Uptake of antigen-antibody complexes by human dendritic cells.

Fc receptors specific for IgG (FcγR) potentiate the immune response by facilitating the interaction between myeloid cells and antibody-coated targets (1-3). Monocyte and neutrophil FcyR engagement can lead to the induction of lytic-type mechanisms associated with innate responses. FcyR triggering can also play a key role in adaptive immune responses. For example, FcyR-directed capture and uptake of antigens (Ag) by dendritic cells (DC) results in processing and presentation to naive Ag-specific T cells, leading to their expansion and maturation into effector T-cell populations. This chapter describes methodology currently in use to explore and manipulate antigen-antibody (Ag-Ab) uptake by FcyR expressed on DC.

Human dendritic cells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c(+) blood dendritic cells (DCs) express TRAIL after stimulation with either interferon (IFN)-gamma or -alpha and acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-3 receptor (R)alpha(+) blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells, demonstrating a clear functional difference between the CD11c(+) DC and IL-3Ralpha(+) pre-DC subsets. These results indicate that TRAIL may serve as an innate effector molecule on CD11c(+) DCs for the elimination of spontaneously arising tumor cells and suggest a means by which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen presented by the DCs.

The leukocyte immunoglobulin-like receptors (LIRs): a new family of immune regulators.

Identification of a counterstructure for the human cytomegalovirus-encoded major histocompatibility complex class I-related gene product, UL18, has led to the discovery of a novel family of immunoreceptors, termed leukocyte immunoglobulin-like receptors (LIRs). The LIRs are differentially expressed in cells of the dendritic cell, monocytic and lymphocytic lineages, and appear to mediate diverse roles in immune regulation. This review summarizes the expression, distribution, and signaling capacities of the LIRs and discusses possible roles of the LIRs in both inhibition and activation of the cellular responses.

Human cytomegalovirus, MHC class I and inhibitory signalling receptors: more questions than answers.

The human cytomegalovirus UL18 protein, an MHC class I homologue, has been shown to bind to leucocyte immunoglobulin-like receptor (LIR)-1, a member of a family of nine closely related immunoglobulin superfamily receptors expressed on leucocytes. The LIRs are related to the natural killer (NK)-cell immunoglobulin-like receptors and to several other immunoreceptors. Three groups of LIR molecules have been defined: those containing cytoplasmic domain inhibitory signalling motifs, those with short cytoplasmic domains and a charged residue within the transmembrane domain, and a secreted molecule. LIR-1 and LIR-2 bind to a broad spectrum of cellular MHC class I antigens, including HLA-A, -B and -C alleles. LIR-2 is expressed by all monocytes and dendritic cells, whereas LIR-1 is additionally expressed by B cells and subsets of T and NK cells. Upon tyrosine phosphorylation, LIR-1 and LIR-2 associate with the tyrosine phosphatase, SHP-1, and have been shown to inhibit Fc gamma RI signalling when co-crosslinked in monocytes. Evidence for and against a role of UL18 as an inhibitor of NK-cell function is discussed, as are possible functional outcomes of UL18-LIR-1 interactions in monocytic cells.

Monocyte-mediated tumoricidal activity via the tumor necrosis factor-related cytokine, TRAIL.

TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) is a molecule that displays potent antitumor activity against selected targets. The results presented here demonstrate that human monocytes rapidly express TRAIL, but not Fas ligand or TNF, after activation with interferon (IFN)-gamma or -alpha and acquire the ability to kill tumor cells. Monocyte-mediated tumor cell apoptosis was TRAIL specific, as it could be inhibited with soluble TRAIL receptor. Moreover, IFN stimulation caused a concomitant loss of TRAIL receptor 2 expression, which coincides with monocyte acquisition of resistance to TRAIL-mediated apoptosis. These results define a novel mechanism of monocyte-induced cell cytotoxicity that requires TRAIL, and suggest that TRAIL is a key effector molecule in antitumor activity in vivo.

The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes.

The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and -2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcgamma receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor gamma chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.

Expanding dendritic cells in vivo enhances the induction of oral tolerance.

The intestine is under perpetual challenge from both pathogens and essential nutrients, yet the mucosal immune system is able to discriminate effectively between harmful and innocuous Ags. It is likely that this selective immunoregulation is dependent on the nature of the APC at sites where gut Ags are processed and presented. Dendritic cells (DC) are considered the most potent of APC and are renowned for their immunostimulatory role in the initiation of immune responses. To investigate the role of DC in regulating the homeostatic balance between mucosal immunity and tolerance, we treated mice with Flt3 ligand (Flt3L), a growth factor that expands DC in vivo, and assessed subsequent systemic immune responsiveness using mouse models of oral tolerance. Surprisingly, mice treated with Flt3L to expand DC exhibited more profound systemic tolerance after they were fed soluble Ag. Most notably, tolerance could be induced in Flt3L-treated mice using very low doses of Ag that were ineffective in control animals. These findings contrast with the generally accepted view of DC as immunostimulatory APC and furthermore suggest a pivotal role for DC during the induction of tolerance following mucosal administration of Ag.

