mRNA abundance, rather than differences in subunit assembly, determine differential expression of HLA-DR beta 1 and -DR beta 3 molecules

The class II major histocompatibility molecules HLA-DR are formed by the association of a single DR alpha chain with two nonallelic DR beta chains. In DR3 cells one DR beta chain is severalfold more abundant than the other. We have studied the mechanism that controls the differential expression of these DR beta genes. We determined the amino-terminal sequences of the two expressed DR beta chains. Comparison of these sequences with the nucleotide sequences of the DR3B1 and DRB3a genes indicates that the abundant chain is the B1 gene product. Supporting this conclusion, an informative mutant, 9.4.3, was found to have lost the abundant beta chain and beta 1 mRNA. This mutant expresses normal cell surface levels of the DR beta 3 chain and exhibits no significant dosage compensation of its beta 3 chain. The unchanged level of DR beta 3 dimer on the cell surface suggests that free DR alpha chains are not the limiting factor in the surface expression of the beta 3 chain and, further, that the differential regulation of the surface expression of the two DR beta chains occurs at a step prior to DR assembly. Quantitation of DR beta mRNAs by locus-specific oligonucleotide probes showed that beta 1 mRNA is 4.5-fold more abundant than beta 3 mRNA, strongly indicating that the greater surface expression of DR beta 1 is a direct consequence of greater beta 1 mRNA abundance.     

Unassembled HLA-DR beta monomers are degraded rapidly by a nonlysosomal mechanism.

We previously observed that in a mutant B lymphoblastoid cell line which has a homozygous HLA-DR alpha deletion, DR beta-chains appeared to be unstable. In the present study, we have studied the pathway that leads to degradation of unassembled DR beta-chains. Unassembled DR beta-chains are degraded rapidly in the DR alpha deletion mutant cells, compared with the assembled DR heterodimers present in non-mutant cells. Accelerated DR beta turnover in 9.22.3 cells is specific; class I molecules in these DR alpha-deficient cells turned over slowly. DR beta-chains assemble with Ii in the DR alpha deficient cell line, but this did not protect DR beta-chains from degradation. The maturation of unassembled beta-chains is arrested before their reaching the medial Golgi compartment, and this degradation proceeds by a nonlysosomal, nonendosomal pathway. Degradation of DR beta-chains is blocked when cells are cultured at 16 degrees C, a temperature known to prevent vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus. Degradation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, a drug that is also known to inhibit protein transport from the ER. The results, taken together, suggest that degradation of unassembled DR beta-chains occurs by a nonlysosomal, nonendosomal pathway which involves transport of DR beta-chains out of the ER.     

A role for calnexin (IP90) in the assembly of class II MHC molecules.

Major histocompatibility complex (MHC) class II antigens consist of alpha and beta chains that associate intracellularly with the invariant (I) chain. The HLA-DR alpha beta I complex assembles in the endoplasmic reticulum (ER) into a nonameric structure via progressive addition of three alpha beta dimers to a core invariant chain trimer. We have examined intracellular association of alpha beta I complexes with the resident ER protein calnexin. Calnexin associates rapidly (within 3 min) with newly synthesized alpha, beta and I chains, and remains associated with the assembling alpha beta I complex until the final alpha beta dimer is added, forming the complete nonamer. Dissociation of calnexin parallels egress of alpha beta I from the ER. These results suggest that calnexin retains and stabilizes both free class II subunits and partially assembled class II-I chain complexes until assembly of the nonamer is complete.     

Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.     

RFLP analysis of HLA-DR and -DQ genes and their linkage relationships in the Pacific

