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.     

Lead influences translational or posttranslational regulation of Ia expression and increases invariant chain expression in mouse B cells.

The molecular mechanisms governing the increased cell surface expression of major histocompatibility complex (MHC) class II molecules (Ia) on lead-treated mouse B cells was investigated. Lead has been shown to directly cause a selective, two-fold increase in the B cell's surface density of both products of the I region of the mouse MHC, I-A and I-E. In the present study, Western blot analysis showed that Pb increases the total cellular pool of I-A beta-chain by twofold. The increase in cellular I-A was not found to be due to increased messenger RNA (mRNA) for either the alpha- or the beta-chain of I-A. Biosynthetic labeling studies showed that Pb increases the translation or the stability of the Ia-associated invariant chain (Ii or gamma) and possibly the beta-chain of Ia. Collectively these results suggest that Pb increases the B cell's surface Ia by influencing translational or posttranslational regulation of Ia and/or Ia-associated chains.     

Lead influences translational or posttranslational regulation of ia expression and increases invariant chain expression in mouse B cells

The molecular mechanisms governing the increased cell surface expression of major histocompatibility complex (MHC) class II molecules (Ia) on lead-treated mouse B cells was investigated. Lead has been shown to directly cause a selective, two-fold increase in the B cell's surface density of both products of the I region of the mouse MHC, I-A and I-E. In the present study, Western blot analysis showed that Pb increases the total cellular pool of I-A β-chain by twofold. The increase in cellular I-A was not found to be due to increased messenger RNA (mRNA) for either the α- or the β-chain of I-A. Biosynthetic labeling studies showed that Pb increases the translation or the stability of the Ia-associated invariant chain (Ii or γ) and possibly the β-chain of Ia. Collectively these results suggest that Pb increases the B cell's surface Ia by influencing translational or posttranslational regulation of Ia and/or Ia-associated chains.     

A single monoclonal anti-Ia antibody inhibits antigen-specific T cell proliferation controlled by distinct Ir genes mapping in different H-2 I subregions

A xenogeneic rat anti-mouse Ia monoclonal antibody, M5/114 (gamma 2b, kappa), was studied for its effects in vitro on T cell proliferative responses. Strain distribution studies revealed that M5/114 could inhibit I-A subregion-restricted T cell responses of the H-2b,d,q,u but not the H-2f,k,s haplotypes, indicating that this xenoantibody recognizes a polymorphic determinant on mouse Ia molecules. This same monoclonal antibody was found to inhibit BALB/c (H-2d) T cell proliferation to both G60A30T10 and G58L38 phi 4. The Ir genes regulating responses to these antigens map to either the I-A subregion (GAT), or the I-A and I-E subregions (GL phi), raising the possibility that M5/114 recognizes both I-A and I-E subregion-encoded Ia glycoproteins. It could be shown, using appropriate F1 responding cells, that M5/114 does in fact affect GAT and GL phi responses by interaction with both the I-A and the I-E subregion products, and not by any nonspecific effect resulting from binding to the I-A subregion product alone. These results are consistent with genetic and biochemical studies directly demonstrating that M5/114 recognizes A alpha A beta and E alpha E beta molecular complexes. The existence of a shared epitope on I-A and I-E subregion products suggests the possibility that these molecules arose by gene duplication. Finally, the precise correlation between the Ia molecules recognized by M5/114 and the ability of this antibody to block T cell responses under Ir gene control strengthens the hypothesis that Ia antigens are Ir gene products.     

Expression of I-A and I-E,C region-coded Ia antigens on functional B cell subpopulations.

Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.     

Chemical cross-linking of Ia alloantigen alpha and beta chains with dimethyl 3,3'-dithiobispropionimidate.

We have examined the polypeptide chain composition of membrane-bound and detergent-solubilized Ia antigens using the chemical cross-linking reagent dimethyl 3,3'-dithiobispropionimidate (DTBP). Products of the I-E/C subregion of the major histocompatibility complex, which were solubilized from spleen cells with the detergent NP-40 and partially purified by affinity chromatography on lentil lectin-agarose, could be almost completely cross-linked by DTBP. Thus, the characteristic 33,000 m.v. (alpha) and 28,000 (beta) polypeptide chains seen on sodium dodecylsulfate polyacrylamide gels disappeared and a major new species of 60,000 m.w. appeared after cross-linking. When isolated and reduced with 2-mercaptoethanol, the 60,000 m.w. peak was found to be comprised to alpha and beta chains. Similar results were obtained when I-E/C, as well as I-A, alpha and beta chains were crosslinked on the cell surface. These data demonstrate that the alpha and beta chains of the Ia antigens exist primarily in the form of a dimer both in detergent solution and in situ.     

