Bell, Michael V.

Abstract IgM antibodies, as multivalent ligands, have the capacity to activate cells differentially by cross-linking targeted cell surface signaling molecules. For example, hIgM12 (B7DC Xab) activates dendritic cells (DCs), leading to potent immunomodulation, while hIgM22 binds glial cells and causes remyelination in mouse models of multiple sclerosis. Both antibodies activate common signaling intermediates in their respective cellular targets suggesting a common mechanism of action. Elucidation of these shared mechanisms became possible by targeting both antibodies to a common cell type. Our previous studies demonstrate that B7-DC expression is required to activate downstream signaling in DCs by hIgM12. We utilized a phage display system to identify 7mer peptides bound by each IgM and expressed each peptide attached to a surrogate cell surface carrier on retrovirally tranduced bone marrow-derived DCs from B7-DC -/- mice. Transduced cells were treated with hIgM12, hIgM22 or anti-carrier IgM, and subsequent activity was compared against B7-DC +/+ DCs or B7-DC-reconstituted B7-DC -/- DCs. DC activation by each IgM was specific to expression of their conjugate peptide construct. Activation by hIgM22 was indistinguishable from activation by hIgM12, while anti-carrier IgM caused no activation. This indicates that while their target specificity differs, once bound these IgMs activate cells via common mechanisms.

Abstract To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.

Although interleukin 1 (IL-1) receptor signaling events in T helper lymphocytes are incompletely characterized, events associated with translocation of the transcription factor NF-kappa B are receptor-proximal assays of ligand-initiated responses. In this report we demonstrate that the transient nature of IL-1-induced NF-kappa B nuclear translocation occurs as a consequence of ligand-induced receptor desensitization. Other receptor-initiated events including induction of I kappa B alpha phosphorylation, expression of c-jun and junB mRNA, and costimulatory effects on IL-2 synthesis also are altered by IL-1 receptor desensitization. IL-1 receptor desensitization is not initiated by tumor necrosis factor, which also stimulates NF-kappa B translocation, and is not a consequence of alterations in either IL-1 receptor expression or binding affinity. In the absence of IL-1, the effects of desensitization are completely reversed within 18 h. Since IL-1 desensitization is initiated under conditions of low receptor occupancy, it is likely that receptor desensitization results from alterations to a receptor-proximal transducer, rather than from direct modification of the IL-1 receptor. These results suggest that the cyclic nature of the events in the T helper lymphocyte activation program can be controlled, in part, by the reversible desensitization of cell surface IL-1 receptors.