NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model.

Inhibitory receptors that engage self-MHC class I molecules enable NK cells to detect disease-associated loss of MHC class I on surrounding cells. Previous studies showed that some NK cells lack all receptors for self-MHC class I, yet fail to exhibit autoimmunity because they are generally hyporesponsive to stimulation. We asked whether NK cells exist in only two states, responsive and hyporesponsive, corresponding to cells that express or fail to express inhibitory receptors for self-MHC class I. The alternative model is that NK cells vary continuously in their responsiveness, based on variations in the number of different inhibitory and stimulatory receptors they express, which is known to vary. In this study, we show in the murine system that NK cell responsiveness increases quantitatively with each added self-MHC-specific inhibitory receptor. Genetic analysis demonstrated that interactions of each of the receptors with self-MHC class I were necessary to observe augmented responsiveness. These findings suggest that NK cell responsiveness is comparable to a rheostat: it is tuned to an optimal set point depending on the inhibitory and stimulatory interactions encountered in the normal environment, so as to ensure self-tolerance and yet optimize sensitivity to changes in normal cells.

Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses.

Distinct genes encode 6 human receptors for IgG (hFcgammaRs), 3 of which have 2 or 3 polymorphic variants. The specificity and affinity of individual hFcgammaRs for the 4 human IgG subclasses is unknown. This information is critical for antibody-based immunotherapy which has been increasingly used in the clinics. We investigated the binding of polyclonal and monoclonal IgG1, IgG2, IgG3, and IgG4 to FcgammaRI; FcgammaRIIA, IIB, and IIC; FcgammaRIIIA and IIIB; and all known polymorphic variants. Wild-type and low-fucosylated IgG1 anti-CD20 and anti-RhD mAbs were also examined. We found that (1) IgG1 and IgG3 bind to all hFcgammaRs; (2) IgG2 bind not only to FcgammaRIIA(H131), but also, with a lower affinity, to FcgammaRIIA(R131) and FcgammaRIIIA(V158); (3) IgG4 bind to FcgammaRI, FcgammaRIIA, IIB and IIC and FcgammaRIIIA(V158); and (4) the inhibitory receptor FcgammaRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other hFcgammaRs. We also identified parameters that determine the specificity and affinity of hFcgammaRs for IgG subclasses. These results document how hFcgammaR specificity and affinity may account for the biological activities of antibodies. They therefore highlight the role of specific hFcgammaRs in the therapeutic and pathogenic effects of antibodies in disease.

Natural killer cell differentiation driven by Tyro3 receptor tyrosine kinases.

Although understanding of the function and specificity of many natural killer (NK) cell receptors is increasing, the molecular mechanisms regulating their expression during late development of NK cells remain unclear. Here we use representational difference analysis to identify molecules required for late NK cell differentiation. Axl protein tyrosine kinase, together with the structurally related receptors Tyro3 and Mer, were essential for NK cell functional maturation and normal expression of inhibitory and activating NK cell receptors. Also, all three receptors were expressed in maturing NK cells, the ligands of these receptors were produced by bone marrow stromal cells, and recombinant versions of these ligands drove NK cell differentiation in vitro. These results collectively suggest that Axl, Tyro3 and Mer transmit signals that are essential for the generation of a functional NK cell repertoire.

Selection of a human anti-RhD monoclonal antibody for therapeutic use: impact of IgG glycosylation on activating and inhibitory Fc gamma R functions.

The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety concerns. Since it has been suggested that FcgammaR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcgammaR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcgammaRIII and inhibitory FcgammaRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD+ RBCs and a potent FcgammaRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.

A subset of natural killer cells achieves self-tolerance without expressing inhibitory receptors specific for self-MHC molecules.

It is widely believed that self-tolerance of natural killer (NK) cells occurs because each NK cell expresses at least one inhibitory receptor specific for a host major histocompatibility complex (MHC) class I molecule. Here we report that some NK cells lack all known self-MHC-specific inhibitory receptors, yet are nevertheless self-tolerant. These NK cells exhibit a normal cell surface phenotype and some functional activity. However, they respond poorly to class I-deficient normal cells, tumor cells, or cross-linking of stimulatory receptors, suggesting that self-tolerance is established by dampening stimulatory signaling. Thus, self-tolerance of NK cells in normal animals can occur independently of MHC-mediated inhibition, and hyporesponsiveness plays a role in self-tolerance of NK cells, as also proposed for B and T cells.

Regression of AK7 malignant mesothelioma established in immunocompetent mice following intratumoral gene transfer of interferon gamma.

Malignant mesothelioma (MM) is a lethal tumor linked with a prior exposure to asbestos in which limited progress has been made so far using conventional therapies. MM is an example of a "nonimmunogenic" tumor characterized by a fibrous stroma and an absence of infiltrating T lymphocytes. High levels of transforming growth factor-beta (TGF-beta) produced by mesothelioma cells have been related to the immune tolerance towards the tumor. In order to evaluate the effect of local delivery of cytokines such as interferon gamma (IFN-gamma) by gene transfer, we characterized and used a murine model, AK7, which appeared very similar to human mesothelioma. AK7 cells expressed low levels of major histocompatibility class I and class II antigens and secreted high levels of latent TGF-beta. The TGF-beta pathway in AK7 cells is operative but inefficient because endogenous TGF-beta is predominantly inactive. Treatment of pre-established AK7 tumors by direct intratumoral injection of an adenovirus vector expressing murine IFN-gamma, Ad.mIFN-gamma, led to significant tumor regression. Peripheral tumor infiltration by CD4+ and CD8+ T lymphocytes in the treated tumors appeared to be because of the induction of an immune response. Tumor relapse was observed, which could be due to local TGF-beta secretion by remaining tumor cells.

Dendritic cells (DC) promote natural killer (NK) cell functions: dynamics of the human DC/NK cell cross talk.

Dendritic cells (DC) were originally found critical in the setting of cognate immune responses. We first demonstrated that DC can also induce mouse NK cell activation and NK cell dependent-antitumor effects in mice. Here we analyzed the dynamics between DC and NK cells in human in vitro model systems. In the absence of LPS, DC do not trigger resting NK cells. Conversely, in the presence of LPS, resting bulk NK cells interacting with DC acquire CD25 and CD69 surface expression, produce high levels of IFN-gamma and lyse DAUDI cells. On activated IL-2 dependent NK cell lines, regardless of their differentiation stage, DC maintain or enhance NK cell proliferation and effector functions in the absence of exogenous cytokines. While IL-12, IL-15 and IL-18 are not critical, a direct cell-to-cell contact is mandatory for NK activation by DC and required for optimal proliferation. These data imply that DC also modulate human NK cell innate effector functions.