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Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca2+. Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca2+-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (Ipr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient Ipr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4−CD8−TCRαβ+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.

γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress.

IL-6 promotes the differentiation of a subset of naïve CD8+ T cells into IL-21–producing B helper CD8+ T cells., IL-6 is known to contribute to the differentiation of CD4+ T cells into different subsets of effector T helper cells. Less is known about the potential of IL-6 in regulating CD8+ T cell effector function. Here, we identify IL-6 as a master regulator of IL-21 in effector CD8+ T cells. IL-6 promotes the differentiation of a subset of naive CD8+ T cells that express IL-6R into a unique population of effector CD8+ T cells characterized by the production of high levels of IL-21 and low levels of IFN-γ. Similar to CD4+ T follicular helper (Tfh) cells, IL-21–producing CD8+ T cells generated in the presence of IL-6 directly provide help to B cells to induce isotype switching. CD8+ T cell–derived IL-21 contributes to the production of protective virus-specific IgG antibodies during influenza virus infection. Thus, this study reveals the presence of a new mechanism by which IL-6 regulates antibody production during viral infection, and a novel function of effector CD8+ T cells in the protection against viruses.

Summary Caspase-8 serves two paradoxical roles in T lymphocytes: it initiates apoptosis following death receptor engagement, and is also indispensible for proliferation following T-cell antigen receptor (TCR) signalling. These opposing processes appear to be controlled by both spatial and quantitative differences in caspase-8 activation. Given differences in the turnover of T-cell subsets, we compared caspase activity and susceptibility to cell death following TCR restimulation in murine CD4 + and CD8 + ab T cells and cd T cells. We observed a spectrum of caspase activity in non-dying effector T cells in which CD4 + T cells manifested the lowest levels of active caspases whereas cd T cells manifested the highest levels. Further analysis revealed that most of the difference in T-cell subsets was the result of high levels of active caspase-3 in non-dying effector cd T cells. Despite this, cd T cells manifested little spontaneous or CD3 restimulation-induced cell death as the result of confinement of active caspases to the cell membrane. By contrast, CD4 + T cells were highly sensitive to CD3-induced cell death, associated with the appearance of active caspases in the cytoplasm and cleavage of the caspase substrates Bid and ICAD. Hence, the location and amount of active caspases distinguishes effector T-cell subsets and profoundly influences the fate of the T-cell response.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Caspase activity is required not only for the death of T cells, but also for their activation. A delicate balance of caspase activity is thus required during T cell activation at a level that will not drive cell death. How caspase activity is initiated and regulated during T cell activation is not known. One logical candidate for this process is cellular FLIP long form (c-FLIPL), because it can block caspase-8 recruitment after Fas (CD95) ligation as well as directly heterodimerize with and activate caspase-8. The current findings demonstrate that after T cell activation, caspase-8 and c-FLIPL associate in a complex enriched for active caspases. This occurs coincidently with the cleavage of two known caspase-8 substrates, c-FLIPL and receptor interacting protein 1. Caspase activity is higher in wild-type CD8+ than CD4+ effector T cells. Increased expression of c-FLIPL results in augmented caspase activity in resting and effector T cells to levels that provoke cell death, especially of the CD8 subset. c-FLIPL is thus not only an inhibitor of cell death by Fas, it can also act as a principal activator of caspases independently of Fas.

Cellular FLIP long form (c-FLIPL) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIPL in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIPL with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIPL-transgenic mice. In this study we show that activated CD4+ T cells from c-FLIPL-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIPL-transgenic CD4+ T cells parallels impaired NF-κB activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-γ and increased Th2 cytokines. The Th2 bias of c-FLIPL-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIPL can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.

Mitogen-activated protein (MAP) kinases, which include the extracellular response kinases, p38 and c-Jun amino terminal kinases (JNK), play a significant role in mediating signals triggered by cytokines, growth factors and environmental stress. The JNK and p38 MAP kinases have been involved in growth, differentiation and cell death in different cell types. In the present paper, we describe how the JNK and p38 MAP kinase signalling pathways are regulated and their role during thymocyte development and the activation and differentiation of T cells in the peripheral immune system. The results from these studies demonstrate that the JNK and p38 MAP kinase signalling pathways regulate different aspects of T-cell mediated immune responses.

The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is strictly regulated during the differentiation of CD4−CD8− thymocytes. Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25+CD44− stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice. Inactivation of p38 MAP kinase is required for further differentiation of these cells into CD4+CD8+ thymocytes. The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation. Therefore, the p38 MAP kinase pathway is a critical regulatory element of differentiation and proliferation during the early stages of in vivo thymocyte development.

