OX40 costimulatory signals potentiate the memory commitment of effector CD8+ T cells

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8(+) T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8(+) T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8(+) T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1(low) MPECs, and, subsequently, markedly impaired the generation of memory CD8(+) T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1(low) MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8(+) T cells.     

CD40-40L signaling in vascular inflammation

Ligation of CD40 in circulating cells or in the vessel wall may promote mononuclear cell recruitment, participate in the weakening of the plaque, and contribute to thrombosis. This process appears to be redox-sensitive, but the precise signaling mechanism by which the interaction between CD40L and its receptor CD40 mediates inflammatory secretion is unclear. Our previous studies have shown that the CD40-CD40L interaction modulates release of reactive oxygen species (ROS) and the current findings demonstrate that in endothelial cells CD40L dose dependently induces intracellular CD40L and MCP1 release in a redox sensitive manner. Pharmacological inhibition of phosphatidylinositol 3-kinase and p38 MAPK as well as adenovirus-mediated inactivation of Akt and p38 MAPK inhibited CD40L effects on endothelial cells. Akt, in particular, appeared to mediate CD40L-induced CD40L synthesis and MCP1 release by endothelial cells in a redox sensitive manner via NFkappaB activation. In addition, using confocal microscopy, exogenous addition of recombinant CD40L or adenoviral mediated CD40L overexpression was found to stimulate nuclear translocation of NFkappaB, which was further augmented by Akt overexpression and inhibited by Akt inactivation. These data support a mechanism whereby redox-sensitive CD40-CD40L interactions induce activation of Akt and p38 MAPK, leading to stimulation of NFkappaB and enhanced synthesis of CD40L and MCP1. Increased CD40L and MCP1 may contribute to the adherence of CD40-positive cells, such as platelets and monocytes, to the vessel wall modulating atherothrombosis.     

Enzymatic reduction of oxysterols impairs LXR signaling in cultured cells and the livers of mice

Liver X receptors (LXRs) are nuclear receptors that play crucial roles in lipid metabolism in vivo and are activated by oxysterol ligands in vitro. The identity of the ligand that activates LXRs in vivo is uncertain. Here we provide two lines of evidence that oxysterols are LXR ligands in vitro and in vivo. First, overexpression of an oxysterol catabolic enzyme, cholesterol sulfotransferase, inactivates LXR signaling in several cultured mammalian cell lines but does not alter receptor response to the nonsterol agonist T0901317. Adenovirus-mediated expression of the enzyme in mice prevents dietary induction of hepatic LXR target genes by cholesterol but not by T0901317. Second, triple-knockout mice deficient in the biosynthesis of three oxysterol ligands of LXRs, 24S-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, respond to dietary T0901317 by inducing LXR target genes in liver but show impaired responses to dietary cholesterol. We conclude that oxysterols are in vivo ligands for LXR.     

CD40L启动CD4^＋T细胞缺乏小鼠免疫反应的研究

随着HIV感染者及各类医疗措施导致的免疫受损者的增多,探讨一种适合免疫缺陷人群的预防机会感染的策略越来越受到重视。研究表明,CD4^＋T细胞是抵抗肺孢子菌等机会感染的最主要因素,但不是唯一的因素。其中CD40配体（CD40L）被认为是一种可以启动B细胞和CD8^＋T细胞反应的关键因子。为探讨CD4^＋L是否能在缺乏CD4^＋T细胞的小鼠体内启动免疫反应,本文研究了用卵白蛋白（OVA）作为模型抗原,联合应用CD40L引起的免疫反应。结果显示,同时应用OVA和CD40L,可使CD4^＋T细胞耗竭小鼠体内抗OVA IgG抗体和抗原特异性IFN明显增多,提示在CD4^＋T细胞缺乏的宿主体内,CIMOL可以启动B细胞和CD8^＋T细胞类免疫反应。该结果为抗肺孢子菌等机会性感染的免疫预防研究提供可贵的资料。     

