Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2).

CTLA-4 expressed on activated T cells binds to CD80 (B7-1) and CD86 (B7-2) molecules present on APC with high avidity and appears to deliver a negative regulatory signal to the T cell. We have investigated the kinetics of CTLA-4 binding to CD80 and CD86, together with the effects of selected CTLA-4 mutations on binding activity. The dissociation constants (Kd) for binding of CTLA-4-Ig to CD80 and CD86 transfectants were 8.1 and 6.7 nM, respectively. Surface plasmon resonance was used to determine kinetic parameters of CTLA-4-Ig binding to CD80-Ig and CD86-Ig fusion proteins and revealed enhanced association (ka) and dissociation (kd) rate constants for CD86-Ig compared with CD80-Ig. Furthermore, CD80-Ig and CD86-Ig fusion molecules demonstrated variable abilities to cross-compete for binding to several modified forms of CTLA-4-Ig. Differential binding of CD80 and CD86 to CTLA-4 was further revealed by analysis of 10 discrete CTLA-4 mutants. Five single amino acid substitutions within the CTLA-4 MYPPPY domain exerted modest effects on CD80 binding, but each of these substitutions completely abrogated CD86 binding. In addition, substitutions just N-terminal of the MYPPPY region, and within the CDR1-like region of CTLA-4, eliminated both CD80 and CD86 binding. Hence, CD80 and CD86 bind with different association/dissociation kinetics to similar, but distinct, sites on CTLA-4.

Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation.

T cell activation involves not only recognition of antigen presented by the MHC, but also nonspecific interactions termed "costimulation." The costimulatory molecules B7-1 and B7-2 are ligands on antigen-presenting cells for the CD28 and CTLA-4 receptors on T cells. Previously, a fusion protein consisting of human CTLA-4 linked to human Fc was shown to bind B7-1 and B7-2 with high avidity and to prevent specific T cell activation. Here we investigated the effects of a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 bound to mouse IgG2a Fc (CTLA-4-Fc) upon experimental autoimmune encephalomyelitis, a T cell-mediated disease that serves as a model for multiple sclerosis. CTLA-4-Fc prevented experimental autoimmune encephalomyelitis in 26 of 28 CTLA-4-Fc-treated mice (median maximum score 0), whereas 28 of 30 mice treated with control mouse IgG2a developed disease (median maximum score 2.75). Less inflammation and virtually no demyelination or axonal loss occurred in CTLA-4-Fc-treated compared with control-treated mice. Activated splenocytes from CTLA-4-Fc-treated mice were able to transfer disease adoptively to naive recipients. These results indicate a key role for the B7/CD28 system in the development of actively induced murine experimental autoimmune encephalomyelitis, suggesting an area of investigation with therapeutic potential for multiple sclerosis.

Delivery of nascent MHC class II-invariant chain complexes to lysosomal compartments and proteolysis of invariant chain by cysteine proteases precedes peptide binding in B-lymphoblastoid cells.

The intracellular trafficking, proteolysis, and dissociation of invariant chain (li) associated with nascent class II molecules was examined in B-lymphoblastoid cells. Metabolic labeling and Percoll gradient centrifugation was used to assess the kinetics of delivery and processing of class II-li complexes within the endocytic pathway. Catabolism of class II-li complexes rapidly followed their delivery from post-Golgi compartments to dense lysosome-like compartments distinct from early and late endosomes. Direct peptide binding assays revealed that class II molecules associated with even small N-terminal fragments of li failed to bind peptide. Cysteine protease inhibitors alone blocked li proteolysis/dissociation and accumulation of class II-li biosynthetic intermediates within lysosome-containing compartments. Active-site labeling of cysteine proteases in B cells was used to identify cysteine proteases capable of mediating li proteolysis within endosomal compartments. Our results indicate rapid, possibly direct, transport of nascent class II-li complexes from the Golgi/trans-Golgi network to dense lysosomal compartments wherein cysteine protease(s), likely including cathepsin B, mediate complete removal of li. Inhibition of cysteine protease activity results in the accumulation of incompletely processed class II-li complexes, which lack peptide binding ability, within lysosomal compartments.

Peptide binding to surface class II molecules is the major pathway of formation of immunogenic class II-peptide complexes for viable antigen presenting cells.

Although studies on fixed APCs have demonstrated that peptide can bind to cell surface class II molecules, the mechanisms by which peptide-class II complexes are formed in viable cells is largely unexplored. To explore the possibility that peptide loading of class II molecules was occurring after endocytosis of peptides as well as by surface binding, we utilized an immunogenic hemagglutinin peptide (HAP 128-145) from the influenza strain A/Japan/57, and studied the appearance of surface complexes of HAP 128-145 bound to HLA-DRw11 molecules on human B-lymphoblastoid cells (BLCLs). Detection of the bound peptide was made possible by a rabbit anti-serum (alpha HAP) raised against HAP 128-145, which recognizes both the free peptide as well as peptide bound to DRw11 on living APCs. Pretreatment of the BLCLs with a variety of inhibitors of protein synthesis and intracellular trafficking failed to decrease the levels of HAP 128-145/DRw11 surface complexes. However, significant inhibition in the appearance of these complexes was caused by a decrease in the temperature at which the cells were incubated with peptide. Temperature-specific inhibition was also observed for fixed DRw11-positive APCs and purified DRw11 molecules indicating that the effect of temperature was directly on the class II molecules. We conclude that surface binding of peptide by class II molecules on human B cells is a major pathway of formation of immunogenic class II-peptide complexes for at least some soluble antigenic peptides, and that endocytosis of soluble peptides with subsequent binding of peptide by intracellular class II molecules plays little if any role in the formation of such complexes. Moreover, class II molecules have evolved to stably bind peptide optimally at physiologic temperatures, independent of cell metabolism.