CD2 is a cell surface glycoprotein expressed on most T lymphocytes that is generally viewed as a cell adhesion molecule and, in this capacity, contributes to T cell receptor (TCR) signaling. CD2 has a relatively long cytoplasmic tail which associates with the src family tyrosine kinases, p56(lck) and p59(fyn), and could potentially signal directly. Down-modulation of CD2 on T cells has been shown to result in diminished proliferative capacity and interleukin (IL)-2 production. Furthermore, re-expression of CD2 can result in the restoration of these functions. This suggests that CD2 can influence the intensity of TCR signaling. As TCR signal intensity is pivotal to the induction of T cell apoptosis, we considered the hypothesis that the level of CD2 on the T cell surface may influence its propensity toward apoptosis. Using an anti-CD2 antibody, CD2 was down-modulated in vivo on mouse T lymphocytes without affecting the levels of surface CD3, TCR alphabeta, CD4 or CD8. Deletion of superantigen-responsive T cells was delayed in mice with down-modulated CD2 following the administration of staphylococcal enterotoxin B (SEB). This was paralleled by diminished apoptosis of SEB-responsive cells. The findings suggest a model whereby the level of CD2 expression influences the intensity of TCR signaling and the ability to undergo apoptosis.

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co-stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas-induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf-1/MAPK pathway versus JNK activation by Fas.

γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function and manifesting cytolytic activity. Their numbers often increase during infections, such as human immunodeficiency virus, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we observed that despite more rapid proliferation in vitro by human Lyme arthritis synovial γδ T cells of the Vδ1 subset, they have reduced surviving cell numbers compared with αβ T cells because of increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and because signaling is more efficient through T cell receptor (TCR)-γδ than through TCR-αβ, we examined the levels of active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than in comparable αβ T cells. This was paralleled by greater spontaneous cell death and TCR restimulation-induced cell death of the γδ T cells, which was caspase dependent. Our current findings thus are consistent with a model in which human γδ T cells evolved to function quickly and transiently in an innate fashion.

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are γδ T cells. γδ T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human γδ T cells of the Vδ1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine γδ T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of γδ T cells, are both caspase-dependent.

Effective stimulation of NF-kappaB in T cells following TCR ligation requires the activity of caspase-8. The active caspase-8 complex includes the paracaspase, MALT1, and Bcl-10, which connect to the NF-kappaB pathway. It has been less clear what regulates the level of caspase-8 activity during T cell activation. A likely candidate is cellular FLIP (c-FLIP), an enzymatically inert caspase-8 homologue. Two alternatively spliced forms of c-FLIP exist, a long form (c-FLIP(L)) and a short-form (c-FLIP(S)). The latter lacks the C-terminal caspase-like domain. c-FLIP(L) can heterodimerize with and activate caspase-8 through an activation loop in the C terminus of c-FLIP(L). Here we show that, in contrast to c-FLIP(L), c-FLIP(S) inhibits activation of caspase-8 in T cells, and consequently reduces recruitment of MALT1 and Bcl-10 to the active caspase complex. This results in reduced activity of NF-kappaB. Consequently, T cells from c-FLIP(S)-transgenic mice undergo more rapid cell death both spontaneously and after activation. The findings suggest that c-FLIP(S) functions to reduce the expansion of T cells during an immune response.

Lyme disease represents a complex response toBorrelia burgdorferithat involves both bacterial factors as well as host responses. This results in an inflammatory reaction at several sites, including the synovial lining of joints. Synovial tissues of inflamed joints contain cells expressing high levels of Fas and Fas ligand (FasL). Although Fas stimulation is typically associated with cell death, it can also transmit stimulatory signals to certain cell types. Among these are dendritic cells and macrophages, which are abundant in inflamed synovium. To better assess the role of FasL in the pathogenesis of Lyme arthritis, we evaluated the response toB. burgdorferiinfection in C3H/HeJgldmice that bear a nonfunctional mutation in FasL. Compared to wild-type C3H+/+mice, C3Hgldmice had a similar bacterial burden and antibody response 2 weeks and 4 weeks following infection, but they manifested a significantly reducedBorrelia-specific cytokine response. In addition, C3Hgldmice developed a greatly reduced incidence and severity of arthritis. The findings document a contribution of FasL to the host inflammatory response toB. burgdorferi.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Here we discuss the possibility that the way cells utilize fuel(s) for energy confers the properties that can be recognized by the immune system and, reciprocally, that recognition by the immune system can alter the balance of the cell's energy metabolism. We propose that immune recognition, of somatic cells via MHC can alter the their energy metabolism and induce a metabolic shift. We demonstrate the reciprocal relationship that inducing a shift in metabolism toward glycolysis by supplying glucose and insulin results in the upregulation of immunologically recognizable molecules such as cell surface Fas. Thus, immune recognition can induce metabolic deviation. Metabolic deviation can result in altered immune recognition and ultimately in cell proliferation, cell differentiation, or cell death.

Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4(+) T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and TH17 polarization. TH17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4(+) T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4(+) T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4(+) T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4(+) T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4(+) T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.