I-A region genetic restriction in the production and action of an antigen-specific T-helper factor which bears I-A region determinant(s)

Antigen-specific T-helper factor (ThF) augments the contact sensitivity reaction induced by the injection of small numbers of picrylated cells into mice. ThF was produced by injected picrylated spleen cells into the footpads and taking the 24-hr culture supernatant of the regional lymph node cells. Analysis showed that there was a genetic restriction in the induction of ThF, between these picrylated cells and the mouse making the ThF, which maps to the I-A region. This finding suggests that the receptor on the T cell which makes ThF, and by implication ThF itself, bears recognition site(s) for both antigen and I-A. ThF was then prepared from mice painted on the skin with picryl chloride, and the genetic restriction in its action was investigated. There was a requirement for genetic matching between the mouse producing the ThF and the final recipient which mapped to the I-A region. The genotype of the picrylated cell used as a source of antigen was unimportant. This I-A genetic restriction, together with the finding that ThF bears at least some I-A determinants, suggests that ThF may act by binding to the picrylated cells used as a source of antigen through its antigen-binding site and hence provide the I-A determinants needed for the recognition of antigen in the context of self-MHC. The present findings add to the list of antigen-specific factors which have a two-chain structure and show genetic restriction in their induction, action, and in the interaction between their chains which maps to the same region as the MHC-related determinant(s) which they bear.     

Assignment of antigenic determinants to separated I-A kappa chains

The alpha- and beta-chains of the I-A kappa antigen from the AKTB-1b B cell lymphoma were separated by ion-exchange chromatography on CM-Sephadex in the presence of propionic acid and urea. Removal of the denaturants by dialysis produced isolated chains that regained a significant amount of their native configuration. These materials were used with a battery of monoclonal antibodies in a direct binding assay to localize specific alloantigenic determinants to the A alpha kappa or A beta kappa chains. This method allowed the assignment of the nominal specificity Ia. 17 and at least one epitope of the specificity Ia.2 to the A beta kappa chain. Finally, the I-A kappa antigen from the B cell lymphoma AKTB-1b was shown to be identical, by the criterion of tryptic peptide analysis, to that derived from normal B10.BR splenocytes. This constitutes the first demonstration that the polypeptide portion of a tumor-derived class II MHC antigen is identical to that derived from a normal tissue.     

Murine interstitial nephritis. IX. Induction of the nephritogenic effector T cell repertoire with an antigen-specific T cell cytokine

The experiments presented in this report describe the biochemical and functional characteristics of a soluble Th cell factor (ThF) which can induce a nephritogenic effector T cell repertoire producing autoimmune interstitial nephritis. The ThF is Ag-specific, I-A-restricted, and comprises two chains noncovalently linked as a heterodimer. One chain at approximately 78,000 Mr is related to the TCR/Id and expresses a framework determinant (14-30) common to Ag-binding factors, and the other chain at approximately 82,000 Mr is I-A+. Together these chains can replace their parent cell by providing cognate help to precursor effector T lymphocytes in the presence of accessory cells, tubular Ag, and IL-2.     

Cell type-dependent collagen-type recognition by cell receptors.

Affinity chromatography of a number of cell types on collagens I and III reveals three proteins with M(R)of 250, 170 and 140 kDa. These proteins are able to discriminate between types I and III, but not types III and IV. Collagen-type recognition is therefore characteristic for cells of connective tissue origin. Polyclonal antibodies (Ab) raised against 170 and 140 kDa polypeptides and used in immunofluorescence show membrane localisation for both, with their distribution being similar to each other and to the distribution of the integrin beta1 chain. Ab p140 and commercial monoclonal antibodies against alpha(2)chain stain a band of the same molecular mass as from purified collagen binding proteins from liver cells, indicating that the 140 kDa protein is probably the alpha(2)integrin chain. The alpha(2)chain containing integrins are therefore able to discriminate collagen types I and III and collagen type recognition by this receptor is cell-type dependent.     

Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.     

Structural comparison of I-A antigens produced by a cloned murine T suppressor cell line with B-Cell-Derived I-A

A cloned, antigen-specific T suppressor cell line derived from a CBA mouse expresses large amounts of I-A and I-E antigens. Comparative two-dimensional polyacrylamid gel electrophoresis of biosynthetically labeled I-A antigens immunoprecipitated with a variety of monoclonal I-Ak-specific antibodies suggested that alpha, beta and Ii polypeptide chains are identical with B-cell-derived I-A. Dimeric complexes formed by I-A chains derived from B or T suppressor cells were also similar with two major exceptions. Pulse-labeled T-cell-derived Ia antigen was complexed with two additional unknown components of about 31K. These components were not visible in pulse-chased (processed) materials. In addition, T suppressor-cell-derived I-A antigens did not contain S-S linked dimers consisting of processed alpha and beta chains, which are usually formed during solubilization of B cells. We consider the possibility that in T cells these chains are associated with other structures, thus preventing S-S linkage between alpha and beta chains.     

T suppressor efferent circuit which affects contact sensitivity to picryl chloride: the late-acting, second nonspecific T suppressor factor bears I-A determinants which are responsible for the I-A genetic restriction in its interaction with its target cell

The T suppressor efferent circuit in the picryl (TNP) system, which inhibits the passive transfer of contact sensitivity, involves at least two antigen-nonspecific factors. The second nonspecific T suppressor factor (ns-2) bears I-A determinants of both the alpha and the beta chain as shown by affinity chromatography on immobilized anti-I-A monoclonal antibodies. Sequential absorption shows that the determinants of the alpha and beta chain occur on the same molecular complex. No absorption was obtained with anti-I-E antibody. There are two genetic restrictions associated with ns-2--the first is in its release from the second T suppressor efferent cell (on exposure to antigen) and the second is in its inhibitory interaction with its target cell. Both are MHC restricted and matching in I-A (but not I-E, or I-J) is sufficient. The question was asked whether the I-A of the ns-2 was directly responsible for the I-A genetic restriction in its action. F1 TsF was made in (H-2k X H-2b)F1 mice by injecting picrylated parental cells intravenously and triggering the release of ns-2 with the corresponding picrylated parental cells. Both I-Ak- and I-Ab-positive ns-2 were produced and were separated by affinity chromatography on immobilized anti-I-A monoclonal antibody. The I-A phenotype of these separated ns-2 of F1 origin determines the genetic restriction in their action; i.e., I-Ak+ ns-2 only inhibits passive transfer by H-2k cells and I-Ab+ ns-2 only acts on H-2b cells. In contrast, the I-A haplotype of the picrylated cell used to induce the Ts cell which makes ns-2 is unimportant. It was concluded that the I-A on the ns-2, and not a possible recognition site for I-A, serves as a restriction element. This finding suggests that ns-2 may act directly on the I-A-restricted T cell which mediates contact sensitivity.     

Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella

NH2-terminal analysis of the alpha and beta heavy chain polypeptides (Mr greater than 400,000) from the outer arm dynein of sea urchin sperm flagella, compared with that of the 230,000- and 200,000-Mr peptides formed upon photocleavage of dynein by irradiation at 365 nm in the presence of vanadate and ATP, shows that the NH2 termini of the intact chains are acetylated and that the 230,000- and 200,000 Mr peptides constitute the amino- and carboxy-terminal portions of the heavy chains, respectively. Tryptic digestion of the beta heavy chain is known to separate it into two particles, termed fragments A and B, that sediment at 12S and 6S (Ow, R. A., W.-J. Y. Tang, G. Mocz, and I. R. Gibbons, 1987. J. Biol. Chem. 262:3409-3414). Immunoblots against monoclonal antibodies specific for epitopes on the beta heavy chain, used in conjunction with photoaffinity labeling, show that the ATPase-containing fragment A is derived from the amino-terminal region of the beta chain, with the two photolytic sites thought to be associated with the purine-binding and the gamma-phosphate-binding areas of the ATP-binding site spanning an approximately 100,000 Mr region near the middle of the intact beta chain. Fragment B is derived from the complementary carboxy-terminal region of the beta chain.     

