HLA-DR7-Specific monoclonal antibodies and a chimpanzee anti-DR7 serum detect different epitopes on the same molecule

We describe here two monoclonal antibodies with HLA-DR7 serologic specificity. The antibodies, SFR16-DR7M, a cytotoxic rat IgM antibody of high affinity, and SFR16-DR7G, a noncytotoxic antibody of the rat IgG 2a class, react with only DR7-positive cells in radioimmunoassay. The cytotoxic activity of SFR16-DR7M correlates completely with the presence of the DR7 specificity, and segregates with the DR7-bearing haplotype in a family. SFR16-DR7M precipitates a class II molecule with the electrophoretic characteristics of DR molecules from LG-10, an HLA-DR7 homozygous cell line. SFR16-DR7G completely inhibits the cytotoxicity of SFR16-DR7M, but only partially inhibits the cytotoxicity of a chimpanzee antiserum with DR7 specificity, Gay/Swei. In binding-inhibition studies, binding of SFR16-DR7M to LG-10 cells is only partially inhibited by the chimpanzee antiserum and vice versa. Both SFR16-DR7M and Gay/Swei reciprocally deplete the same class II molecules from a ³⁵S-methionine-labeled detergent-solubilized membrane preparation of the LG-10 cell line. The chimpanzee serum Gay contains antibodies reactive with epitopes on separated DR7 beta chains, while both SFR16-DR7M and SFR16-DR7G bind only to DR7 alpha-beta complexes. These data suggest that at least two allogeneic epitopes exist which result in the same serologic specificity, and that these epitopes differ in their requirement for alpha-beta complex formation.     

Complexity of the supertypic HLA-DRw53 specificity: two distinct epitopes differentially expressed on one or all of the DR beta-chains depending on the HLA-DR allotype

The supertypic HLA-DRw53 specificity is associated with three allelic class II specificities defined by alloantisera: HLA-DR4, -DR7, and DRw9. The present study demonstrates the complexity of this supertypic DR specificity by comparing two DRw53-related determinants defined by the monoclonal antibodies PL3 and 109d6. For every HLA-DR4 cell line tested, both monoclonal antibodies were found to bind to the same subpopulation of DR molecules. This PL3+, 109d6+ DR subpopulation is also found on most, but not all, DR7+ cell lines with a beta-chain pattern that is identical to the beta-chain pattern of the PL3+, 109d6+ subpopulation on DR4 cell lines. However, some DR7+ cells which carry the HLA haplotype Bw57, DR7, DRw53, DQw3 were also found which completely lack the expression of the 109d6 determinant, but continue to express the PL3 determinant and some of the DRw53 determinants recognized by alloantisera. This results from the fact that the PL3 determinant is expressed on all of the DR molecules found on DR7 cells, including the distinct subpopulation of molecules that carry the HLA-DR7 determinant recognized by the monoclonal antibody SFR16-DR7. This PL3+, SFR16-DR7+ subpopulation does not carry the 109d6 determinant, demonstrating that the PL3 and 109d6 DRw53-related determinants are distinct and can be expressed on a different number of DR molecules, depending on the allotype of the cells. Blocking studies were also performed by using these monoclonal antibodies with alloreactive HLA-DR7-specific cytotoxic T cell clones. In these studies, the T cell-defined HLA-DR7 determinants were found to be carried by the same subpopulation of DR molecules recognized by the HLA-DR7-specific monoclonal antibody and not carried by the DR molecules recognized by 109d6. The DR7+ cell lines which do not express the 109d6 determinant also fail to express another supertypic determinant recognized by the monoclonal antibody IIIE3 carried on this molecule. Furthermore, no additional allelic forms of this unique DR beta-chain were found associated with the nonpolymorphic DR alpha-chain on these cells, suggesting that this DR beta-chain gene is not expressed. These cells also behave as homozygous typing cells for the Dw11 subtype of DR7 in HLA-D typing in the mixed lymphocyte culture assay. This suggests that the lack of expression of a specific class II gene may contribute additional genetic polymorphism within the known HLA-DR allotypes.     