Antibody recognition of an immunogenic influenza hemagglutinin-human leukocyte antigen class II complex.

The A/Japan/57 influenza hemagglutin (HA) peptide HA 128-145, when bound by human histocompatibility leukocyte antigen-DRw11 cells, is recognized by the human CD4+ T cell clone V1. A rabbit antiserum has been raised against HA 128-145 which recognizes not only the free peptide, but also the HA 128-145/DRw11 complex on a solid matrix, in solution, or on the surface of viable cells. The detection of these complexes on viable cells was shown to be class II specific, DRw11 restricted, and commensurate with the level of DRw11 expression. The identity of DRw11 as the cell surface molecule binding HA 128-145 was confirmed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping. Using this antiserum HA 128-145/DRw11 complexes could be detected on the cell surface as soon as 30 min after the peptide was added, and increased up to 24 h. Dissociation kinetics showed these complexes were long-lived, with a half-life of approximately 14 h. This anti-HA peptide antiserum represents the first direct means of studying antigenic peptide-human leukocyte antigen class II complexes on the surface of living cells without the addition of a non-amino acid moiety to the peptide. The properties of this antiserum thus provide the potential to study naturally processed antigenic peptides as well as the mechanism of processing itself in a physiologically relevant system.

Human Ia alpha- and beta-chains are sulfated.

The human Ia antigens (DR, DQ, and DP), determined by genes with the HLA complex, are heterodimers consisting of a 34,000-Da alpha-chain glycoprotein and a 29,000-Da beta-chain glycoprotein. During the course of studies characterizing a recently described sulfated proteoglycan that is specifically associated with Ia, we discovered that there were also nonproteoglycan sulfated components present in the Ia immunoprecipitates. One-dimensional sodium dodecyl sulfate-gel analysis of these latter sulfated components derived from both DR and DQ immunoprecipitates indicated that these components have mobilities indistinguishable from conventional Ia alpha and beta glycoproteins. Two-dimensional gel analysis confirmed these findings and revealed that Ia-associated invariant proteins are sulfated as well. The sulfate moiety was not removed by endoglycosidase F treatment, suggesting that the protein portion of the molecule was sulfated. These results indicate that Ia alpha-, beta-, and invariant chains can be sulfated and raise the possibility that sulfation may play a role in the physiology of Ia molecules.

The human invariant chain is the core protein of the human class II-associated proteoglycan.

The human class II-associated chondroitin sulfate proteoglycan (CSPG) was analyzed biochemically and immunologically to determine a possible relationship with the human invariant chain (gamma 1) and its related components. The CSPG was purified by a three-step procedure involving associative ion-exchange chromatography, immunoprecipitation, and dissociative ion-exchange chromatography. Treatment of the CSPG with chondroitinase revealed core proteins of Mr approximately 46,000, 38,000, and 28,000, with the 38,000 species most highly represented. Tryptic peptide analysis revealed identity of the peptides of the 38,000 Mr core protein and gamma 1, and of the 28,000 Mr species and p25. The CSPG and its core proteins were shown to react directly with the mouse anti-human invariant chain monoclonal antibody VIC-Y1 and a rabbit antiserum produced against a synthetic peptide corresponding to the C-terminal end of invariant chain. These results demonstrate that the invariant chain is the core protein of the class II-associated CSPG. In addition, virtually all the CSPG was shown to be present on the cell surface.

Invariant chain is the core protein of the Ia-associated chondroitin sulfate proteoglycan.

The murine Ia-associated chondroitin sulfate proteoglycan (CSPG) was studied both biochemically and immunochemically to determine the nature of its core protein. Chondroitinase ABC or chondroitinase AC treatment of the CSPG digested the chondroitin sulfate glycosaminoglycan, yielding a core protein that migrated with an apparent molecular weight of 38,000. Comparative V8 protease digestion of the CSPG core protein and conventional invariant glycoproteins yielded homologous peptides, indicating that the core protein and invariant chain were structurally similar. The purified CSPG and its core protein were both shown to react directly with the monoclonal anti-invariant chain antibody, In-1. Comparative tryptic peptide analysis by high performance liquid chromatography demonstrated coelution of the majority of the peptides from the invariant chain and the CSPG core protein. Collectively, these results indicate that the CSPG is an alternatively processed form of invariant chain.