Expression of the lymphotoxin beta receptor on follicular stromal cells in human lymphoid tissues.

The lymphotoxin beta receptor (LTbetaR), and its ligand, LTalpha1beta2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbetaR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbetaR is detected on some myeloid leukemic lines. In the developing thymus LTbetaR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbetaR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbetaR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTalpha1beta2 complex. These results support the concept that directional interactions between LTalpha1beta2 bearing lymphocytes and LTbetaR bearing stromal cells are involved in the organization of lymphoid tissue.

A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules.

The human cytomegalovirus UL18 gene product is a homolog of cellular major histocompatibility (MHC) class I antigens. UL18 has been proposed to protect virus-infected cells against natural killer (NK) cell cytotoxicity by engaging NK cell killer inhibitory receptors (KIR) for MHC class I. UL18 binds to a novel immunoglobulin superfamily glycoprotein, designated Leukocyte Immunoglobulin-like Receptor (LIR-1). This protein is distinct from, but related to, known KIRs and binds cellular MHC class I antigens. The cytoplasmic domain of LIR-1 contains four putative immunoreceptor tyrosine-based inhibitory motifs. Upon tyrosine phosphorylation, LIR-1 associates with the tyrosine phosphatase SHP-1. In contrast to KIRs, LIR-1 is expressed predominantly on monocytic and B lymphoid cell types, suggesting a distinct biological function.

Activation of human T cells by major histocompatability complex class II expressing neutrophils: proliferation in the presence of superantigen, but not tetanus toxoid.

The primary function of polymorphonuclear neutrophils (PMN) in the immune response appears to be acute phagocytic clearance of foreign pathogens and release of inflammatory mediators. Consistent with their assumed lack of major histocompatibility complex (MHC) class II expression, PMN have not been considered to play a role in antigen presentation and T-cell activation. However, recent reports have shown that human PMN can express MHC class II molecules both in vitro and in vivo after stimulation with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). Thus, under appropriate conditions, PMN could play a significant role in immune regulation, including T-cell activation. In this report, we demonstrate that human class II-expressing PMN can serve as accessory cells in superantigen (SAg)-mediated T-cell activation. This accessory activity for SAg presentation was present only after induction of MHC class II expression, and was especially pronounced following culture of PMN with GM-CSF plus IFN-gamma, which acted synergistically to induce MHC class II molecules on PMN. Moreover, the level of MHC class II expression and the magnitude of SAg-induced T-cell responses were found to be highly correlated and distinctly donor dependent, with PMN from some donors repeatedly showing fivefold higher responses than PMN from other donors. On the other hand, culture of PMN with GM-CSF plus IFN-gamma under conditions that resulted in optimal MHC class II expression did not enable them to function as antigen-presenting cells for either intact tetanus toxoid (TT) or for a TT peptide. These results delineate a new pathway for T-cell activation by SAg that may play an important role in the severity of SAg-induced inflammatory responses. They also identify a donor-specific polymorphism for induction of PMN MHC class II expression which may be of significance for therapies involving GM-CSF and IFN-gamma.

Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules.

Leukocyte Ig-like receptors (LIRs) are a newly discovered family of immunoreceptors expressed on monocytes and B cells and at lower levels on dendritic cells and NK cells. The amino acid sequences in the extracellular regions of eight of these receptors show between 63 and 84% identity to the prototypic LIR-1 sequence. LIRs contain either two or four Ig domains and fall into three classes: those with cytoplasmic domains containing two, three, or four immunoreceptor tyrosine-based inhibitory motif-like motifs; those with a short cytoplasmic domain and no immunoreceptor tyrosine-based inhibitory motif-like motifs; and those with no transmembrane domain represented by a single LIR molecule that is presumably secreted. The LIRs are structurally related to the human Fc(alpha)R and the killer inhibitory receptors and map to the same region of chromosome 19 as these genes. Like killer inhibitory receptors, at least two LIRs bind to MHC class I Ags, but their different cellular distribution suggests a distinct role in immune system modulation.

Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells.

Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.

Antigen targeting to myeloid-specific human Fc gamma RI/CD64 triggers enhanced antibody responses in transgenic mice.

Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.