Antibody-reliant destruction of tumor cells by immune effector cells is mediated by antibody-dependent cellular cytotoxicity, in which Fc receptor (FcR) engagement is crucial. This study documents an important role for the ␤ 2 integrin Mac-1 (CD11b/CD18) in FcR-mediated protection against melanoma. CD11b-deficient mice, those that lack Mac-1, were less protected by melanoma-specific monoclonal antibody TA99 than wild-type (WT) mice. Significantly more lung metastases and higher tumor loads were observed in Mac-1 ؊/؊ mice. Histologic analyses revealed no differences in neutrophil infiltration of lung tumors between Mac-1 ؊/؊ and WT mice. Importantly, Mac-1 ؊/؊ phagocytes retained the capacity to bind tumor cells, implying that Mac-1 is essential during actual FcR-mediated cytotoxicity. In summary, this study documents Mac-1 to be required for FcR-mediated antimelanoma immunity in vivo and, furthermore, supports a role for neutrophils in melanoma rejection. IntroductionAntibody (Ab)-dependent cellular cytotoxicity (ADCC) is considered crucial for Ab-mediated tumor cell degradation. Specific Ab-Fc receptor (FcR) interactions establish close contacts between tumor targets and immune effector cells, which triggers cytotoxicity and cytokine release. Neutrophils, monocytes, macrophages, and natural killer (NK) cells can mediate ADCC via activating FcRs, which include Fc␥RIa (CD64), Fc␥RIIa (CD32), Fc␥RIIIa (CD16), and Fc␣RI (CD89) in man, and Fc␥RI and Fc␥RIII in mice. [1][2][3][4] Although Abs may affect tumor growth via FcR-unrelated mechanisms (such as complement-dependent lysis, blockade of growth factor receptors, or via induction of apoptosis), 5 in vivo antitumor effects of Abs have been documented to depend on immune activation through FcRs. [6][7][8] Numerous studies in cancer immunology focused on melanoma and melanoma-specific differentiation antigens that induce immune responses. 9 If tolerance is broken, melanosomal proteins can be recognized by T cells, which may provide B-cell help and participate in Ab production. Actual tumor rejection seems dependent on phagocytes, which may be activated by CD4 ϩ or NK cells. [10][11][12] Improved clinical outcome has, furthermore, been correlated with the presence of melanoma-specific Abs in patients. 13 Ab-mediated protection in the murine B16F10 melanoma model is well established. Monoclonal antibody (mAb) TA99, specific for melanoma differentiation antigen gp75 (brown locus protein, or TRP-1), is effective in preventing and eradicating early established metastases. 11 Studies with mice deficient in the FcR ␥ chain, lacking expression of Fc␥RI and Fc␥RIII, revealed activating FcR to be critical in TA99-mediated tumor rejection. 6 Further evidence supporting FcR dependence in Ab-mediated melanoma rejection was established by (1) the documented inability of F(abЈ) 2 fragments to mediate protection, 12 (2) lack of Ab effects on tumor cells in the absence of effector cells, 12 and (3) enhancement of antitumor immunity in Fc␥RII (inhibitory murine FcR) knock-out mice. ...

To achieve a correct cellular immune response toward pathogens, interaction between FcR and their ligands must be regulated. The Fc receptor for IgA, FcαRI, is pivotal for the inflammatory responses against IgA-opsonized pathogens. Cytokine-induced inside-out signaling through the intracellular FcαRI tail is important for FcαRI-IgA binding. However, the underlying molecular mechanism governing this process is not well understood. In this study, we report that PP2A can act as a molecular switch in FcαRI activation. PP2A binds to the intracellular tail of FcαRI and, upon cytokine stimulation, PP2A becomes activated. Subsequently, FcαRI is dephosphorylated on intracellular Serine 263, which we could link to receptor activation. PP2A inhibition, in contrast, decreased FcαRI ligand binding capacity in transfected cells but also in eosinophils and monocytes. Interestingly, PP2A activity was found crucial for IgA-mediated binding and phagocytosis of Neisseria meningitidis. The present findings demonstrate PP2A involvement as a molecular mechanism for FcαRI ligand binding regulation, a key step in initiating an immune response.

Effectiveness of bispecific-monoclonal-antibody (BsMAb)-mediated cellular anti-tumour activity was evaluated in vitro and in vivo in relation to the additional need for T-cell activation in a new immunocompetent rat tumour model. L37 tumour cells, derived from a squamous-cell carcinoma of the lung of Wag/Rij rats, were transfected with the cDNA coding for the human 38-kDa transmembrane pan-carcinoma-associated antigen EGP-2. Intravenous inoculation of EGP-2-positive L37 cells resulted in a rapid outgrowth of EGP-2-positive tumour nodules in the lungs. A BsMAb BIS-19, recognizing EGP-2 on the transfected tumour cells and the T-cell receptor of the rat, was made and allowed specific lysis of EGP-2-transfected L37 tumour cells by activated rat T lymphocytes in vitro. In vivo T-cell activation, assessed by up-regulation of IL-2-receptor expression, could be induced by daily injection of rat rIL-2. Intravenous treatment of tumour-bearing EGP-2-positive L37 tumour with BIS-19 together with rat rIL-2 resulted in almost complete disappearance of established tumour. In contrast, animals treated with BIS-19 alone, IL-2 alone or a combination of anti-EGP-2, anti-TcR and IL-2 showed much less or no tumour reduction. These results show effectiveness of systemic treatment with BsMAbs to induce anti-tumour activity in established tumours. Immune activation prior to or during treatment with BsMAbs, as achieved with IL-2, appears to be a prerequisite for successful treatment.

Summary In this report we describe the role of apoptosis in the process of tumour cell killing by bispecific monoclonal antibody (BsMAb)-redirected cytolytic T cells. The BsMAb used, BIS-1, has dual specificity for the CD3 complex on T cells and the pancarcinoma-associated 38 kDa transmembrane antigen EGP-2. BIS-1 allows activated T cells to specifically recognise and kill EGP-2-positive but not EGP-2-negative target cells. An assay was developed to quantify apoptosis in cells by separation of 3H-thymidine-labelled low-molecular, i.e. fragmented, from high-molecular, i.e. non-fragmented DNA. The presence of low molecular weight DNA was measured both within the target cells and in the cell-free supernatant. After exposure to BIS-1-redirected, -activated T cells, apoptosis was observed in EGP-2-positive target cells but not in EGP-2-negative target cells. Also no DNA fragmentation proved to be induced in the activated effector cells during assay. The degree of EGP-2-positive target DNA fragmentation depended on the concentration of BsMAb, the E/T ratio and the incubation time. Using a low E/T ratio (1/1), DNA fragmentation in and 5'Cr release from target cells showed similar characteristics and kinetics. At higher E/T ratio (20/1), the 5"Cr release from the target cells increased to a greater extent than the percentage fragmented target cell DNA. Inhibitors of DNA fragmentation added to the cytotoxicity assay inhibited not only DNA fragmentation, but also the release of chromium-51 from the target cells, suggesting that apoptosis and cell lysis are closely related in BsMAbmediated cell killing.