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.     

Recombinant human laminin-10 (alpha5beta1gamma1). Production, purification, and migration-promoting activity on vascular endothelial cells

The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.     

Identification of tissue histiocytes on paraffin sections by a new monoclonal antibody

A mouse monoclonal antibody (MAC 387) with specificity for monocytes and tissue histiocytes was produced by immunization of a BALB/c mouse with peripheral blood monocyte components derived by affinity chromatography of detergent-solubilized monocyte material on Sepharose 4B coupled to rabbit anti-monocyte antibodies. MAC 387 strongly stained the cytoplasm of cells of the monocyte/macrophage series on paraffin sections after controlled trypsinization of sections. The antibody showed broad reactivity for a variety of tissue histiocytes, including infiltrating and reactive histiocytes, alveolar macrophages, Kupffer cells, follicle-center macrophages, splenic red pulp macrophages, tumor-infiltrating macrophages, sinus histiocytes, epithelioid giant cells (variably), and cases of histiocytosis X and dermatopathic lymphadenopathy. Molecular weight data obtained by Western blotting, immunoprecipitation, and immunoaffinity-purification revealed that the antigen was present in different forms in the monocyte and granulocyte. In the granulocyte, free alpha (Mr 12 KD) and beta (Mr 14 KD) chains expressing the MAC 387 epitope were found together with associations of one alpha and one beta chain linked by disulfide bonds to yield a heterodimer of Mr 26 KD. In the monocyte, free alpha and beta chains are not found, but instead the heterodimer and associations of two (Mr 56 KD) and four (Mr 112 KD) heterodimers are disulfide-linked together. This new monoclonal reagent should have particular value for identification of tissue histiocytes in routine paraffin sections and particularly for demonstration of histiocytes in malignant lymphomas.     

The leukotoxin of Pasteurella haemolytica binds to β2 integrins on bovine leukocytes

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the alpha chains of beta(2) integrins), and CD18 (the beta chain of beta(2) integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the beta(2) integrins on bovine leukocytes, very likely via CD18.     

Studies on the assembly and secretion of fibrinogen.

cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.     

Skeletal myoblasts utilize a novel beta 1-series integrin and not alpha 6 beta 1 for binding to the E8 and T8 fragments of laminin.

The E8 fragment of laminin stimulates myoblast attachment and locomotion. Myoblast attachment to laminin/E8 was blocked by anti-integrin antibodies against beta 1-chains but not by antibodies against alpha 6-chains. By contrast, other cell lines (e.g. B16, HT1080, P19, F9, Pys2, 3T3, and 3T6) were blocked both by anti-beta 1 and anti-alpha 6. All cells tested also bound to approximately 125-kDa C-terminal fragments of E8 (T8 and T8'). Immunoprecipitation of surface-iodinated myoblasts revealed beta 1-, alpha 3-, and alpha 5-integrin chains and a novel chain that co-precipitated with anti-beta 1 antibodies running at approximately 95 kDa (reduced). I125-alpha 6 beta 1 was immunoprecipitated from cells whose attachment to E8 was blocked by anti-alpha 6 antibodies. By contrast, little alpha 6 beta 1 could be immunoprecipitated from myoblasts. beta 1-Integrin and the novel alpha-chain (alpha'), Mr approximately 120,000/approximately 95.000 (nonreduced/reduced), from myoblast lysates were retained during affinity chromatography on Engelbreth-Holm-Swarm-laminin affinity columns. beta 1, alpha 1, and the novel alpha' were retained from Rugli cell lysates on Engelbreth-Holm-Swarm-laminin columns. alpha 3 was not bound. When E8 was used as affinity matrix, only beta 1 and alpha' were retained. The N-terminal sequence of Rugli alpha' was homologous to alpha-chains of beta 1-series integrins and was most similar to alpha 6 (9 identical residues out of 14). However, there were distinctive differences; in particular, 2 residues were deleted in comparison with alpha 6.     

Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.     

The assignment of chain specificities for anti-Ia monoclonal antibodies using L cell transfectants

The chain specificities of 18 Ak and 26 Ab-reactive anti-Ia monoclonal antibodies have been determined. L cells were transfected with haplotype-matched (A alpha k:A beta k, A alpha b:A beta k) or haplotype-mismatched (A alpha k:A beta b, A alpha b:A beta k) cDNA pairs, lines expressing high levels of surface A complex were selected, and antibody reactivity with a panel of reagents was assessed by cytofluorimetric analysis. Most of the antibodies recognized a determinant specified by one chain, either alpha or (more commonly) beta. A few examples of more complex determinants were also observed. A knowledge of the chain specificities of anti-Ia monoclonal antibodies should prove useful for a variety of studies aimed at dissecting Ia structure-function relationships.     

Isolation and characterization of two molecular variants of myosin heavy chain from rabbit ventricle. Change in their content during normal growth and after treatment with thyroid hormone

We have prepared monoclonal antibodies specific for cardiac myosin heavy chain. These antibodies were used for the separation and characterization of the molecular variants of myosin heavy chain present in the rabbit heart. Two molecular forms of myosin heavy chain, HC alpha and HC beta, were isolated from the euthyroid rabbit heart by affinity chromatography. Their reactivity with our antibodies indicated that the primary structures of HC alpha and HC beta differ in at least four and share at least two antigenic determinants. Differences in the primary structure of HC alpha and HC beta were confirmed by analysis of the peptides produced by limited chymotryptic digestion of the two heavy chains. Thirteen peptide differences were consistently found. The HC alpha and HC beta variants are shown by immunologic analysis and in chymotryptic peptide profiles to be identical with the predominant forms of myosin heavy chain synthesized in the hearts of hyperthyroid and adult euthyroid rabbits, respectively. During development and maturation of the euthyroid rabbit heart, HC alpha comprises approximately 50% of the ventricular myosin between birth and 4 weeks of age; it diminishes to 20-30% by 8 weeks and to 10-20% by 12 weeks of age. Cardiac myosin from a 1-year-old rabbit is composed almost entirely of HC beta. Cardiac myosin from embryonic animals at 20 days gestation contained 20% HC alpha. These results show that HC alpha occurs normally in the euthyroid rabbit heart and that the relative proportions of HC alpha and HC beta depend on both the developmental stage and the thyroid state of the animal.     

Biochemical characterization of Ia antigens. I. Characterization of the 31K polypeptide associated with I-A subregion Ia antigens

Procedures are presented for the preparative isolation of murine Ia antigens directly from splenocyte detergent extracts with monoclonal immunoadsorbents. Utilizing these procedures, three Ia (I-A subregion) polypeptides (alpha, 31K, beta) were isolated and their m.w. and pI values characterized. Evidence is presented that indicates that: 1) the 31K polypeptide probably does not associate with the Ia alpha and beta chain complex during the Ia isolation procedure; 2) the 31K polypeptide is not tightly bound to the alpha/beta Ia complex and can be selectively removed by freezing and thawing and by washing the Ia-immunoadsorbent with buffers containing pyrrolidinone (a polar solvent); and (3) unlike the alpha and beta chains, the 31K polypeptide is not intrinsically radiolabeled with 3H fucose and 3H glucosamine, indicating that the 31K polypeptide either contains a carbohydrate structure that is different from that of the alpha and beta chains or it is not a glycopeptide. These data suggest that although Ia antigens are probably comprised of three polypeptides in the intact cell, only two (alpha and beta) are required to maintain alloantigenic determinants.