Human genomic DNA samples from Melanesians, Micronesians, and Caucasoids of known HLA-DR type were examined with cDNA probes for HLA-DR alpha, -DR beta, -DQ alpha, and -DQ beta chain genes. DR beta hybridizations with TaqI-digested DNA did not detect any new DR specificities in the Pacific. However, within the DR5 specificity a common DNA subtype was found in Pacific Islanders that was not seen in Caucasoids. Altogether, four DNA subtypes of DR5 are described. With the DQ alpha and DQ beta probes, significantly more variation could be demonstrated between populations. For example, DR2 was associated with a DQ beta TaqI pattern in the Pacific that was very rare in Caucasoids and additional RFLP analysis with other enzymes showed that this pattern is probably associated with the Dw12 subtype of DR2. DRw8-positive samples showed two different DQ alpha TaqI patterns, and these correlated with DQw1 and DQw3 specificities. DR alpha hybridizations with BglII-digested DNA also revealed different linkage relationships of the HLA-class II region genes between Pacific and Caucasoid specimens. The different population linkage disequilibrium relationships have permitted tentative assignment of TaqI fragments to either the DR beta 1 or DR beta 2 genes and are highly suggestive that the DQw1 specificity is encoded by the DQ alpha chain gene. This study shows the value of population comparisons in contributing to knowledge of the genetic organization of the genome.     

Lack of detectable endocytosis of B lymphocyte MHC class II antigens using an antibody-independent technique

MHC class II complexes may intercept ingested foreign Ag as the nascent class II molecules exit the cell or as mature complexes recycle between the outer membrane and an internal compartment. To examine endocytosis of HLA-DR, we radiolabeled B lymphoblastoid cells, warmed the cells to allow internalization of membrane proteins, and then subjected viable cells to neuraminidase (NANAse)3 digestion. DR complexes were precipitated and analyzed by two-dimensional PAGE for shifts in isoelectric point signifying sensitivity to or protection from NANAse treatment. DR molecules were completely sensitive to the enzyme with or without specific antibody in the medium during the 37 degrees C incubation, suggesting that no detectable endocytosis had occurred. Control transferrin receptors, which readily internalize, showed marked protection. Assay sensitivity was measured by proportionate mixing of mock and NANAse-treated lysates; precipitated sialylated molecules were detectable at the 10% level. Monensin treatment to block recycling also failed to reveal any internally accumulated. NANAse-protected DR molecules. Measurements of turnover for surface-labeled DR complexes revealed a t 1/2 of approximately 36 h. We conclude that no large endocytically-derived pool of HLA-DR molecules exists within cells and suggest that the majority of mature DR molecules do not actively undergo internalization but reside at the cell surface after synthesis/transport for significantly long periods of time.     

Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II- associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild- type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.     

Polymorphic HLA-DR7 beta 1 chain residues that are involved in T cell allorecognition

The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule.     

The antigen-processing mutant T2 suggests a role for MHC-linked genes in class II antigen presentation.

.174xCEM.T2 (T2) is a human cell hybrid that has a large homozygous deletion within the MHC, including all of the functional class II genes. We have generated stable HLA-DR3 and H-2 I-Ak transfectants of T2 that express parental levels of class II molecules at the cell surface. T2.Ak transfectants fail to stimulate a hen egg lysozyme (HEL)-specific, I-Ak-restricted T cell when incubated with intact HEL. However, stimulation occurs if the appropriate HEL peptide is provided. The T2 cell line therefore has a defect in class II-restricted Ag processing. Biosynthetic studies demonstrate that the kinetics of I-Ak transport in T2.Ak are similar to the parental rates of transport, although the percentage of I-Ak molecules transported appears somewhat lower. I-Ak glycoproteins in T2.Ak associate normally with the I-chain, which appears to be proteolytically cleaved after transport through the Golgi apparatus in a similar fashion to that in the parent cell line, .174xCEM.T1 (T1). The DR alpha beta heterodimers in T2 differ from the parental phenotype in two ways. First, HLA-DR3 expressed in T2 does not have the epitope recognized by the DR3-specific mAb 16.23, although DR3 expressed in the parent does have the epitope. Second, the alpha beta subunits in the parent remain associated when exposed to SDS at room temperature, although those in T2 dissociate.     

Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression.

We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.     

Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM alphaW62A,betaW120A (DM(W62A/W120A)) double mutant was expressed in HLA-DR(+) HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.     

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.     

