The long pentraxin (PTX) 3 is produced by macrophages and myeloid dendritic cells in response to Toll-like receptor agonists and represents a nonredundant component of humoral innate immunity against selected pathogens. We report that, unexpectedly, PTX3 is stored in specific granules and undergoes release in response to microbial recognition and inflammatory signals. Released PTX3 can partially localize in neutrophil extracellular traps formed by extruded DNA. Eosinophils and basophils do not contain preformed PTX3. PTX3-deficient neutrophils have defective microbial recognition and phagocytosis, and PTX3 is nonredundant for neutrophil-mediated resistance against Aspergillus fumigatus. Thus, neutrophils serve as a reservoir, ready for rapid release, of the long PTX3, a key component of humoral innate immunity with opsonic activity.

Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-γ, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7− effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.

IntroductionCirculating monocytes are precursors that can differentiate into a variety of tissue-resident macrophages (M⌽s) or dendritic cells (DCs), and even osteoclasts. 1 M⌽s exhibit a variety of activities, some of which are in opposition (ie, proinflammatory versus anti-inflammatory, immunostimulatory versus immunosuppressive, and tissue destructive versus reconstructive). 1 The functional heterogeneity of M⌽s depends, at least in part, on the local microenvironment. 2,3 In analogy with the Th1/Th2 dichotomy of T-cell responses, M⌽s exposed to IFN␥ or IL-4 have been referred to as M1s or M2s (also called alternatively activated M⌽s), respectively. 4 M1s produce IL-12 and TNF␣ and are potent killers of microorganisms (especially intracellular pathogens) and tumor cells. M2s produce IL-10 but not IL-12, scavenge debris, tune inflammatory responses, and promote humoral immunity and tissue repair. 5 The detection in cancer patients of tumor-specific T cells that kill ex vivo autologous tumor cells demonstrates that numerous tumor-cell types are potentially immunogenic. However, spontaneous clearance of established tumors by immune mechanisms is rare and active antitumor immunotherapy usually has poor clinical efficacy. 6 It is now largely documented that established tumors propagate conditions that favor their immune escape. 6 Tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) accumulate at tumor sites and maintain immune tolerance that contributes to defeating tumor immunity. 6,7 TAMs are far more abundant than Tregs and, in various solid tumors, constitute the major components of the leukocyte infiltrate. In most cases, especially breast, prostate, cervical, and ovarian cancers, TAM density is correlated with poor prognosis. [8][9][10] Strong evidence suggests that TAMs also promote cancer progression and metastasis. 8,11,12 TAMs are polarized M2 cells with potent immunosuppressive functions. They have poor antigen-presenting capacity, prevent T-cell activation, and may contribute to suppressing DC functions. 4,13,14 They also promote the recruitment of Tregs and Th2 cells (through CC chemokine ligand 17 [CCL17] and CCL22 secretion) and naive T cells (through CCL18). Naive T-cell activation, in an environment dominated by immature DCs and TAMs, is likely to induce anergy. 10,15 In addition, TAM production of growth and angiogenic factors (ie, vascular endothelial growth factor [VEGF] and platelet-derived endothelial cell growth factor [PDGF]), proteases (ie, matrix metalloproteinase 9 [MMP9]), and chemokines (eg, CCL2) favors tumor-cell proliferation, angiogenesis, dissolution of connective tissues, and metastasis. 8,12,14,16 The origin of TAMs has mostly been studied in mice in terms of precursor recruitment, survival, and proliferation. TAMs derive from circulating monocytes that are recruited into tumors by chemotactic factors, such as monocyte-colony-stimulating factor Submitted February 19, 2007; accepted August 29, 2007. Prepublished online as Blood First Edition paper, September 11, 2...

Ciliary neurotrophic factor (CNTF) is a cytokine supporting the differentiation and survival of various cell types in the peripheral and central nervous systems. Its receptor complex consists of a non-signaling alpha chain, CNTFR, and two signaling beta chains, gp130 and the leukemia inhibitory factor receptor (LIFR). Striking phenotypic differences between CNTF- and CNTFR-deficient mice suggest that CNTFR serves as a receptor for a second, developmentally important ligand. We have identified this factor as a stable secreted complex of cardiotrophin-like cytokine (CLC) and the soluble receptor cytokine-like factor-1 (CLF). CLF expression was required for CLC secretion, and the complex acted only on cells expressing functional CNTF receptors. The CLF/CLC complex activated gp130, LIFR and signal transducer and activator of transcription 3 (STAT3) and supported motor neuron survival. Our results indicate that the CLF/CLC complex is a second ligand for CNTFR with potentially important implications in nervous system development.

Outer membrane protein A (OmpA) is a conserved major component of the outer membrane of Enterobacteriaceae. Here, we report that OmpA from Klebsiella pneumoniae (KpOmpA) activates macrophages and dendritic cells (DCs) in a TLR2-dependent way. However, TLR2 does not account for binding of KpOmpA to innate immune cells. KpOmpA binds the scavenger receptors (SRs) LOX-1 and SREC-I, but not other members of the same family. LOX-1 colocalizes and cooperates with TLR2 in triggering cellular responses. The TLR2-activated functional program includes production of the long pentraxin PTX3, a soluble pattern recognition receptor involved in resistance against diverse pathogens. PTX3, in turn, binds KpOmpA but does not affect recognition of this microbial moiety by cellular receptors. KpOmpA-elicited in vivo inflammation is abrogated in TLR2(-/-) mice and significantly reduced in PTX3(-/-) mice. Thus, SR-mediated KpOmpA recognition and TLR2-dependent cellular activation set in motion a nonredundant PTX3-mediated humoral amplification loop of innate immunity.

