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-b, 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. T he differentiation of T cells in response to infectious agents constitutes an essential step in effective immune responses. Minimally, Ag-stimulated T cells can take on one of four programs of cytokine production and effector function: IFN-g–secreting Th1 cells effective against viruses and intracellular microbes, IL-4–secreting Th2 cells most effective against hel-minths, IL-17–secreting Th17 cells effective against extracellular microbes, and induced regulatory T cells (iTregs) that limit inflammation and autoimmunity (1). These differentiation programs are engaged in response to cytokines and the conditions of Ag presentation, but the T cell-intrinsic signaling pathways that are operative are not entirely understood. The signaling pathway from membrane-bound receptors to the activation of Erk is found in every cell type in metazoan species. Erk interacts with many substrates and regulators to modulate signal transduction in the cytoplasm, as well as to program gene expression in the nucleus (2, 3). It constitutes a highly connected node in the matrix of cell signaling. The effects include cell-cycle progression, lineage specification and cellular differentiation, survival , chemotaxis, learning and memory, and the elaboration of cell-type–specific functions. The two Erk isoforms appear to act additively in cell cycle control (4); a loss of Erk1 (Mpk3) function reveals few phenotypic changes, whereas even a hemizygous loss of Erk2 (Mpk1) causes embryonic lethality in some strains of mice (5–8). The origin of Erk2 loss-of-function lethality was due, in part, to failure of the polar …

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-β, 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.

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.

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.

This paper provides evidence for a role of caspase 8 in skin homeostasis. Loss of epidermal caspase 8, an important mediator of apoptosis, promotes several phases of the wound healing response in mice, including epidermal cell proliferation and inflammation. The findings illustrate how the loss of caspase 8 can have an impact beyond programmed cell death to affect the local microenvironment and elicit processes common to wound repair and many neoplastic skin disorders. This paper provides evidence for a role of caspase 8 in skin homeostasis. Loss of epidermal caspase 8 promotes epidermal cell proliferation and inflammation, and authors propose that caspase 8 expression by keratinocytes in the granular layer limits the release of preformed IL1α. Tissue homeostasis and regeneration are regulated by an intricate balance of seemingly competing processes—proliferation versus differentiation, and cell death versus survival1. Here we demonstrate that the loss of epidermal caspase 8, an important mediator of apoptosis2, recapitulates several phases of a wound healing response in the mouse. The epidermal hyperplasia in the caspase 8 null skin is the culmination of signals exchanged between epidermal keratinocytes, dermal fibroblasts and leukocytic cells. This reciprocal interaction is initiated by the paracrine signalling of interleukin 1α (IL1α), which activates both skin stem cell proliferation and cutaneous inflammation. The non-canonical secretion of IL1α is induced by a p38-MAPK-mediated upregulation of NALP3 (also known as NLRP3), leading to inflammasome assembly and caspase 1 activation. Notably, the increased proliferation of basal keratinocytes is counterbalanced by the growth arrest of suprabasal keratinocytes in the stratified epidermis by IL1α-dependent NFκB signalling. Altogether, our findings illustrate how the loss of caspase 8 can affect more than programmed cell death to alter the local microenvironment and elicit processes common to wound repair and many neoplastic skin disorders.

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.

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.

The NF-kappa B signaling system has important and distinct roles in determining cell fate decisions, such as cell proliferation and cell death. Specifically, recent evidence indicates that NF-B regulates several types of programmed cell death, such as apoptosis, necroptosis, necrosis, as well as cellular senescence, but its precise rote in these is not fully understood. Distinguishing these cell fates experimentally is therefore important, and several techniques are available to researchers. We summarize experimental strategies and protocols that reveal changes in nuclear morphology and cell shrinkage, exposure of phosphatidylserine, compromised membrane integrity, DNA fragmentation, and altered mitochondrial membrane potential. Together, these may discriminate distinct cell death pathways and lead to a better understanding of the underlying regulatory mechanisms.

