymic pre-T cells to memory T cells, may condition the imbalanced immunoregulation and tolerance in NOD T cells. More generally, this amplification of small initial differences in signal intensity may explain how T cells discriminate between closely related ligands and adopt strongly delineated cell fates.
Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death-independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8-deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type-specific manner and acts uniquely in DCs to maintain tolerance.
The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation.
Particle-based delivery of encapsulated antigens has great potential for improving vaccine constructs. In this study, we show that antigen-loaded, pH-sensitive hydrogel microparticles are taken up and presented by bone marrow-derived dendritic cells (BMDCs) in vitro and are taken up by dendritic cells (DCs) and monocytes in vivo. This uptake is irrespective of targeting antibodies. BMDCs in vitro and DCs in vivo also display upregulation of activation markers CD80 and CD86 when treated with microparticles, again with no difference in conjugated antibodies, even the agonistic CD40 antibody. We further show that these particles induce enhanced expansion of cytokine-producing CD8 T cells in response to challenge with ovalbumin-expressing vesicular stomatitis virus, in both an accelerated vaccination strategy using pre-loaded BMDCs and a traditional mouse immunization setting.
The forkhead O transcription factors (FOXO) integrate a range of extracellular signals, including growth factor signaling, inflammation, oxidative stress, and nutrient availability, to substantially alter the program of gene expression and modulate cell survival, cell cycle progression, and many yet to be unraveled cell type-specific responses. Naive antigen-specific CD8(+) T cells undergo a rapid expansion and arming of effector function within days of pathogen exposure. In addition, by the peak of expansion, they form precursors to memory T cells capable of self-renewal and indefinite survival. Using lymphocytic choriomeningitis virus Armstrong to probe the response to infection, we found that Foxo1(-/-) CD8(+) T cells expand normally with no defects in effector differentiation, but continue to exhibit characteristics of effector T cells long after antigen clearance. The KLRG1(lo) CD8(+) T cells that are normally enriched for memory-precursor cells retain Granzyme B and CD69 expression, and fail to up-regulate TCF7, EOMES, and other memory signature genes. As a correlate, Foxo1(-/-) CD8(+) T cells were virtually unable to expand upon secondary infection. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing the post-effector memory program that is essential to forming long-lived memory cells capable of immune reactivation.
Cell populations are regulated in size by at least two forms of apoptosis. More recently, necroptosis, a parallel, nonapoptotic pathway of cell death, has been described, and this pathway is invoked in the absence of caspase 8. In caspase 8-deficient T cells, necroptosis occurs as the result of antigen receptor-mediated activation. Here, through a genetic analysis, we show that necroptosis in caspase 8-deficient T cells is related neither to the programmed necrosis as defined by the requirement for mitochondrial cyclophilin D nor to autophagy as defined by the requirement for autophagy-related protein 7. Rather, survival of caspase 8-defective T cells can be completely rescued by loss of receptor-interacting serine-threonine kinase (Ripk) 3. Additionally, complementation of a T cell-specific caspase 8 deficiency with a loss of Ripk3 gives rise to lymphoproliferative disease reminiscent of lpr or gld mice. In conjunction with previous work, we conclude that necroptosis in antigen-stimulated caspase 8-deficient T cells is the result of a novel Ripk1- and Ripk3-mediated pathway of cell death.
