John D. Pound

IgG-Fc-mediated effector functions : molecular definition of interaction sites for effector ligands and the role of glycosylation

Summary: The Fr region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions often of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter‐CH2‐CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x‐ray crystal structures for Fc and Fc‐ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is “disordered”, suggesting “internal mobility”. We propose a model in which such “internal mobility” results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Modulation of MHC antigen expression by viruses and oncogenes

Abstract It is becoming increasingly clear that regulation of MHC antigen expression by viruses and oncogenes, leading to either immune evasion or autoimmunity, is widespread and important in disease. At a recent meeting, which brought together workers interested in tumour immunology, viral infection and the MHC, a number of mechanisms for the regulation of MHC antigen expression were revealed and the importance of balanced expression of MHC gene products to effective immunity was underlined.

Molecular definition of interaction sites on human IgG for Fc receptors (huFcγR)

Abstract Evidence from several experimental approaches allows us to conclude that the primary amino acid sequence of the lower hinge region (residues 234–237) of human IgG molecules determines recognition by human FcγRI, FcγRII and FcγRIII. Glycosylation of the CH2 domain is also essential, although the carbohydrate is not accessible for direct interaction with ligands. The role of the carbohydrate moiety may be to maintain a protein conformation that allows accessibility to amino acid side chains essential for ligand recognition and binding. It appears logical that the evolutionarily-related Fcγ R molecules should interact with overlapping non-identical sites on the IgG molecule.

Multiple binding sites on the CH2 domain of IgG for mouse FcγR11

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (FcγR). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fcγ R. This domain was mapped for interaction with mouse Fcγ R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human FcγR1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237 → Ala, Asn 297 → Ala, and Glu 318 → Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse FcγR11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by FcγR11 of the P388D1 cell line.

Epitope-dependent synergism and antagonism between CD40 antibodies and soluble CD40 ligand for the regulation of CD23 expression and IgE synthesis in human B cells

Background: The induction of IgE synthesis in naïve B cells requires two T‐cell‐derived signals: one delivered through CD40 and the other via interleukin‐4 (IL‐4). The natural counterstructure to CD40 is the CD40 ligand (CD40L). We have asked about the interplay between CD40L and CD40 mAb that recognize distinct epitopes in delivering signals for regulating IL‐4‐dependent IgE synthesis and the expression of CD23, the low‐affinity IgE receptor, in resting B cells.

Aglycosylated chimaeric human IgG3 can trigger the human phagocyte respiratory burst

This study investigates the capacity of a complexed aglycosylated chimaeric human IgG3 antibody to induce the respiratory burst in human monocyte-like U937 cells. It demonstrates that the aglycosylated antibody, prepared by cell culture in tunicamycin, retains significant capacity to trigger this effector function which was assayed as superoxide generation. Erythrocytes sensitized with near maximal levels of aglycosylated IgG3 were able to trigger > 80% of the superoxide generation triggered by the glycosylated antibody from U937 cells induced to differentiate by interferon gamma and the aglycosylated IgG3 gave half maximal responses at sensitization levels only 72% higher than those required by the glycosylated form. Aglycosylated IgG3 was, however, much less effective in triggering superoxide generation by interferon gamma treated U937 cells at low sensitization levels as threshold responses required only 60 glycosylated IgG3 molecules per erythrocyte compared with 16,000 aglycosylated molecules. In addition, these studies indicate significant differences between the target cell to effector cell ratios which permit IgG sensitized erythrocytes to stimulate the respiratory burst and those which stimulate ADCC in the same effector cell type.