Although human CD56 ؉ CD3 ؊ natural killer (NK) cells participate in immune responses against microorganisms, their capacity to directly recognize and be activated by pathogens remains unclear. These cells encode members of the Tolllike receptor (TLR) family, involved in innate cell activation on recognition of pathogen-associated molecular patterns (PAMPs). We therefore evaluated whether the 2 bacterial protein PAMPs, the outer membrane protein A from Klebsiella pneumoniae (KpOmpA) and flagellin, which signal through TLR2 and TLR5, respec- IntroductionNatural killer (NK) cells are CD56 ϩ CD3 Ϫ lymphocytes that spontaneously mediate cytotoxicity and are critical in the initial stages of immune defense. 1 They kill transformed and infected cells characterized by modified, down-regulated, or absent host major histocompatibility complex (MHC) class 1 molecules. 2 During microbial infections, they are activated by cytokines produced by surrounding innate cells and, in turn, synthesize a large panel of cytokines that tailor innate and adaptive immune responses. CD3 ϩ T lymphocytes expressing the NK cell receptor CD56 (NKR-expressing T cells) 3 are also involved in resistance to infected and transformed cells. 4,5 CD56 ϩ cells are primary producers of interferon-␥ (IFN-␥), which has a protective role against many intracellular pathogens. 6 Nevertheless, there is no evidence that these cells can in themselves recognize and be directly activated by bacterial components. In fact, NK cells are difficult to study; they are especially difficult to isolate when highly purified, but contaminated cells impede clear identification of NK cell functions. Numerous articles report a role for NK cell defense against bacterial infections, but never in a direct fashion because most purification strategies do not allow the isolation of pure NK cells. Consequently, whether NK cells are stimulated directly 7,8 or in response to signals generated by activated bystander cells is still a matter of debate. 9,10 Macrophages, dendritic cells, and some epithelial cells recognize pathogen-associated molecular patterns (PAMPs) expressed by microorganisms 11 through cell surface receptors (called patternrecognition receptors [PRRs]) involved in endocytosis or in cell activation (members of the Toll-like receptor [TLR] family). 12 Most mammalian TLRs have a key role in initiating inflammatory responses. 13 The TLR repertoire and the functional consequences of signaling through TLR have been mainly studied on antigenpresenting cells and epithelial cells. 13 Recently, TLR mRNA expression has been reported in B and T lymphocytes 9,14 and in NK cells. 9 We analyzed their role in NK cell activation. As TLR ligands, we used 2 bacterial protein PAMPs, the outer membrane protein A from Klebsiella pneumoniae (KpOmpA), which activates DCs and macrophages through TLR2, 15,16 and Escherichia coli flagellin, a major component of bacterial flagella that induces cytokine production by epithelial cells and monocytes through TLR5. 17,18 In response to microbial in...

Neutrophils are professional phagocytes that migrate early, in high number, to the infection sites. Our study has analyzed how neutrophils cross-present antigens and influence CD8 ؉ T-cell responses. By using highly purified neutrophils from peritoneal exudates and bone marrow, we have shown that neutrophils crosspresent ovalbumin to a CD8 ؉ T-cell hybridoma and to naive CD8 ؉ T cells from OT1 transgenic mice. Cross-presentation by neutrophils was TAP and proteasome dependent and was as efficient as in macrophages. Moreover, it actually occurred earlier than in professional antigenpresenting cells. Peritoneal exudate neutrophils from mice injected intraperitoneally with ovalbumin also cross-presented ovalbumin, proving that neutrophils take up and present exogenous antigens into major histocompatibility complex I (MHC I) molecules in vivo. We then evaluated the in vivo influence of antigen crosspresentation by neutrophils on CD8 ؉ Tcell response using ␤2-microglobulindeficient mice transferred with OT1 CD8 ؉ T cells and injected with ovalbuminpulsed neutrophils. Four days after neutrophil injection, OT1 cells proliferated and expressed effector functions (IFN-␥ production and cytolysis). They also responded efficiently to a rechallenge with ovalbumin-pulsed dendritic cells in CFA. These data are the first demonstration that neutrophils cross-prime CD8 ؉ T cells in vivo and suggest that they may constitute, together with professional antigenpresenting cells, an attractive target to induce cytotoxic T cells in vaccines. IntroductionAlthough it was originally thought that peptides presented into major histocompatibility complex I (MHC I) molecules to CD8 ϩ T cells derived exclusively from endogenous proteins, it is now admitted that, under certain circumstances, professional antigenpresenting cells (APCs; dendritic cells [DCs] and macrophages) can present some exogenous antigens in the MHC I molecules. 1,2 This process, called antigen cross-presentation, can occur by 2 different mechanisms. One is dependent on the transporter associated with antigen processing (TAP) that includes proteasomedependent and -independent pathways, and the other one is TAP independent. 3 Due to their ability (1) to migrate from peripheral tissues to the lymph nodes, where they encounter recirculating naive T cells, and (2) to express high levels of costimulatory molecules, DCs are usually considered as the only APC able to prime naive T cells. 4,5 This property has recently been extended to murine macrophages. 6 Activated DCs can present some exogenous proteins to naive CD8 ϩ T cells and induce their differentiation into effector cytotoxic T cells (CTLs). This process, referred to as cross-priming, allows the in vivo generation of protective cytotoxic responses against microorganisms that do not infect DCs or that infect DCs but inhibit their properties. 7 Cross-priming by DCs has opened new perspectives in vaccines based on CTL induction. 7,8 In the absence of licensing, DCs stimulate an abortive response that leads to cross-tolerance...