Margarita Salcedo

Triggering of Natural Killer Cells by the Costimulatory Molecule CD80 (B7-1)

NK cell-mediated cytotoxicity is influenced by triggering as well as inhibitory signals. The identification of inhibitory signals provided by MHC class I molecules has recently attracted significant attention. Much less is known about putative triggering signals. Using purified populations of mouse NK cells, we demonstrate that the CD80 (B7-1) gene product functions as a triggering signal for NK cell-mediated cytotoxicity. The strength of this response is such that it overrides the protection mediated by MHC class I molecules. Triggering of mouse NK cells by B7-1 occurred even in the absence of CD28 and could not be blocked by either anti-CD28 or anti-CTLA-4 antibodies. NK cells may thus, at least in part, use receptors other than CD28 and CTLA-4 in their interaction with B7-1. Furthermore, we demonstrate that bone marrow-derived macrophages and dendritic cells are highly susceptible to lysis by autologous NK cells.

Host MHC class I gene control of NK-cell specificity in the mouse

Summary: The missing self model predicts chat NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host, Transgenic and gene knock‐out mice have provided conclusive evidence chat MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the POSSIBLE Mechanisms behind it. starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK‐cell functional specificity as well as Lγ49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain “useful” NK cells, while avoiding “harmful” and “useless” ones? The available data can be fitted with in each of two mutually non‐exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as energy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

Ex vivo-activated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibody-dependent cellular cytotoxicity and impact of human serum.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the mechanisms of tumor killing during antibody (Ab) immunotherapy, and a role for myeloid cells as effectors has been observed in several models. We are developing immunotherapy approaches based on administration of large numbers of ex vivo interferon-γ–activated macrophages to cancer patients. With a quantitative assay measuring killing of nonproliferating tumor cells, we evaluated whether, in physiologic conditions, these macrophages synergize with the anti-CD20 Ab rituximab for killing primary B-cell chronic lymphocytic leukemia (B-CLL) cells. ADCC reached levels of 70% to 80% at effector to target ratios as low as 1:1. Macrophage recruitment by Ab-opsonized tumor cells did not result in enhanced cytokine secretion, suggesting that the cytokine shower observed in rituximab-treated patients is not caused by macrophage activation, and that cytokines have no role in CLL killing. We observed that uptake of tumor material by macrophages was not directly correlated to tumor killing. Nonetheless, experiments in the presence of cytochalasin D showed that ADCC occurred mainly by phagocytosis. Tumor killing was largely mediated by FcγRI and inhibited by increasing concentration of serum. Importantly, complement deposition on B-CLL cells did not seem to enhance macrophage ADCC in this model, as complement-depleted and complement-repleted human plasmas exerted comparable inhibition.

Distinct Roles for IFN Regulatory Factor (IRF)-3 and IRF-7 in the Activation of Antitumor Properties of Human Macrophages

When properly activated, macrophages can be tumoricidal, thus making them attractive additions to standard cancer therapies. To this end, tolerance and activity of human autologous IFN-gamma-activated macrophages, produced in large scale for clinical use (MAK cells), have been assessed in pilot trials in cancer patients. In the present study, we tested the hypothesis that activation of IFN regulatory factor (IRF)-3 and IRF-7, with subsequent type I IFN production, may be involved in the acquisition of new antitumor functions by macrophages. Adenoviral vectors were generated for the delivery of constitutively active forms of IRF-3 (Ad-IRF-3) or IRF-7 (Ad-IRF-7) into primary human macrophages. Cell death was observed in Ad-IRF-3-transduced macrophages, whereas Ad-IRF-7-transduced macrophages produced type I IFNs and displayed increased expression of genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand, interleukin (IL)-12, IL-15, and CD80, persisting for at least 96 hours. Expression of iNOS, TNF-alpha, FasL, IL-1, and IL-6 genes was unaltered by Ad-IRF-7 transduction. Interestingly, Ad-IRF-3 or Ad-IRF-7 transduction negatively regulated the transcription of protumorigenic genes encoding vascular endothelial growth factor and matrix metalloproteinase-2. Furthermore, Ad-IRF-7-transduced macrophages exerted a cytostatic activity on different cancer cell lines, including SK-BR-3, MCF-7, and COLO-205; the latter cells were shown previously to be insensitive to MAK cells. In conclusion, transduction of active forms of IRF-3 or IRF-7 differentially modulate the apoptotic and antitumor properties of primary macrophages, with active IRF-7 leading to the acquisition of novel antitumor effector functions.

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2‐microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild‐type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I‐deficient mice were tolerant to autologous MHC class I‐deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon‐inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self‐tolerant state of MHC class I‐deficient NK cells was broken by in vitro stimulation with IL‐2 for 4 days. Under these conditions, NK cells from the MHC class I‐deficient mice killed autologous MHC class I‐deficient cells while MHC class I‐positive targets were spared. The C‐type lectin inhibitory receptor Ly49C has a specificity for H‐2Kb and is expressed on a subset of NK1.1+ cells in B6 mice. Wild‐type and all MHC class I‐deficient mice had similar numbers of Ly49C‐positive NK1.1+ cells. However, Ly49C expression was markedly down‐regulated on NK1.1+ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL‐2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.

Analysis and characterization of antitumor T-cell response after administration of dendritic cells loaded with allogeneic tumor lysate to metastatic melanoma patients.