Two-dimensional gel analysis of a second family of class II molecules by polymorphic HLA-DR4,5, and w9 monoclonal antibodies

Human Ia-like, class II molecules were isolated by immunoprecipitation with monoclonal antibodies from various HLA-D/DR homozygous cell lines and were analyzed by two-dimensional gel electrophoresis. The monoclonal antibody PLM12 reacted with B cells carrying DR4, DR5, DRw6.2, and DRw9 phenotypes, and its reactivity perfectly correlated with the previously defined TB21 (MB3-like) specificity. Class II molecules detected by PLM12 were structurally distinct from those precipitated by the anti-DR monoclonal antibody NC1 on all HLA-DR4, DR5, DRw6.2, and DRw9 homozygous cell lines and showed polymorphism in heavy and light chains among these cell lines. The monoclonal antibodies PLM2 and PLM9 only reacted with B cells carrying DR5 and DRw6.2 and also detected a distinct set of class II molecules from those precipitated by NC1 but identical to those of PLM12. Thus, PLM2 and PLM9 serologically detected a new subtypic antigen of the PLM12-reactive class II molecules. Furthermore, the antibody NC1 precipitated two light chains and one heavy chain from HLA-DRw6.2 homozygous cell line EBV-Sh. The result indicated the presence of three sets of class II molecules: two in a DR family and another carrying the polymorphic determinants detected by PLM2, PLM9, and PLM12 in a second family.     

Characterization of specific HLA-DQ alpha allospecificities by genomic, biochemical, and serologic analysis

Bgl II restriction endonuclease digestion of genomic DNA from lymphoblastoid cell lines homozygous for HLA DR and DQ serological specificities, followed by hybridization with a DQ alpha cDNA probe, identified a genomic polymorphism characterized by two reciprocal patterns, one associated with DR 3, 5 and 8 and the other with DR 1, 2, 4, 7, and 9. The former pattern corresponded precisely to the reactivity of monoclonal antibody SFR20-DQ alpha 5, shown by Western blotting to react with isolated alpha-chains, but not with beta-chains. Additional variants of the DQ alpha genes were identified by using a locus-specific oligonucleotide probe for the DQ alpha gene, indicating differences among the DQ alpha 5-negative set of alleles. This analysis defines a set of DQ alpha allelic markers that are distinct from the well-established DQ serologic specificities DQw1, 2, 3 or "blank." Although most DQ alpha 5+ cells carry the DRw52 specificity associated with the DR beta 2 gene, analysis of DQ alpha polymorphisms on DR5, DQw1; DR8, DQw1; and DRw13, DQw1 cells verified that this DQ alpha family of alleles was not invariably linked to the DR beta 2 locus.     

Biochemical and functional evidence that an MT3 supertypic determinant defined by a monoclonal antibody is carried on the DR molecule on HLA-DR7 cell lines

A cytotoxic monoclonal antibody, PL3, was produced by immunizing mice with a cell line homozygous for the HLA class II antigenic specificity DR7. The serologic specificity of PL3 was completely concordant with the MT3 supertypic specificity, which is tightly associated with HLA-DR4, -DR7, and -DRw9. This was confirmed by the finding that F(ab')2 fragments of PL3 blocked the cytotoxicity of anti-MT3 alloantisera. Although PL3 bound to each of the MT3-positive cell lines, it showed significantly weaker binding to HLA-DR4 and -DRw9 cells relative to -DR7 cells, both in titration and in quantitative absorption assays. This differential pattern of binding was not found for the polyclonal MT3 alloantisera, suggesting that the PL3 determinant may be one of several closely related determinants that comprise the MT3 allospecificity. To identify which of the subpopulations of class II molecules carry the PL3 determinant, several approaches have been used. F(ab')2 fragments of PL3 which block the anti-MT3 alloantisera were also tested with anti-MB2 and anti-DR7 sera. Binding of the PL3 F(ab')2 fragments to DR7 homozygous target cells had no effect on the anti-MB2 sera, but significantly enhanced the cytotoxic reactivity of some anti-DR7 sera. This finding suggested that the PL3 determinant is distinct from the DR7 determinant, but is carried on the same molecule. PL3 was also used in blocking studies with allocytotoxic T cell clones which only recognize DR7-positive cell lines. Binding of PL3 to the DR7-positive target cells was found to completely inhibit these T cell clones. Complete blocking was also found with a monoclonal antibody, PL8, which recognizes a monomorphic determinant found on the DR subpopulation of class II molecules. This finding suggested that the PL3 determinant is carried on the same molecule that carries these T cell-defined DR7 allodeterminants. In biochemical studies with DR7-positive cell lines, PL3 and PL8 were found to immunoprecipitate the same subpopulation of class II molecules recognized by other DR-specific antibodies, SG157 and TAL-1B5. Two-dimensional gel analysis demonstrated that the pattern of alpha- and beta-chains immunoprecipitated by PL3, PL8, and TAL-1B5 were identical. In sequential immunoprecipitation studies, both PL3 and TAL-1B5 were capable of removing the same DR subpopulation of molecules recognized by PL3, PL8, TAL-1B5, or SG157 while leaving the additional class II molecules (DS) recognized by SG171 on DR7 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The clinical immunogenetics of viral hepatitis C in a Caucasoid population of western Siberia