Dysfunction of the intestinal epithelium is believed to result in the excessive translocation of commensal bacteria into the bowel wall that drives chronic mucosal inflammation in Crohns disease, an incurable inflammatory bowel disease in humans characterized by inflammation of the terminal ileum. In healthy individuals, the intestinal epithelium maintains a physical barrier, established by the tight contact of cells. Moreover, specialized epithelial cells such as Paneth cells and goblet cells provide innate immune defence functions by secreting mucus and antimicrobial peptides, which hamper access and survival of bacteria adjacent to the epithelium. Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as a possible pathogenic mechanism driving Crohns disease in humans. However, the regulation of epithelial cell death and its role in intestinal homeostasis remain poorly understood. Here we demonstrate a critical role for caspase-8 in regulating necroptosis of intestinal epithelial cells (IECs) and terminal ileitis. Mice with a conditional deletion of caspase-8 in the intestinal epithelium (Casp8(?IEC)) spontaneously developed inflammatory lesions in the terminal ileum and were highly susceptible to colitis. Casp8(?IEC) mice lacked Paneth cells and showed reduced numbers of goblet cells, indicating dysregulated antimicrobial immune cell functions of the intestinal epithelium. Casp8(?IEC) mice showed increased cell death in the Paneth cell area of small intestinal crypts. Epithelial cell death was induced by tumour necrosis factor (TNF)-?, was associated with increased expression of receptor-interacting protein 3 (Rip3; also known as Ripk3) and could be inhibited on blockade of necroptosis. Lastly, we identified high levels of RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patients with Crohns disease, suggesting a potential role of necroptosis in the pathogenesis of this disease. Together, our data demonstrate a critical function of caspase-8 in regulating intestinal homeostasis and in protecting IECs from TNF-?-induced necroptotic cell death.
Recent studies have highlighted a fundamental role for Forkhead box O (Foxo) transcription factors in immune system homeostasis. Initial reports designed to dissect function of individual Foxo isoforms in the immune system were based on in vitro overexpression systems, and these experiments suggested that Foxo1 and Foxo3 are important for growth factor withdrawal-induced cell death. Moreover, Foxo factors importantly regulate basic cell cycle progression, and so the implication was that these factors may control lymphocyte homeostasis, including a critical function in the termination and resolution of an immune response. Most recently, cell-type-specific loss mutants for the different Foxo isoforms have revealed unexpected and highly specialized functions in the control of multiple cell types in the immune system, but they have yet to reveal a role in cell death or proliferation. This review will focus on the recent advances made in the understanding of the many ways that Foxo factors regulate the immune system, including a discussion of how the specialized versus redundant functions of Foxo transcription factors impact immune system homeostasis.
Programmed cell death (PCD) occurs widely in species from every kingdom of life. It has been shown to be an integral aspect of development in multicellular organisms, and it is an essential component of the immune response to infectious agents. An analysis of the phylogenetic origin of PCD now shows that it evolved independently several times, and it is fundamental to basic cellular physiology. Undoubtedly, PCD pervades all life at every scale of analysis. These considerations provide a backdrop for understanding the complexity of intertwined, but independent, cell death programs that operate within the immune system. In particular, the contributions of apoptosis, autophagy, and necrosis in the resolution of an immune response are considered.
Foxo transcription factors integrate extrinsic signals to regulate cell division, differentiation and survival, and specific functions of lymphoid and myeloid cells. Here, we showed the absence of Foxo1 severely curtailed the development of Foxp3(+) regulatory T (Treg) cells and those that developed were nonfunctional in vivo. The loss of function included diminished CTLA-4 receptor expression as the Ctla4 gene was a direct target of Foxo1. T cell-specific loss of Foxo1 resulted in exocrine pancreatitis, hind limb paralysis, multiorgan lymphocyte infiltration, anti-nuclear antibodies and expanded germinal centers. Foxo-mediated control over Treg cell specification was further revealed by the inability of TGF-? cytokine to suppress T-bet transcription factor in the absence of Foxo1, resulting in IFN-? secretion. In addition, the absence of Foxo3 exacerbated the effects of the loss of Foxo1. Thus, Foxo transcription factors guide the contingencies of T cell differentiation and the specific functions of effector cell populations.