Involvement of CD40-CD40L signaling in postischemic lung injury

Our studies show that ischemia-reperfusion (I/R) in the isolated rat lung causes retention of lymphocytes, which is associated with increased microvascular permeability, as determined by quantitative measurement of the microvascular filtration coefficient (K(f,c)). Immunoneutralization of either CD40 or CD40L, cell surface proteins important in lymphocyte-endothelial cell proinflammatory events, results in significantly lower postischemic K(f,c) values. Antagonism of CD40-CD40L signaling also results in attenuation of I/R-elicited macrophage inflammatory protein-2 production. Rat lymphocytes activated ex vivo with phorbol 12-myristate, 13-acetate increased K(f,c) in isolated lungs independently of I/R, and this increase was prevented by pretreating lungs with anti-CD40. In addition to lymphocyte involvement via CD40-CD40L interactions, our studies also show that I/R injury is potentiated by antagonism of IL-10 produced locally within the postischemic lung, whereas exogenous, rat recombinant IL-10 provided protection against I/R-induced microvascular damage. Thus acute lymphocyte involvement in lung I/R injury involves CD40-CD40L signaling mechanisms, and these events may be influenced by local IL-10 generation.     

Differential regulation of soluble and membrane CD40L proteins in T cells

CD40 ligand is an important immunoregulatory protein expressed by T cells. This protein exists as two isoforms, a membrane glycoprotein and a truncated soluble form. Here we demonstrate that membrane and soluble CD40L (sCD40L) are differentially regulated depending upon the activation stimulus. In T cell receptor activated cells, both membrane and sCD40L proteins are expressed and CD28 costimulation further increases their expression. The dissection of TCR generated signals into calcium and PKC-dependent pathways demonstrates that calcium is sufficient to induce membrane CD40L yet insufficient for sCD40L. In contrast, sCD40L is preferentially induced by PKC. Moreover, sCD40L production is blocked by Zn(2+)-dependent metalloproteinase inhibitors while membrane CD40L is concurrently increased. This profile suggests the potential involvement of the ADAM-10 protease which was subsequently shown to cleave membrane CD40L to generate sCD40L. Given the role of sCD40L in numerous disease pathologies and its ability to activate proximal and distal immune responses, the regulated cleavage of CD40L may likely contribute to disease mechanisms.     

IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells.

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1alpha/beta-deficient (IL-1(-/-)) mice and enhanced in IL-1R antagonist(-/-) mice. The intrinsic functions of T, B, and APCs were normal in IL-1(-/-) mice. However, we showed that IL-1(-/-) APCs did not fully activate DO11.10 T cells, while IL-1R antagonist (-/-) APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1(-/-) mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.     

Inhibition of terminal differentiation of B cells mediated by CD27 and CD40 involves signaling through JNK

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.     

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, 1C10) can actually promote the in vitro generation of CTL activity by CD8⁺ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220⁺ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220⁺ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220⁺ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8⁺ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220⁺ cells. Received: 23 December 1998 / Accepted: 22 February 1999     

CD40L co-stimulation from CD8+ to CD4+ effector memory T cells supports CD4+ expansion

Effector memory T cells (T(EM)) have an important role in immunity against infection. However, little is known about the factors regulating T(EM) maintenance and proliferation. In this study, we investigated the role of direct interactions between CD4(+) and CD8(+) T cells (TC) for human T(EM) expansion. Proliferation of separated or mixed CD4(+) and CD8(+)T(EM) populations was analyzed after polyclonal stimulation in vitro. Compared to each isolated subset mixed T(EM) populations showed increased proliferation and expansion of both CD4(+) and CD8(+)T(EM) subpopulations. Combined activation of CD4(+) and CD8(+) memory T cells (Tmem) induced an increased expression of CD40L and CD40 on both populations. Subsequently, CD40/CD40L caused a bi-directional stimulation of CD40(+)CD4(+)T(EM) by CD40L(+)CD8(+)T(EM) and of CD40(+)CD8(+)T(EM) by CD40L(+)CD4(+)T(EM). Blocking of CD40L on activated CD8(+)T(EM) selectively inhibited proliferation of CD4(+)T(EM), while blocking of CD40L on CD4(+)T(EM) abrogated proliferation of CD8(+)T(EM). Taken together, we demonstrate for the first time that the expression of CD40L is exploited on the one hand by CD8(+)T(EM) to increase the proliferation of activated CD4(+)T(EM) and on the other hand by CD4(+)T(EM) to support the expansion of activated CD8(+)T(EM). Thus, efficient T(EM) expansion requires bi-directional interactions between CD4(+) and CD8(+)T(EM) cells.     

HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells

Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here, we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction and that HIV utilizes the Galphai-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin-depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a postentry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.     