The analysis of the immunogenetic studies on hepatitis C patients among the Caucasoid population of western Siberia has revealed a significant increase in the detection rate of antigens HLA-A10 and HLA-DR5, the combinations of DR2-DR5, DR5-DR7, DR1-B27 and the complete absence of antigen HLA-DR4, which is indicative of the fact that susceptibility and resistance to the development of the disease is associated with the genes of the main histocompatibility complex. In hepatitis of mixed etiology, B and C, a significant increase in the occurrence of HLA antigens: -A1, -B8, -DR1 and -DR3, as well as the combinations of A1-DR1, A1-DR3, A3-DR3, A9-A10, DR1-DR3, B8-DR3 is noted; at the same time a decrease in the occurrence of antigen DR4 and its combination with antigen HLA-A2 is observed.     

A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death

Like anti-Fas monoclonal antibodies, some monoclonal antibodies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors have tumoricidal activity too. In this article we report a novel mouse anti-human DR5 monoclonal antibody, AD5-10, that induces apoptosis of various tumor cell lines in the absence of second cross-linking in vitro and showed strong tumoricidal activity in vivo. AD5-10 does not compete with TRAIL for binding to DR5 and synergizes with TRAIL to induce apoptosis of tumor cells. AD5-10 induces both caspase-dependent and caspase-independent cell death in Jurkat cells, whereas TRAIL induces only caspase-dependent cell death. We show for the first time that DR5 can mediate caspase-independent cell death, and DR5 can mediate distinct cell signals when interacting with different extracellular proteins. Studies on AD5-10 help us to understand more on the functions of DR5 and may provide new ideas for cancer immunotherapy.     

Quantitative expression of epitopes defined by murine monoclonal antibodies on DR molecules from different HLA haplotypes

Monoclonal antibodies 50D6 and 21r5, reactive with human class II molecules, were analyzed quantitatively by flow cytometry and cellular radioimmunoassay for their binding to cells of different HLA-DR types. Monoclonal antibody 50D6 bound equally to cells of all DR types tested except DR7, where no reactivity was observed. Monoclonal antibody 21r5 was reactive with all cells. However, the percentage of DR molecules at the cell surface expressing 21r5 epitope varied with the DR type and increased as follows: DR3 = DR7 less than DR2 less than DR5 less than DR4 less than DR1. MAb 50D6 reacted with an epitope spatially related to but distinct from the 21w4 epitope present on all DR molecules. The 50D6 epitope was shown to be present on isolated DR1 molecules.     

Structural relationships between the DR beta 1 and DR beta 2 subunits in DR4, 7, and w9 haplotypes and the DRw53 (MT3) specificity

The class II molecules of DR4, DR7, and DRw9 haplotypes were analyzed by immunoprecipitation, followed by two-dimensional gel electrophoresis and N-terminal amino acid sequencing. By using HLA-DR chain-specific monoclonal antibodies, two distinct DR beta-chains were identified. One beta-chain, designated DR beta 2, had a characteristic acidic mobility. In all three DR types the DR beta 2-chains were indistinguishable by two-dimensional gel electrophoresis and partial N-terminal sequencing. A second DR beta-chain designated beta 1 had a more basic mobility on two-dimensional gel electrophoresis, and differed from the DR beta 2-chains by the consistent presence of phenylalanine at position 18. In contrast to the DR beta 2-chains, the DR beta 1-chains were clearly polymorphic, with specific amino acid sequence differences characteristic of each DR type. The monoclonal antibodies 109d6 and 17-3-3S, recognizing distinct polymorphic epitopes similarly correlated with the DRw53 allospecificity, were found to react with different DR beta-chains. The epitope recognized by monoclonal antibody 109d6 was identified on the DR beta 2-chain in the prototypic DR4, DR7, and DRw9 cell lines. However, the DR7, Dw11, DQw3 cell line BEI was unreactive with antibody 109d6 by either immunofluorescence or immunoprecipitation despite the presence of the DRw53 allodeterminant on this cell line. The other DRw53-like monoclonal antibody, 17-3-3S, reacted with the DR beta 1-rather than the DR beta 2-chain in all DR4 and DR7 cell lines tested, including the cell line BEI. However, antibody 17-3-3S did not react with the DRw53-positive DRw9 cell line ISK. These studies suggest that the DRw53 allospecificity is more complex than previously thought and may comprise a number of distinct epitopes encoded by two different DR beta loci.     

