The natural killer (NK) cell receptor NKp30 is involved in the recognition of tumor and dendritic cells (DCs). Here we describe the influence of three NKp30 splice variants on the prognosis of gastrointestinal sarcoma (GIST), a malignancy that expresses NKp30 ligands and that is treated with NK-stimulatory KIT tyrosine kinase inhibitors. Healthy individuals and those with GIST show distinct patterns of transcription of functionally different NKp30 isoforms. In a retrospective analysis of 80 individuals with GIST, predominant expression of the immunosuppressive NKp30c isoform (over the immunostimulatory NKp30a and NKp30b isoforms) was associated with reduced survival of subjects, decreased NKp30-dependent tumor necrosis factor-α (TNF-α) and CD107a release, and defective interferon-γ (IFN-γ) and interleukin-12 (IL-12) secretion in the NK-DC cross-talk that could be restored by blocking of IL-10. Preferential NKp30c expression resulted partly from a single-nucleotide polymorphism at position 3790 in the 3' untranslated region of the gene encoding NKp30. The genetically determined NKp30 status predicts the clinical outcomes of individuals with GIST independently from KIT mutation.

Interleukin (IL)-10 and glucocorticoids (GCs) inhibit the ability of antigen-presenting dendritic cells (DCs) to stimulate T lymphocytes. We show that induction of GILZ (GC-induced leucine zipper) is involved in this phenomenon. IL-10, dexamethasone (DEX), and transforming growth factor (TGF)beta stimulate GILZ production in human immature DCs derived from monocytes and from CD34+ cells. GILZ is necessary and sufficient for DEX, IL-10, and TGFbeta modulation of CD80, CD83, CD86, immunoglobulin-like transcript (ILT)-3, and B7-H1 expression by DCs, and alteration of DC functions. GILZ stimulates the production of IL-10 by immature DCs and prevents the production of inflammatory chemokines by CD40L-activated DCs. In contrast, GILZ does not prevent CD40 ligand-mediated inhibition of phagocytosis, indicating that it affects some but not all aspects of DC maturation. GILZ prevents DCs from activating antigen-specific T lymphocyte responses. Administration of GCs to patients stimulates GILZ expression in their circulating antigen-presenting cells, and this contributes to the weak lymphocyte responses of GC-treated patients. Thus, regulation of GILZ expression is an important factor determining the decision of DCs whether or not to stimulate T lymphocytes, and IL-10, GCs, and TGFbeta share this mechanism for influencing DC functions and the balance between immune response and tolerance.

Interleukin-2 (IL-2) withdrawal is a physiologic process inducing cell death in activated T lymphocytes. Glucocorticoidinduced leucine zipper (GILZ) has recently been identified as a protein modulating T-cell receptor activation by repressing various signaling pathways. We report here that IL-2 deprivation leads to expression of GILZ in T lymphocytes. We then characterized the human gilz promoter and showed that FoxO3 (Forkhead box class O3) binding to the Forkhead responsive elements identified in the promoter is necessary for induction of gilz expression upon IL-2 withdrawal. To assess the functional consequences of this induction, we used 2 strategies, GILZ overexpression and GILZ silencing in murine IL-2-dependent CTLL-2 cells. GILZ overexpression protects CTLL-2 cells from IL-2 withdrawal-induced apoptosis, whereas cell death is accelerated in cells unable to express GILZ. Concomitantly, the expression of Bim is inhibited in GILZ-overexpressing cells and enhanced when GILZ expression is impaired. Furthermore, GILZ inhibits FoxO3 transcriptional activity that leads to inhibition of Bim expression but also to down-regulation of GILZ itself. Therefore, GILZ is a transiently expressed protein induced upon IL-2 withdrawal that protects T cells from the onset of apoptosis. ( IntroductionA complex network of cytokines controls the development, maturation, homeostasis, and responses of the immune system. Immune responses typically involve clonal expansion of activated T cells, which must be eliminated at the end of the response to preserve homeostasis. 1 Interleukin-2 (IL-2) is secreted by activated T cells and plays a role in their survival and proliferation. IL-2 is also involved in the regulation of homeostasis of the lymphoid tissue, as IL-2 induces apoptosis of activated T cells in an "active" way involving regulation of Fas and Fas ligand (FasL) expression and in a "passive" way due to deprivation of this survival factor. 2 Binding of IL-2 to its receptor triggers a signaling cascade that induces, among others, the phosphoinositide 3-kinase (PI3K) pathway involved in T-cell proliferation and survival. 3,4 One of the downstream effectors of PI3K signaling is protein kinase B (PKB)/Akt. PKB is a serine/threonine kinase promoting cell survival by targeting proapoptotic proteins. 5 PKB exerts one of its inhibitory effects by phosphorylating members of the Forkhead family of transcription factors (Forkhead box class O1 [FoxO1], FoxO3, and FoxO4), which contain 3 RXRXXS/T consensus phosphorylation sites for PKB. 6 Interruption of the PI3K/PKB pathway following IL-2 deprivation results in dephosphorylation of FoxO proteins leading to their translocation to the nucleus and transcription of target genes.Brunet et al 7 were the first to ascribe a role to FoxO proteins in programmed cell death, reporting that overexpression of a constitutive active form of FoxO3 induces apoptosis of Jurkat T cells in a Fas-dependent manner. Fas-independent mechanisms of apoptosis induced by FoxO proteins have also been observed in othe...