The primary goal of cancer vaccines is to induce CD8+ T cells specific for tumor-associated antigens (TAA) but the characterization of these cells has been difficult because of the low sensitivity of ex vivo assays. Here, we focused on TAA-specific CD8+ T-cell responses in melanoma patients after vaccination with autologous dendritic cells loaded with lysates derived from allogeneic tumor-cell lines (Lysate-DC). Out of 40 patients treated, 16 patients developed immune response to tumor-cell lysate and/or CD8+ T cells specific for differentiation and cancer-testis antigens. TAA-specific CD8+ T-cell responses were detected by interferon (IFN)-γ enzyme-linked immunospot after in vitro sensitization and were, either transient during the treatment period or delayed, that is, observed after completion of all vaccinations. We could not correlate these immune responses to clinical data as none of the patients achieved an overall objective response according to Response Evaluation Criteria in Solid Tumors criteria. Three patients were reported as stable disease and 10 patients presented evidence of antitumor activity. We found that TAA-specific T cells characterized in 4 patients produced perforin ex vivo, but no IFN-γ in enzyme-linked immunospot. Differential expression of IFN-γ and perforin was also observed for viral-specific T cells. Altogether, our results show that Lysate-DC therapy elicited tumor-specific CD8+ T cells nonlimited to human leukocyte antigen-A2+ patients, with some T cells secreting perforin ex vivo and IFN-γ only after restimulation. The differential expression of perforin and IFN-γ by antitumor and antiviral CD8+ T cells supports that the sole use of IFN-γ production to monitor T cells overlooks functional T-cell subpopulations triggered by vaccines.

Involvement of TBK1 and IKKepsilon in lipopolysaccharide-induced activation of the interferon response in primary human macrophages

Interferon (IFN) is an important effector of the innate immune response, induced by different viral or bacterial components through Toll‐like receptor‐dependent and ‐independent mechanisms. In human macrophages and macrophage‐activated killer cells, we demonstrate that (i) the type I IFN response to lipopolysaccharide (LPS) is weak compared to the host response to virus infection; (ii) there is a temporal difference in the induction of tank‐binding kinase‐1 (TBK1) and IkappaB kinase (IKK)‐related kinase epsilon (IKKϵ) kinase activities in response to LPS, with TBK1 activated early and IKKϵ induced in the late phase of IFN induction; and (iii) interferon regulatory factor (IRF)‐7 is induced following LPS treatment, but there is no evidence that IRF‐7 becomes activated by phosphorylation in vivo. Specifically, TBK1 kinase activity is rapidly increased after LPS stimulation (15 min) whereas IKKϵ activation occurs at 8 h. RNA interference‐mediated inhibition of TBK1 and IKKϵ expression in macrophages interfere with IFNB and IRF7 gene expression following LPS activation. Macrophage priming with rIFN‐α increased IRF‐7 expression, led to a sharp up‐regulation of the IFNB gene and to a rapid induction of IFNA2 upon LPS stimulation. These data support a differential role of TBK1 and IKKϵ in the downstream response mediated by IRF‐3 and IRF‐7 to LPS in primary human macrophages.

IL-2 Production by Virus- and Tumor-Specific Human CD8 T Cells Is Determined by Their Fine Specificity

Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-γ, whereas effector memory CD8 cells (CD45RAdullCCR7−) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-γ. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.

Major histocompatibility complex class I-independent killing of xenogeneic targets by rat allospecific natural killer cells

Major histocompatibility complex class I molecules can inhibit mouse as well as human natural killer (NK) cell cytotoxicity. In contrast, antigens encoded in the RT1.C region of the rat MHC gene complex have been suggested to trigger, rather than inhibit, rat NK cells. In an attempt to analyze rat NK cell specificity, with respect to the cross-species difference that may exist in NK cell-mediated cytotoxicity, we investigated the ability of interleukin 2-activated, allospecific rat NK cells to recognize MHC class I-positive and -deficient target cells of mouse and human origins. Recognition of xenogeneic target cells by rat allospecific NK cells was found to be MHC class I independent; target cell MHC class I was not required for killing, and expression of different sets of mouse and human MHC class I molecules did not influence the cytotoxic response. These results indicate that rat NK cells can recognize xenogeneic nontransformed cells by mechanisms not related to target cell MHC class I expression, and that mouse and human MHC class I molecules, at least among those tested in this study, are unable to confer inhibition of rat NK cells.

Triggering of Natural Killer Cell Mediated Cytotoxicity by Costimulatory Molecules

Natural killer (NK) cell mediated cytotoxicity is affected by both triggering and inhibitory signals (see e.g., Gumperz and Parham 1995; Lanier and Philips 1996; Raulet 1996). In several models target cell major histocompatibility complex (MHC) class I molecules have been demonstrated to be able to turn off NK cells (Ljunggren and Karre 1990; Karre 1995) by delivering inhibitory signals to MHC class I binding receptors (Karlhofer et al. 1992; Yokoyama and Seaman 1993). The latter include members of the Ly-49 receptors in mouse and the killer-cell inhibitor receptors (KIR) in man (Gumperz and Parham 1995; Lanier and Philips 1996; Raulet 1996). While much attention has been focused on the role of MHC class I inhibition of NK cell mediated cytotoxicity in recent years, significantly less attention has been devoted to receptor-ligand interactions that may trigger cytotoxicity. This review discusses new insights into the ability of costimulatory molecules to trigger NK cell mediated cytotoxicity.