Structure, biosynthesis, and polymorphism of chicken MHC class II (B-L) antigens and associated molecules

Chicken MHC class II (B-L) antigens were immunoprecipitated by the monoclonal antibody TaP1 from inbred chicken splenic leukocytes and a lymphoblastoid B cell line (RP9), and were studied by two dimensional gel electrophoresis. B-L antigens are composed of one alpha and one beta chain that are noncovalently bound at the cell surface. In all haplotypes studied, a single acidic 34,000 dalton non-polymorphic chain was observed, whereas two polymorphic chains could be distinguished, differing in both pH and m.w. The alpha-beta heterodimer is associated during its maturation in the cytoplasm with several basic invariant molecules with m.w. ranging from 30,000 to 42,000 daltons. Treatment of cells with tunicamycin and treatment of immunoprecipitated molecules with several glycosidases revealed a complex process of maturation for all of these molecules. The alpha and beta chains undergo a N-glycosylation of complex type, whereas the invariant molecules bear N-linked high mannose glycans, and perhaps also O-linked glycans in the RP9 lymphoblastoid line. Overall, the B-L antigens appear very similar to the HLA-DR and I-E antigens.     

Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins

Susceptibility to multiple sclerosis (MS) is associated with certain MHC class II haplotypes, in particular HLA-DR2. Two DR beta chains, DRB1*1501 and DRB5*0101, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP 84-102) to MBP-specific T cells from MS patients. We have determined the crystal structure of HLA-DR2a complexed with MBP 86-105 to 1.9 A resolution. A comparison of this structure with that of HLA-DR2b complexed with MBP 85-99, reported previously, reveals that the peptide register is shifted by three residues, such that the MBP peptide is bound in strikingly different conformations by the two MHC molecules. This shift in binding register is attributable to a large P1 pocket in DR2a, which accommodates Phe92, in conjunction with a relatively shallow P4 pocket, which is occupied by Ile95. In DR2b, by contrast, the small P1 pocket accommodates Val89, while the deep P4 pocket is filled by Phe92. In both complexes, however, the C-terminal half of the peptide is positioned higher in the binding groove than in other MHC class II/peptide structures. As a result of the register shift, different side-chains of the MBP peptide are displayed for interaction with T cell receptors in the DR2a and DR2b complexes. These results demonstrate that MHC molecules can impose different alignments and conformations on the same bound peptide as a consequence of topological differences in their peptide-binding sites, thereby creating distinct T cell epitopes.     

Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., B?chinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the empty DR2-derived RTLs. These novel molecules represent a new class of small soluble ligands for modulating the behavior of T cells and provide a platform technology for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.     

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.     

Isoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments

Newly synthesized class II molecules of the major histocompatibility complex must be transported to endosomal compartments where antigens are processed for presentation to class II-restricted T cells. The invariant chain (Ii), which assembles with newly synthesized class II alpha- and beta-chains in the endoplasmic reticulum, carries one or more targeting signals for transport to endosomal compartments where Ii dissociates from alpha beta Ii complexes. Here we show that the transport route of alpha beta Ii complexes is regulated selectively by two forms of Ii (p33 and p35) that are generated by the use of alternative translation initiation sites. Using a novel quantitative surface arrival assay based on labeling with [6-3H]-D-galactose combined with biochemical modification at the cell surface with neuraminidase, we demonstrate that newly synthesized alpha beta Ii molecules containing the Ii-p33 isoform can be detected on the cell surface shortly after passage through the Golgi apparatus/trans-Golgi network. A substantial amount of these alpha beta Ii complexes are targeted to early endosomes either directly from the trans-Golgi network or after internalization from the cell surface before their delivery to antigen processing compartments. The fraction of alpha beta Ii complexes containing the p35 isoform of Ii with a longer cytosolic domain was not detected at the cell surface as determined by iodination of intact cells and the lack of susceptibility to neuraminidase trimming on ice. However, treatment with neuraminidase at 37 degrees C did reveal that some of the alpha beta Ii-p35 complexes traversed early endosomes. These results demonstrate that a fraction of newly synthesized class II molecules arrive at the cell surface as alpha beta Ii complexes before delivery to antigen processing compartments and that class II alpha beta Ii complexes associated with the two isoforms of Ii are sorted to these compartments by different transport routes.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.