Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.     

TLR agonists downregulate H2-O in CD8alpha- dendritic cells

Peptide loading of MHC class II (MHCII) molecules is catalyzed by the nonclassical MHCII-related molecule H2-M. H2-O, another MHCII-like molecule, associates with H2-M and modulates H2-M function. The MHCII presentation pathway is tightly regulated in dendritic cells (DCs), yet how the key modulators of MHCII presentation, H2-M and H2-O, are affected in different DC subsets in response to maturation is unknown. In this study, we show that H2-O is markedly downregulated in vivo in mouse CD8α(-) DCs in response to a broad array of TLR agonists. In contrast, CD8α(+) DCs only modestly downregulated H2-O in response to TLR agonists. H2-M levels were slightly downmodulated in both CD8α(-) and CD8α(+) DCs. As a consequence, H2-M/H2-O ratios significantly increased for CD8α(-) but not for CD8α(+) DCs. The TLR-mediated downregulation was DC specific, as B cells did not show significant H2-O and H2-M downregulation. TLR4 signaling was required to mediate DC H2-O downregulation in response to LPS. Finally, our studies showed that the mechanism of H2-O downregulation was likely due to direct protein degradation of H2-O as well as downregulation of H2-O mRNA levels. The differential H2-O and H2-M modulation after DC maturation supports the proposed roles of CD8α(-) DCs in initiating CD4-restricted immune responses by optimal MHCII presentation and of CD8α(+) DCs in promoting immune tolerance via presentation of low levels of MHCII-peptide.     

Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.     

Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active site structures than mAb 4-4-20.     

Molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody.

Anti-metatype antibodies have been described as antibodies which recognize ligand-induced conformational changes in the antibody variable region. Additionally, anti-metatype antibodies, produced by multiple immunizations with liganded high affinity monoclonal anti-fluorescein antibody 4-4-20, enhanced the lifetime of monoclonal antibody 4-4-20-fluorescein complex. To better understand the mechanism of the delayed dissociation rate, deuterium oxide was used to probe the liganded active site. The rate and extent of deuterium oxide-mediated fluorescence enhancement of bound ligand served to monitor the conformational dynamics of the active site in the presence and absence of anti-metatype antibodies. Results showed that anti-metatype antibodies reduced the rate and extent of deuterium oxide-mediated fluorescence enhancement of 4-4-20, a single-chain derivative of 4-4-20 (consisting of the variable domains and a polylinker), and idiotypically related monoclonal anti-fluorescein antibodies suggesting that anti-metatype stabilized the liganded active site. Size exclusion liquid chromatography was utilized to isolate the liganded antibody-anti-metatype complex. Liganded single chain antibody 4-4-20 was mixed with 10-fold molar excess anti-metatype Fab fragments, and a major complex eluted with an apparent M(r) 249,000. The apparent molecular weight of this complex inferred that one liganded single chain antibody was bound by five antimetatype Fab fragments. Spectral analysis confirmed these results and the characteristic delayed rate of ligand dissociation was also observed for the isolated complex. The results suggest that anti-metatype antibodies stabilize the liganded conformation by forming a large, stable, macromolecular complex.     

Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.

This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.     

CDw78 defines MHC class II-peptide complexes that require Ii chain-dependent lysosomal trafficking, not localization to a specific tetraspanin membrane microdomain

MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.     

Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody

Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.     

Expression patterns of H2-O in mouse B cells and dendritic cells correlate with cell function

In the endosomes of APCs, the MHC class II-like molecule H2-M catalyzes the exchange of class II-associated invariant chain peptides (CLIP) for antigenic peptides. H2-O is another class II-like molecule that modulates the peptide exchange activity of H2-M. Although the expression pattern of H2-O in mice has not been fully evaluated, H2-O is expressed by thymic epithelial cells, B cells, and dendritic cells (DCs). In this study, we investigated H2-O, H2-M, and I-A(b)-CLIP expression patterns in B cell subsets during B cell development and activation. H2-O was first detected in the transitional 1 B cell subset and high levels were maintained in marginal zone and follicular B cells. H2-O levels were down-regulated specifically in germinal center B cells. Unexpectedly, we found that mouse B cells may have a pool of H2-O that is not associated with H2-M. Additionally, we further evaluate H2-O and H2-M interactions in mouse DCs, as well as H2-O expression in bone marrow-derived DCs. We also evaluated H2-O, H2-M, I-A(b), and I-A(b)-CLIP expression in splenic DC subsets, in which H2-O expression levels varied among the splenic DC subsets. Although it has previously been shown that H2-O modifies the peptide repertoire, H2-O expression did not alter DC presentation of a number of endogenous and exogenous Ags. Our further characterization of H2-O expression in DCs, as well as the identification of a potential free pool of H2-O in mouse splenic B cells, suggest that H2-O may have a yet to be elucidated role in immune responses.