Targeting a Novel N-terminal Epitope of Death Receptor 5 Triggers Tumor Cell Death

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu⁶ was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.     

Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.     

Distribution of antigenic determinants recognized by three monoclonal antibodies (Q2/70, Q5/6 and Q5/13) on human Ia-like alloantigens and on their subunits

The distribution of antigenic determinants recognized by the anti-Ia-like antigen monoclonal antibodies (MoAb) Q2/70, Q5/6 and Q5/13 on molecules coded for by the DR locus and by non-DR loci was investigated using a binding assay with 125I-labeled Ia-like antigens isolated from four B lymphoid cell lines. The determinants reacting with the MoAb Q2/70 and Q5/13 are expressed on all DR alloantigens tested and on BR4X7 specificities, while those reacting with the MoAb Q5/6 are not detectable on DRw7 and BR4X7 molecules. None of the monoclonal antibodies reacted with DC1 molecules. The MoAb Q5/6 and Q5/13 reacted with the isolated beta subunit of the Ia-like antigenic complex, while the MoAb Q2/70 did not react with the isolated chains.     

Optimized combination therapy using bortezomib, TRAIL and TLR agonists in established breast tumors

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptosis in various tumor cells by engaging the receptors, DR4 and DR5. Bortezomib (Velcade) is a proteasome inhibitor that has been approved for patients with multiple myeloma. There is some experimental evidence in preclinical models that bortezomib can enhance the susceptibility of tumors to TRAIL-induced apoptosis. In this study, we investigated the effects of TRAIL-induced death using an agonistic antibody to the TRAIL receptor DR5 (α-DR5) in combination with bortezomib administered to mice previously injected with breast cancer cells (TUBO). This combination had some beneficial therapeutic effect, which was significantly enhanced by the co-administration of a Toll-like receptor 9 agonist (CpG). In contrast, single agent treatments had little effect on tumor growth. In addition, we evaluated the effect of combination with α-DR5, bortezomib, and CpG in the prevention/treatment of spontaneous mammary tumors in Balb-neuT mice. In this model, which is more difficult to treat, we observed dramatic antitumor effects of α-DR5, bortezomib and CpG combination therapy. Since such a mouse model more accurately reflects the immunological tolerance that exists in human cancer, our results strongly suggest that these combination strategies could be directly applied to the therapy for cancer patients.     

Eradication of established tumors in mice by a combination antibody-based therapy

Tumor-cell apoptosis is the basis of many cancer therapies, and tumor-specific T cells are the principal effectors of successful antitumor immunotherapies. Here we show that induction of tumor-cell apoptosis by an agonistic monoclonal antibody to DR5, the apoptosis-inducing receptor for TNF-related apoptosis-inducing ligand (TRAIL), combined with T-cell activation by agonistic monoclonal antibodies to the costimulatory molecules CD40 and CD137, potently and rapidly stimulated tumor-specific effector CD8+ T cells capable of eradicating preestablished tumors. Primary fibrosarcomas initiated with the carcinogen 3-methylcholanthrene (MCA), multiorgan metastases and a primary tumor containing as many as 90% tumor cells resistant to DR5-specific monoclonal antibody were rejected without apparent toxicity or induction of autoimmunity. This combination therapy of three monoclonal antibodies (trimAb) rapidly induced tumor-specific CD8+ T cells producing interferon (IFN)-gamma in the tumor-draining lymph node, consistent with a crucial requirement for CD8+ T cells and IFN-gamma in the tumor rejection process. These results in mice indicate that a rational monoclonal antibody-based therapy that both causes tumor-cell apoptosis through DR5 and activates T cells may be an effective strategy for cancer immunotherapy in humans.     

Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5

Activation of the proapoptotic receptor death receptor5 (DR5) in various cancer cells triggers programmed cell death through the extrinsic pathway. We have generated a fully human monoclonal antibody (Apomab) that induces tumor cell apoptosis through DR5 and investigated the structural features of its interaction with DR5. Biochemical studies showed that Apomab binds DR5 tightly and selectively. X-ray crystallographic analysis of the complex between the Apomab Fab fragment and the DR5 ectodomain revealed an interaction epitope that partially overlaps with both regions of the Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand binding site. Apomab induced DR5 clustering at the cell surface and stimulated a death-inducing signaling complex containing the adaptor molecule Fas-associated death domain and the apoptosis-initiating protease caspase-8. Fc crosslinking further augmented Apomab's proapoptotic activity. In vitro, Apomab triggered apoptosis in cancer cells, while sparing normal hepatocytes even upon anti-Fc crosslinking. In vivo, Apomab exerted potent antitumor activity as a single agent or in combination with chemotherapy in xenograft models, including those based on colorectal, non-small cell lung and pancreatic cancer cell lines. These results provide structural and functional insight into the interaction of Apomab with DR5 and support further investigation of this antibody for cancer therapy.     

Epitope mapping of an HLA-DR-specific monoclonal antibody produced by using human-mouse transfectant cells

A polymorphic HLA-DR mAb, TAL15.1, was produced against L cells transfected with DR alpha- and beta-chain cDNA from a cell line homozygous for HLA-DRw8. This antibody reacted with DRw8 plus all other DR types except DR3 and DRw52. DR3 and DRw52 differ uniquely from other other DR antigens at position 77 in the beta 1-domain of their beta-chains where there is asparagine instead of threonine. In Western blots the antibody reacted with DR alpha/beta-dimer but not with free alpha- or beta-chains. Two-dimensional gel analysis of a DRw11, DRw52 cell line showed that TAL15.1 immunoprecipitated the DR products. Although it also coprecipitated the DQ beta chain products, flow microfluorimetric analysis with various transfectant cell lines showed that TAL15.1 failed to bind the DQ or DP products tested. We conclude that TAL15.1 is a DR-specific polymorphic antibody whose activity correlates with a specific residue. It has already proved to be a valuable reagent for distinguishing DR3 homozygotes from DR3, DRw6 heterozygotes, which have in the past been difficult to separate.     

Testicular germ cell sensitivity to TRAIL-induced apoptosis is dependent upon p53 expression and is synergistically enhanced by DR5 agonistic antibody treatment

The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 μg/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32°C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL. This work was supported, in part, by grants from the National Institute of Environmental Health Sciences/NIH (ES09145, JHR), Toxicology Training grant (ES T32 ES007247, CM), NIH Center Grant (P30 ES07784^, JHR) and the Center for Molecular and Cellular Toxicology (CMCT).     

High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR

Immunogenic peptides have been shown to bind detergent-solubilized class II (Ia) molecules from mice. In this investigation, we report that highly purified HLA-DR (DR) molecules in detergent solution are capable of binding a synthetic peptide (HAp) derived from the influenza hemagglutinin sequence. Although the presentation of this peptide has been demonstrated only to DR1-restricted Th cells, the association rate constants for the formation of HAp-DR1, -DR5, and -DR8 complexes were essentially identical (ka = 1.1 x 10(2) to 1.6 x 10(2) M-1 s-1). By contrast, the value of the rate constants for the dissociation of preformed HAp-DR1, -DR5, and -DR8 complexes varied nearly threefold (kd = 1.6 x 10(6) to 4.4 x 10(-6) s-1). The value of the equilibrium dissociation constants (KD) derived from these rate constants were 13 nM, 24 nM, and 28 nM, for HAp-DR1, -DR5, and -DR8 complexes, respectively. Scatchard analysis demonstrated that the KD obtained from the rate constants for the HAp-DR1 reaction was in excellent agreement with that obtained under equilibrium conditions. SDS-PAGE confirmed that the HAp-DR complexes were remarkably stable, as HAp remained associated with the DR alpha beta heterodimer after treatment of the complexes with SDS and beta-mercaptoethanol. Steady-state binding studies demonstrated that 18% of all DR1 molecules had bound HAp at equilibrium, whereas only 3.8% of all DR8 molecules had bound HAp under identical conditions. The slight differences in the KD for HAp-DR complexes suggest that differences in the affinity of a peptide for DR alleles alone may not always explain the process of MHC restriction.