Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.

Targeting weak antigens to CD64 elicits potent humoral responses in human CD64 transgenic mice.

Previous studies have documented that targeting foreign Ags to IgG FcgammaR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcgammaR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcgammaRI) and their nontransgenic littermates with Fab' derived from the murine anti-human CD64 mAb m22. The m22 Fab' served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab', which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab')(2) at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab' fragments. Chemical addition of a second murine Fab' (520C9 anti-human HER2/neu) to m22 Fab' multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.

Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages.

Macrophages represent an important effector cell for Ab-mediated tumor therapy. Previous studies have documented that cytokines can influence Fc receptor (FcR) expression and function. In this study we examined the tumoricidal activities of the type I receptors for IgG (Fc gamma RI) and the IgA FcR (Fc alpha RI) on monocyte-derived macrophages (MDM) cultured in the presence of IFN-gamma, M-CSF, or GM-CSF. Bispecific Abs were used to target a Her2/neu breast carcinoma cell line, SKBR-3, to Fc alpha RI or Fc gamma RI on MDM. Although Fc alpha RI and Fc gamma RI share a common signaling pathway contingent on association with the gamma-chain (FcR gamma subunit), a marked difference in their efficiency in mediating tumoricidal functions was seen in response to specific cytokines. M-CSF- and GM-CSF-treated MDM mediated efficient phagocytosis of SKBR-3 cells by Fc alpha RI and Fc gamma RI; however, IFN-gamma-treated MDM phagocytosed tumor cells only with the Fc gamma RI-directed bispecific Abs. Similarly, IFN-gamma-cultured MDM lysed tumor cells more efficiently via Fc gamma RI then by Fc alpha RI as measured in Ab-dependent cellular cytotoxicity assays. Conversely, GM-CSF-treated MDM mediated more efficient lysis of tumor cells via Fc alpha RI than Fc gamma RI, while M-CSF-cultured MDM were relatively less efficient in mediating Ab-dependent cellular cytotoxicity through either receptor. With the exception of IFN-gamma-mediated enhancement of Fc gamma RI expression and Fc gamma RI gamma-chain complexes, the regulation of Fc gamma RI- or Fc alpha RI-mediated activity occurred without significant change in either receptor expression or total complexes with gamma subunit. These data suggest that the efficiency of Ab-mediated tumor therapy, which depends on FcR effector cell functions, may be modified by the use of specific cytokines.

Development of T-cell lines expressing functional HIV-1 envelope glycoproteins for evaluation of immune responses in HIV-infected individuals.

The human T-lymphoid cell line, CEM, was transfected with gp 160 cDNA of human immunodeficiency virus type 1 (HIV-1)pm213. Three clones expressing the envelope glycoproteins (env), designated CEM-213env1, -env4, and -env7, were isolated. These clones expressed high levels of surface gp41 and gp120, as demonstrated by flow cytometry with anti-HIV env monoclonal antibodies. Processing and function of env was shown by induction of syncytia with CD4-expressing HeLa cells and by immunoblot analysis. The env expression resulted in specific down-regulation of surface CD4 levels, supporting the role of HIV env in CD4 modulation. Furthermore, serum samples from nine of nine HIV-1-infected individuals bound specifically to the env-expressing transfectants, substantiating the presence of conserved antigenic determinants. These sera also mediated antibody-dependent cellular cytotoxicity (ADCC) of the env-expressing cell lines. The env-expressing cell lines provide a relevant, safe, and practical model for qualitative and quantitative analysis of humoral and cellular immune responses and their role in HIV-1 pathogenesis and therapy.

Flow cytometric analysis of the cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human natural killer cells.

The goal of this investigation was to determine if human natural killer (NK) cells were susceptible to the cytolytic effects of the Actinobacillus actinomycetemcomitans leukotoxin (LTX). Following treatment with LTX (0-200 ng/ml), NK cell activation by interleukin-2 (IL-2) was evaluated. LTX inhibited the IL-2-induced expression of both CD69 and the IL-2 receptor. Furthermore, the up-regulation of CD56 was also impaired. To determine whether the observed functional deficits were the result of cell death, NK cell viability was evaluated by flow cytometry. Changes in forward and side light scatter patterns consistent with cell death were observed within 60 min. Direct analysis of cell viability by measuring propidium iodide exclusion, however, indicated little change in the viability of LTX-treated NK cells. Electron microscopic analysis of NK cells exposed to LTX revealed early nuclear alterations characterized by hyperchromaticity, nuclear fragmentation, and condensation of nucleoplasm. However, no change in membrane integrity was initially noted. Finally, LTX caused a rapid and sustained elevation in the intracellular levels of Ca2+. These morphological and biochemical changes are consistent with the notion of programmed cell death.

Immune suppression induced by Actinobacillus actinomycetemcomitans: effects on immunoglobulin production by human B cells.

Actinobacillus actinomycetemcomitans produces an immunosuppressive factor (ISF) which has been shown to suppress mitogen- and antigen-induced DNA, RNA, and protein synthesis in human T lymphocytes. In this study, we examined purified A. actinomycetemcomitans ISF for its ability to alter immunoglobulin production by human B cells. The ISF caused a dose-dependent inhibition of pokeweed mitogen (PWM)-induced immunoglobulin G (IgG) and IgM production. Preexposure to ISF was not required to achieve maximal inhibition of immunoglobulin synthesis, as previously observed for its effect on T-cell activation. Nevertheless, the ISF appeared to act by irreversibly affecting the early stages of cell activation. While PWM-induced immunoglobulin production is under the influence of T-regulatory circuits, it appears that the ISF interacts directly with B cells. First, ISF failed to alter either the synthesis of interleukin-2 (IL-2) or the expression of IL-2 receptors on T cells. Second, experiments in which individual purified populations of cells were exposed to ISF, washed, and placed back into tissue culture indicated that when all cells (i.e., T cells, B cells, and monocytes) were exposed to ISF, significant suppression was observed. However, when only one cell population was treated with ISF, suppression of both IgG and IgM synthesis was observed only when the B-cell-enriched population was exposed to ISF. These results in conjunction with our earlier findings suggest that the ISF functions via the activation of a regulatory subpopulation of B lymphocytes, which in turn either directly or indirectly (via suppressor T cells) downregulate both B- and T-cell responsiveness. Furthermore, it is hypothesized that patients who harbor A. actinomycetemcomitans could suffer from local or systemic immune suppression. This suppression may enhance the pathogenicity of A. actinomycetemcomitans itself or that of some other opportunistic organism.