Galectin-9 controls CD40 signaling through a Tim-3 independent mechanism and redirects the cytokine profile of pathogenic T cells in autoimmunity

While it has long been understood that CD40 plays a critical role in the etiology of autoimmunity, glycobiology is emerging as an important contributor. CD40 signaling is also gaining further interest in transplantation and cancer therapies. Work on CD40 signaling has focused on signaling outcomes and blocking of its ligand, CD154, while little is known about the actual receptor itself and its control. We demonstrated that CD40 is in fact several receptors occurring as constellations of differentially glycosylated forms of the protein that can sometimes form hybrid receptors with other proteins. An enticing area of autoimmunity is differential glycosylation of immune molecules leading to altered signaling. Galectins interact with carbohydrates on proteins to effect such signaling alterations. Studying autoimmune prone NOD and non-autoimmune BALB/c mice, here we reveal that in-vivo CD40 signals alter the glycosylation status of non-autoimmune derived CD4 T cells to resemble that of autoimmune derived CD4 T cells. Galectin-9 interacts with CD40 and, at higher concentrations, prevents CD40 induced proliferative responses of CD4(lo)CD40(+) effector T cells and induces cell death through a Tim-3 independent mechanism. Interestingly, galectin-9, at lower concentrations, alters the surface expression of CD3, CD4, and TCR, regulating access to those molecules and thereby redirects the inflammatory cytokine phenotype and CD3 induced proliferation of autoimmune CD4(lo)CD40(+) T cells. Understanding the dynamics of the CD40 receptor(s) and the impact of glycosylation status in immunity will gain insight into how to maintain useful CD40 signals while shutting down detrimental ones.     

CD2 and CD3 associate independently with CD5 and differentially regulate signaling through CD5 in Jurkat T cells.

In T lymphocytes, the CD2 and CD5 glycoproteins are believed to be involved in the regulation of signals elicited by the TCR/CD3 complex. Here we show that CD2 and CD3 independently associate with CD5 in human PBMC and Jurkat cells. CD5 coprecipitates with CD2 in CD3-deficient cells and, conversely, coprecipitates with CD3 in cells devoid of CD2. In unstimulated CD2+ CD3+ Jurkat cells, CD5 associates equivalently with CD2 and CD3 and is as efficiently phosphorylated in CD2 as in CD3 immune complexes. However, upon activation the involvement of CD5 is the opposite in the CD2 and CD3 pathways. CD5 becomes rapidly tyrosine phosphorylated after CD3 stimulation, but is dephosphorylated upon CD2 cross-linking. These opposing effects correlate with the decrease in the activity of the SH2 domain-containing protein phosphatase 1 (SHP-1) following CD3 activation vs an enhanced activity of the phosphatase after CD2 triggering. The failure of CD5 to become phosphorylated on tyrosine residues in the CD2 pathway has no parallel with the lack of use of zeta-chains in CD2 signaling; contrasting with comparable levels of association of CD2 or CD3 with CD5, zeta associates with CD2 only residually and is nevertheless slightly phosphorylated after CD2 stimulation. The modulation of CD5 phosphorylation may thus represent a level of regulation controlled by CD2 in signal transduction mechanisms in human T lymphocytes.     

Act1 modulates autoimmunity through its dual functions in CD40L/BAFF and IL-17 signaling

Coordinated regulation of T and B cell-mediated immune responses plays a critical role in the control and modulation of autoimmune diseases. This review is focused on the adapter molecule Act1 and its regulation of autoimmunity through its impact on both T and B cell-mediated immune responses. Whereas Act1 molecule is an important negative regulator for B cell-mediated humoral immune responses through its function in CD40L and BAFF signaling, recent studies have shown that Act1 is also a key positive signaling component for IL-17 signaling pathway, critical for T(H)17-mediated autoimmune and inflammatory responses. The dual functions of Act1 are evident in Act1-deficient mice that displayed B cell-mediated autoimmune phenotypes (including dramatic increase in peripheral B cells, lymphadenopathy and splenomegaly, hypergammaglobulinemia and Sjogren's disease in association with Lupus Nephritis), but showed resistance to T(H)17-dependent EAE and colitis. Such seemingly opposite functions of Act1 in CD40-BAFFR and IL-17R signaling are orchestrated by different domains in Act1. Whereas Act1 interacts with the IL-17R through the C-terminal SEFIR domain, Act1 is recruited to CD40 and BAFFR indirectly, which is mediated by TRAF3 through the TRAF binding site in Act1. Such delicate regulatory mechanisms may provide a common vehicle to promote balance between host defense to pathogens and tolerance to self.     

