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.

Tumor-associated macrophages (TAMs), the most abundant immunosuppressive cells in the tumor microenvironment, originate from blood monocytes and exhibit an IL-10^highIL-12^low M2 profile. The factors involved in TAM generation remain unidentified. We identify here leukemia inhibitory factor (LIF) and IL-6 as tumor microenvironmental factors that can promote TAM generation. Ovarian cancer ascites switched monocyte differentiation into TAM-like cells that exhibit most ovarian TAM functional and phenotypic characteristics. Ovarian cancer ascites contained high concentrations of LIF and IL-6. Recombinant LIF and IL-6 skew monocyte differentiation into TAM-like cells by enabling monocytes to consume monocyte–colony-stimulating factor (M-CSF). Depletion of LIF, IL-6, and M-CSF in ovarian cancer ascites suppressed TAM-like cell induction. We extended these observations to different tumor-cell line supernatants. In addition to revealing a new tumor-escape mechanism associated with TAM generation via LIF and IL-6, these findings offer novel therapeutic perspectives to subvert TAM-induced immunosuppression and hence improve T-cell–based antitumor immunotherapy efficacy.

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.

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 Toll-like 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, respectively, may directly stimulate human NK cells. These proteins induce interferon-γ (IFN-γ) production by NK cells and synergize with interleukin-2 (IL-2) and proinflammatory cytokines in PAMP-induced activation. Similar results were obtained using CD56⁺CD3⁺ (NKR-expressing) T cells. NK cells from TLR2^-/- mice fail to respond to KpOmpA, demonstrating TLR involvement in this effect. Defensins are antimicrobial peptides expressed mainly by epithelial cells and neutrophils that disrupt the bacterial membrane, leading to pathogen death. We show that NK cells and NKR-expressing T cells constitutively express α-defensins and that KpOmpA and flagellin rapidly induce their release. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to pathogen components through TLRs and evidence defensins as a novel and direct cytotoxic pathway involved in NK cell-mediated protection against microorganisms. (Blood. 2004;104:1778-1783)

Tumor‐associated macrophages (TAM) are M2d‐polarized cells (IL‐10^high, IL‐12^low, ILT3^high, CD86^low) that accumulate in tumor microenvironment. TAM inhibit antitumor T lymphocyte generation and function, contribute to tumor tolerance and are trophic for tumors. In this study, we investigated whether some immunological factors may reverse TAM immunosuppressive properties. Among 32 cytokines, we have identified IFNγ on its ability to switch immunosuppressive TAM into immunostimulatory cells. Upon IFNγ exposure, TAM purified from ovarian cancer ascites recover a M1 phenotype (IL‐10^low, IL‐12^high), express high levels of CD86 and low levels of ILT3, enhance the proliferation of CD4⁺ T lymphocytes and potentiate the cytotoxic properties of a MelanA‐specific CD8⁺ T cell clone. IFNγ‐treated TAM also secreted reduced levels of mediators promoting suppressive T cell accumulation (CCL18) and trophic for tumors (VEGF and MMP9). As TAM derive from the local differentiation of peripheral blood monocytes, we investigated whether IFNγ may also affect TAM generation. In the presence of ovarian ascites, IFNγ skewed monocyte differentiation from TAM‐like cells to M1‐polarized immunostimulatory macrophages. Together, these data show that IFNγ overcomes TAM‐induced immunosuppression by preventing TAM generation and functions. These data highlight that IFNγ used locally at the tumor site could potentiate the efficacy of antitumor immunotherapies based on the generation of effector T cells. © 2009 UICC