T cells enigmatically require caspase-8, an inducer of apoptosis, for antigen-driven expansion and effective antiviral responses, and yet the pathways responsible for this effect have been elusive. A defect in caspase-8 expression does not affect progression through the cell cycle but causes an abnormally high rate of cell death that is distinct from apoptosis and does not involve a loss of NFκB activation. Instead, antigen or mitogen activated Casp8-deficient T cells exhibit an alternative type of cell death similar to programmed necrosis that depends on receptor interacting protein (Ripk1). The selective genetic ablation of caspase-8, NFκB, and Ripk1, reveals two forms of cell death that can regulate virus-specific T cell expansion.

The NF‐κB signaling system has important and distinct roles in determining cell fate decisions, such as cell proliferation and cell death. Specifically, recent evidence indicates that NF‐B regulates several types of programmed cell death, such as apoptosis, necroptosis, necrosis, as well as cellular senescence, but its precise role in these is not fully understood. Distinguishing these cell fates experimentally is therefore important, and several techniques are available to researchers. We summarize experimental strategies and protocols that reveal changes in nuclear morphology and cell shrinkage, exposure of phosphatidylserine, compromised membrane integrity, DNA fragmentation, and altered mitochondrial membrane potential. Together, these may discriminate distinct cell death pathways and lead to a better understanding of the underlying regulatory mechanisms.

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

Caspase-8 is a cysteine protease that is an essential initiator of death receptor-mediated apoptosis. Paradoxically, it has been shown that T cells require Caspase-8 for proliferation following antigenic or mitogenic stimulation. We have generated a T cell-specific conditional deletion of Caspase-8, and found that deletion of Caspase-8 causes profound hypo-proliferation in T cells, both in vitro and in an immune response to LCMV. Further characterization has revealed that T cells deficient in Caspase-8 do not have a defect in cell cycle progression, but rather experience an abnormally high rate of cell death that does not exhibit hallmarks of classic apoptosis. Contrary to published work, the loss of cell viability does not result from a defect in NFkB activation. Instead, we find Caspase-8 deficient T cells die by an alternative form of cell death.

Caspase-8 is an essential component of death receptor-mediated apoptosis. Along with Fas-associated death domain protein, it is also essential for T cell proliferation in response to antigenic or mitogenic stimuli. To determine whether caspase-8 is also required for B cell proliferation, we generated mice with a B cell-specific Casp8 deficiency. Unlike T cells, caspase-8 was not required for Ag receptor-driven proliferation or Ab formation. Rather, Casp8-deficient B cells failed to proliferate in response to dsRNA and LPS, ligands for TLR3 and TLR4, respectively, but responded normally to the TLR9 agonist CpG DNA. Similarly, Ab production to trinitrophenol-LPS was selectively reduced in B cell-specific Casp8-deficient mice. The activation of NF-kappa B or IFN regulatory factor 3 was found to be unaffected by the loss of caspase-8, implicating it in a novel pathway important for some forms of innate immunity mediated by B cells.

Stimulation of the TCR leads to an oscillatory release of free calcium that activates members of the calcium/calmodulin-dependent protein kinase II (CaMKII) family. The CaMKII molecules have profound and lasting effects on cellular signaling in several cell types, yet the role of CaMKII in T cells is still poorly characterized. In this report we describe a splice variant of CaMKIIbeta, CaMKIIbeta'e, in mouse T cells. We have determined its function, along with that of CaMKIIgamma, by introducing the active and kinase-dead mutants into activated P14 TCR transgenic T cells using retroviral transduction. Active CaMKII enhanced the proliferation and cytotoxic activity of T cells while reducing their IL-2 production. Furthermore, it induced a profound state of unresponsiveness that could be overcome only by prolonged culture in IL-2. These results indicate that members of the CaMKII family play an important role in regulation of CD8 T cell proliferation, cytotoxic effector function, and the response to restimulation.