Although humans and chimpanzees share >99% identity in alignable protein sequences, they differ surprisingly in the incidence and severity of some common diseases. In general, humans infected with various viruses, such as HIV and hepatitis C virus, appear to develop stronger reactions and long-term complications. Humans also appear to suffer more from other diseases associated with over-reactivity of the adaptive immune system, such as asthma, psoriasis, and rheumatoid arthritis. In this study, we show that human T cells are more reactive than chimpanzee T cells to a wide variety of stimuli, including anti-TCR Abs of multiple isotypes, l-phytohemagglutin, Staphylococcus aureus superantigen, a superagonist anti-CD28 Ab, and in MLRs. We also extend this observation to B cells, again showing a human propensity to react more strongly to stimuli. Finally, we show a relative increase in activation markers and cytokine production in human lymphocytes in response to uridine-rich (viral-like) ssRNA. Thus, humans manifest a generalized lymphocyte over-reactivity relative to chimpanzees, a finding that is correlated with decreased levels of inhibitory sialic acid-recognizing Ig-superfamily lectins (Siglecs; particularly Siglec-5) on human T and B cells. Furthermore, Siglec-5 levels are upregulated by activation in chimpanzee but not human lymphocytes, and human T cell reactivity can be downmodulated by forced expression of Siglec-5. Thus, a key difference in the immune reactivity of chimp and human lymphocytes appears to be related to the differential expression of Siglec-5. Taken together, these data may help explain human propensities for diseases associated with excessive activation of the adaptive immune system.
Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.
A screen for increased longevity in Caenorhabditis elegans has identified a transcription factor that programs cells for resistance to oxidative stress, DNA repair and cell cycle control. The mammalian orthologs of this factor are referred to as Foxo for Forkhead box, with the second o in the name denoting a subfamily of four members related by sequence. This family of factors is regulated by growth factors, oxidative stress or nutrient deprivation. Thus, it might readily control the inflammatory conflagration associated with infection-driven lymphocyte proliferation. Surprisingly, the first insights into Foxo-mediated immune regulation have instead revealed direct control of highly specialized genes of the adaptive immune system.
A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Because the signaling molecules RAS and ERK1/2 (extracellular signal-regulated kinases 1 and 2) are activated by an LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBPbeta (CCAAT/Enhancer-binding protein-beta) is a critical downstream mediator of ERK1/2 activation. Thus, ERK1/2 and C/EBPbeta constitute an in vivo LH-regulated signaling pathway that controls ovulation- and luteinization-related events.
Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.
Foxo transcription factors have a conserved role in the adaptation of cells and organisms to nutrient and growth factor availability. Here we show that Foxo1 has a crucial, nonredundant role in T cells. In naive T cells, Foxo1 controlled the expression of the adhesion molecule L-selectin, the chemokine receptor CCR7 and the transcription factor Klf2, and its deletion was sufficient to alter lymphocyte trafficking. Furthermore, Foxo1 deficiency resulted in a severe defect in interleukin 7 receptor alpha-chain (IL-7Ralpha) expression associated with its ability to bind an Il7r enhancer. Finally, growth factor withdrawal induced a Foxo1-dependent increase in Sell, Klf2 and Il7r expression. These data suggest that Foxo1 regulates the homeostasis and life span of naive T cells by sensing growth factor availability and regulating homing and survival signals.
The outcome of an infection with any given pathogen varies according to the dosage and route of infection, but, in addition, the physiological state of the host can determine the efficacy of clearance, the severity of infection and the extent of immunopathology. Here we propose that the forkhead box O (FOXO) transcription factor family--which is central to the integration of growth factor signalling, oxidative stress and inflammation--provides connections between physical well-being and the form and magnitude of an immune response. We present a case that FOXO transcription factors guide T cell differentiation and function in a context-driven manner, and might provide a link between metabolism and immunity.
One of the fundamental paradigms in the use of nanoparticles to treat disease is to evade or suppress the immune system in order to minimize systemic side effects and deliver sufficient nanoparticle quantities to the intended tissues. However, the immune system is the bodys most important and effective defense against diseases. It protects the host by identifying and eliminating foreign pathogens as well as self-malignancies. Here we report a nanoparticle engineered to work with the immune system, enhancing the intended activation of antigen presenting cells (APCs). We show that luminescent porous silicon nanoparticles (LPSiNPs), each containing multiple copies of an agonistic antibody (FGK45) to the APC receptor CD40, greatly enhance activation of B cells. The cellular response to the nanoparticle-based stimulators is equivalent to a 30-40 fold larger concentration of free FGK45. The intrinsic near-infrared photoluminescence of LPSiNPs is used to monitor degradation and track the nanoparticles inside APCs.
Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-?, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.