The extrafollicular-to-follicular transition of human B lymphocytes: induction of functional globotriaosylceramide (CD77) on high threshold occupancy of CD40

Amongst lymphocytes, expression of CD77 (globotriaosylceramide, Gb3) is exclusive to B cells of the germinal center (GC). Its acquisition by extrafollicular B cells may thus herald their commitment to a follicular response. Here we show that high threshold occupancy of CD40 by its cognate ligand (CD40L) promotes rapid induction of CD77 expression in non‐GC (CD38lo) B cells. The kinetics of CD77 acquisition mirrored those of GC‐related markers CD95 and CD86 but contrasted with the more delayed increase in CD38 expression. Induction of CD77 was not a simple consequence of cell cycle entry: other conditions of stimulation equally capable of driving proliferation failed to promote CD77 expression. CD77 was functional in that cells were now sensitive to Verotoxin‐1, an Escherichia coli‐derived ligand of Gb3. These data indicate that acquisition by extrafollicular B cells of CD77 results from high threshold occupancy of CD40, a situation that should be reached physiologically only once a critical level of T cell priming has been achieved.

Human Fcγ RI triggering of the mononuclear phagocyte respiratory burst

Abstract This study investigates the role played by Fcγ RI and Fcγ RII in triggering the respiratory burst induced in human monocyte-like U937 cells by monoclonal chimaeric anti-NIP antibodies expressing the human IgG subclass and mouse IgG2b heavy chains. Respiratory burst activity was measured as Superoxide generation. Four separate lines of evidence indicate a predominant role for Fcγ RI in triggering Superoxide generation induced by erythrocytes sensitized with up to the maximum of 100,000 IgG molecules per cell. Firstly, erythrocytes sensitized with mouse IgG2b anti-NIP antibodies which are not recognized by human Fcγ RI, did not induce a response but when residue Glu-235 was replaced by Leu to give the lower hinge sequence of mouse IgG2a which is recognized by Fcγ RI, the mutant bound to Fcγ RI and induced a response equal to 80% of that given by chimaeric human IgG3. Chimaeric human IgG3 antibodies with amino acid substitutions in the lower hinge showed reduced activity and the greatest reductions ( IgG1 > IgG4 ⪢ IgG2. Thirdly, responses induced by chimaeric human IgG were inhibited by concns of monomeric human IgG3 in the nM range. Finally, chimaeric human IgG3 induced responses were inhibited by anti-Fcγ RI, but not anti-Fcγ RII monoclonal antibodies. Consistent with a major role for FcγRI in triggering the responses of U937 cells, erythrocytes sensitized with chimaeric human IgG3 did not induce Superoxide generation by neutrophils which express FcγRII and FcγRIII, or eosinophils which express Fcγ RII, but neither of which expresses Fcγ RI.

Release of clonal block in B cell chronic lymphocytic leukaemia by engagement of co-operative epitopes on CD40

The clonal cells of patients with B-chronic lymphocytic leukaemia (B-CLL)--which essentially reside in a resting configuration--are characterised by a relative refractoriness to the normal signals for B cell growth and differentiation. Previously it has been shown that, using an in vitro culture system where CD40 is hyper-crosslinked by monoclonal antibody (mAb) held on CD32-transfected mouse L cells, the clonal block in B-CLL cells can be released with a resultant high rate of DNA synthesis ensuing. In the present study, we report that such release can be achieved purely with soluble reagents whereby co-operative epitopes on CD40 are targeted by the combined use of mAb and soluble recombinant CD40L. Substantial levels of DNA synthesis were induced under such conditions in 7/18 patients using CD40-targeted reagents alone and in 16/18 patients in the additional presence of interleukin 4. Possible extrapolation of these findings to novel therapeutic modalities could be envisaged.

Signals for Survival and Apoptosis in Normal and Neoplastic B Lymphocytes

B lymphocytes are subject to selection within germinal centers following somatic hyper-mutation on immunoglobulin variable region genes based on their ability to bind antigen with high affinity. Non-selected cells die by apoptosis. Tumors with features of germinal center B cells include follicular center cell lymphoma and Burkitts lymphoma. We have used the latter extensively as a neoplastic model of germinal center cells and have compared directly the behaviour of cell lines derived from biopsy material with that of the normal counterparts. Here we describe some of our findings in the two systems with regard to signals regulating survival and apoptosis.