Protocol for the murine antibody-dependent cellular phagocytosis assay.

Antibody-dependent cellular phagocytosis (ADCP) is a process through which myeloid cells are able to exert their phagocytic function after recognition of opsonized bacteria, viruses, infected cells or any cells targeted by a specific antibody. ADCP of tumor cells represents a potent effector mechanism of monoclonal antibody therapy mediated by tumor associated macrophages (TAM) and other phagocytic cells as an in situ anti-tumor activity. Here we described a protocol based on flow cytometry and immunofluorescence assays enabling extensive comparative studies to address whether a monoclonal antibody engaging Fcγ receptors on macrophages can mediate in vitro ADCP of tumor cells.

Cross-presentation of human melanoma peptide antigen MART-1 to CTLs from in vitro reconstituted gp96/MART-1 complexes.

Heat shock proteins (HSPs) have two unique roles as constituents of tumor vaccines: (i) to shuttle associated tumor antigens into professional antigen-presenting cells (APCs) and (ii) to activate professional APCs. Here we investigated the shuttle function of the HSP gp96 (glycoprotein 96) for a human melanoma peptide antigen MART-1 that was noncovalently bound to gp96 in vitro. This in vitro complexing reaction was optimized using the radioiodinated MART-1 peptide and human gp96. Up to 20% of gp96 molecules could bind the peptide, assuming a 1:1 molar ratio. The binding was temperature-dependent and thus reversible. At -20 degrees C, 95% of the peptide remained complexed after 24 h, but 25% and 60% of the peptide dissociated at 37 degrees C within 6 and 24 h, respectively. This observation suggests that under the physiological conditions in APCs, spontaneous peptide dissociation from gp96 complexes may facilitate the delivery of peptide antigen into antigen presentation pathways. The gp96/MART-1 complexes stimulated an HLA A2-restricted MART-1-specific CTL clone dependent on the amount of complexed peptide and the presence of HLA-A2-positive APCs. The reaction was peptide-specific and could be blocked by an excess of untreated native gp96. These results show for the first time that peptide antigens from in vitro reconstituted gp96/peptide antigen complexes can be cross-presented by human APCs. These findings extend the scientific basis for further evaluating the use of either endogenous or in vitro reconstituted gp96/tumor-antigen complexes as tumor vaccines.

Different efficiency of heat shock proteins (HSP) to activate human monocytes and dendritic cells: superiority of HSP60.

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.