Estrogen deficiency, T cells and bone loss

Estrogen plays a fundamental role in the maintenance of skeletal homeostasis. Although estrogen is established to have direct effects on bone cells, animal studies have identified additional regulatory effects of estrogen centered at the level of the adaptive immune response. Furthermore, a potential role for reactive oxygen species has now been identified in both humans and animals. One of the major challenges has been to integrate a multitude of redundant pathways and cytokines, that all appear capable of playing a relevant role, into a global model of postmenopausal osteoporosis. This review presents our current understanding of the process of estrogen deficiency mediated bone destruction and explores some of the most recent findings and hypotheses to explain estrogen action in bone.     

The duration of signaling through CD40 directs biological ability of dendritic cells to induce antitumor immunity

Although it has been demonstrated that the functions of dendritic cells (DCs), including Ag capture, Ag presentation, and migratory activity, change dynamically with their maturation, the most appropriate conditioning of DCs for anticancer immunotherapy is still unclear. The help signal is one of the most potent stimuli for DC maturation and is provided by the interaction of CD40 expressed on DCs with CD40 ligand on CD4(+) T cells. To elucidate the appropriate conditioning of DCs for anticancer immunotherapy, we examined the biological activity of DCs stimulated with immobilized anti-CD40 Ab. DCs stimulated for 3 h (3h-DCs) still showed an immature phenotype, but exhibited augmented migration toward secondary lymphoid tissues. Subcutaneous injection of 3h-DCs facilitated priming of T cells, which could mediate potent antitumor therapeutic efficacy, in draining lymph nodes and successfully induced protective immunity. In contrast, 24h-DCs showed a mature phenotype with good Ag presentation ability to induce cell killing by adoptively transferred CD8(+) T cells when injected at tumor sites; however, they showed no migratory activity and were unable to induce protective immunity when injected s.c. This is the first report that functionally distinct DCs, either for the priming phase or for the effector phase, could be obtained by conditioning with CD40 stimulation and that the duration of stimulation determines the biological outcome. The usage of DCs conditioned for the priming phase might provide significant advantages in anticancer immunotherapy.     

Cooperativity in the interaction of synthetic CD40L mimetics with CD40 and its implication in cell signaling

CD40 ligand (CD40L) and CD40 are members of the tumor necrosis factor (TNF) and TNF receptor superfamilies, respectively. Their interaction is crucial for the development of a proper immune response. Intervention on this pathway provides an important ground for new treatments targeting autoimmune diseases or helping to fight infection and cancer. We have recently reported on the structure-based design of synthetic molecules with C3 symmetry, named mini-CD40Ls, that can effectively mimic homotrimeric soluble CD40L. Here we show that substitution of a D-prolyl residue for the glycyl within the Lys-Gly-Tyr-Tyr CD40-binding motif leads to a complete loss of cooperativity in the interaction of the mimetic with its cognate receptor as assessed by surface plasmon resonance experiments. The ability of the modified mini-CD40L to induce apoptosis on both human and murine lymphoma cells was not affected by this mutation. However, it was unable to induce the NF-kappaB pathway in the mouse D1 dendritic cell line, which is essential for its complete maturation, but still activated production of IL-12 p40 mRNA. These differential effects might be partly explained by the change in rigidity of the CD40 recognition element. In this study, we not only point out the consequences of the abrogation of the cooperative property in a ligand-receptor interaction on downstream cellular events but also demonstrate the usefulness of synthetic multivalent ligands in dissecting the complex mechanisms implicated in the signalosome.     

CD40-CD40 ligand-independent activation of CD8+ T cells can trigger allograft rejection

In experimental transplantation, blockade of CD40-CD40 ligand (CD40L) interactions has proved effective at permitting long-term graft survival and has recently been approved for clinical evaluation. We show that CD4+ T cell-mediated rejection is prevented by anti-CD40L mAb therapy but that CD8+ T cells remain fully functional. Furthermore, blocking CD40L interactions has no effect on CD8+ T cell activation, proliferation, differentiation, homing to the target allograft, or cytokine production. We conclude that CD40L is not an important costimulatory molecule for CD8+ T cell activation and that following transplantation donor APC can activate recipient CD8+ T cells directly without first being primed by CD4+ T cells.