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Neutrophil extracellular traps (NETs) are extracellular DNA filaments formed during neutrophil activation. This process, called netosis, was originally associated with neutrophil antibacterial properties. However, several lines of evidence now suggest a major role for netosis in thrombosis, autoimmune diseases, and cancer. We demonstrate here that highly purified human blood monocytes are also capable of extracellular trap (ET) release in response to several stimuli. Monocyte ETs display a morphology analogous to NETs and are associated with myeloperoxidase (MPO), lactoferrin (LF), citrullinated histones, and elastase. Monocyte ET release depends on oxidative burst but not on MPO activity, in contrast to neutrophils. Moreover, we demonstrate procoagulant activity for monocyte ETs, a feature that could be relevant to monocyte thrombogenic properties. This new cellular mechanism is likely to have implications in the multiple pathologic contexts where monocytes are implicated, such as inflammatory disorders, infection, or thrombosis.

The chimeric antibodies anti-CD20 rituximab (Rtx) and anti-TNFα infliximab (Ifx) induce antidrug antibodies (ADAs) in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA)-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs) loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.

Dendritic cells (DCs) play an important role in bridging innate and adaptive immunity. These APCs have the ability to recognize specific molecular signatures of pathogens through TLRs. In particular, the intracellular TLR7 and TLR8, mediating the recognition of ssRNA by DCs, play a major role in the immune response during viral infection. Although differences have been identified between TLR7 and TLR8, in terms of cellular expression and functions, the signaling pathways that lead to DC maturation following TLR7 or TLR8 engagement are largely unknown. We compared the signaling pathways involved in human CD34-DC maturation induced by agonists selective for TLR7 (imiquimod) or TLR8 (3M002). TLR7 and TLR8 activation up-regulated CCR7, CD40, CD86, and CD83 expression and IL-6 and IL-12p40 production. However, only TLR8 activation led to IL-12p70 production and il-12p35 mRNA expression. We found that upon TLR7 and TLR8 activation, JNK and NF-kappaB positively regulated the expression of CCR7, CD86, CD83, and CD40 and the production of IL-6 and IL-12p40. However, although p38MAPK participated in the up-regulation of maturation markers in response to TLR7 activation, this kinase exerted an inhibitory effect on CD40 expression and IL-12 production in TLR8-stimulated DCs. We also showed that the Jak/STAT signaling pathway was involved in CD40 expression and cytokine production in TLR7-stimulated DCs but negatively regulated CD83 expression and cytokine secretion in DCs activated through TLR8. This study showed that TLR7 and TLR8 activate similar signaling pathways that play different roles in DC maturation, depending on which TLR is triggered.

After application of haptens on the skin, immature dendritic cells (DC), also named Langerhans cells (LC), migrate to the draining lymph node to sensitize naïve T-lymphocytes. Migration of DC involves many factors including the Cys-Cys chemokine receptor, CCR7. We investigated the effects of two well-known haptens, dinitrochlorobenzene (DNCB) and nickel (NiSO(4)), on the expression of CCR7 on human DC derived from CD34(+) progenitor cells. Both haptens were able to induce CCR7 expression and DC migration in response to Cys-Cys chemokine ligand, CCL19. Since interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha have been shown to participate in LC migration during contact hypersensitivity, we tested the effect of their neutralization on CCR7 expression. Neutralization of IL-1beta activity did not modify CCR7 expression in response to both haptens. CCR7 expression was strongly dependent on TNF-alpha in the case of DNCB, however, neutralization of TNF-alpha only partially reduced CCR7 expression upon NiSO(4) treatment. DNCB, NiSO(4) and TNF-alpha activated p38 mitogen-activated protein kinases (MAPK) and c-jun N-terminal kinase (JNK). Both p38 MAPK and JNK participated to TNF-alpha production induced by DNCB. Inhibition of both p38 MAPK and JNK affected significantly CCR7 expression upon nickel treatment whereas only inhibition of p38 MAPK but not of JNK downregulated CCR7 in the case of TNF-alpha stimulation. These results suggest that MAPK are necessary for haptens to induce CCR7 expression. NiSO(4), however, activates directly CCR7 expression through p38 MAPK and JNK activation whereas DNCB needs TNF-alpha whose secretion is also regulated by p38 MAPK and JNK.