Identity of molecules bearing murine Ia alloantigenic specificities Ia.7 and Ia.22: evidence for a single type of I-E molecule in homozygotes

In homozygous mice bearing I regions derived from haplotype k, only a single type of Ia molecule bearing the alloantigenic specificities Ia.7 and Ia.22 was found using techniques of sequential immune precipitation and tryptic peptide analysis. As suggested at the fourth Ir Gene Workshop (Sachs 1978), Ia.7 is considered here to be an antigenic determinant associated with I-E-subregion-encoded molecules, i.e., it is excluded from the I-C subregion. The I-C subregion is currently defined mainly by functional traits. It is now known that the I-E molecules are composed of an alpha chain encoded in the I-E subregion, and a beta chain encoded in the I-A subregion. Since the I-C subregion is not involved with the determination of these Ia molecules, and since in homozygotes there is apparently only a single type of molecule bearing both specificities Ia.7 and Ia.22, the term "I-E/C" molecule should probably be dropped in favor of the simpler designation I-E.     

Analysis of Ir gene function using monoclonal antibodies: independent regulation of GAT and GLPhe T cell responses by I-A and I-E subregion products on a single accessory cell population

T cell proliferative responses to the synthetic polypeptides GAT and GLPhe are under Ir gene control. GAT responses are regulated by gene(s) in the I-A subregion, and GLPhe responses are controlled by a pair of complementing genes mapping to the I-A and I-E subregions. We demonstrate that monoclonal antibody to the I-A gene product inhibits GAT proliferation but not the GLPhe response, whereas a monoclonal antibody to the I-E associated Ia-7 determinant inhibits GLPhe but not GAT proliferation, which indicates independent involvement of each Ia determinant in antigen presentation for the T cell response to these antigens. Use of the same subregion-specific monoclonal antibodies in complement-dependent lysis demonstrates that the antigen-presenting cells for GAT and GLPhe express both I-A and I-E products. The possibility that an Ia subregion-specific "self-receptor" functions on the reactive T cells as a regulatory element is discussed.     

Ia antigens in mouse skin are predominantly expressed on Langerhans cells.

We have investigated the expression of products of the mouse major histocompatibility complex (MHC) on BALB/c and A/J epidermal cells. By using reagents with specificity for various products of the MHC in an indirect immunofluorescence procedure, we found that H-2 antigens are expressed on the vast majority of epidermal cells. Ia antigens, by contrast, are present on only 2.4 to 6.9% of all epidermal cells. These Ia-bearing cells bear a receptor for the Fc portion of IgG and ultrastructurally exhibit the characteristics of Langerhans cells. Ia antigens on Langerhans cells are encoded for by at least the I-A and I-E/C subregions of the MHC.     

Molecular associations of class II MHC antigens in mitogen-stimulated B cells

Previously, we showed that murine B cell membrane proteins undergo rearrangements in the plasma membrane to form new molecular associations in response to mitogenic stimulation. These complexes were covalently stabilized by photoreactive cross-linking agents and were analyzed by SDS PAGE. We have now identified certain complexes that involve class II MHC products, the Ia antigens. Upon stimulation of B cells with LPS, Ia surface molecules (as identified by radioimmunoprecipitation with polyclonal anti-Ia antiserum) enter into a molecular complex with a 95-kd membrane-associated protein (p95) to form a 200-kd complex that may be stabilized by the cross-linking agent dithiobisphenylazide (DTPA). This molecular association is not observed upon stimulation with mitogenic anti-Ig reagents, nor with the polyclonal B cell activator 8-bromoguanosine. p95 is not a disulfide-linked molecule itself, and by separate immunoprecipitation experiments we have established that it is not a component of surface Ig, transferrin receptor, the B cell Fc receptor, or CR1, the receptor for complement component C3b. Further analysis of the association of Ia antigens with surface proteins, with the use of monoclonal antibodies directed against I-A or I-E, has demonstrated that each subregion gene product forms a unique molecular association. Precipitation of radiolabeled lysates from LPS-activated B cells with anti-I-A reveals the aforementioned association with p95. In contrast, the I-E antigen apparently forms complexes with a multimer of a 15-kd protein to give complexes of 45, 60, 75, and 90 kd. When analyzed by two-dimensional diagonal gels (nonreducing/reducing), only the I-E bands are revealed by autoradiography, indicating that the putative p15 that associates with I-E may not be accessible to surface labeling. The disparate molecular associations for I-A and I-E suggest that the formation of these distinct protein complexes may be functionally related to a different role in the process of cellular activation for each of these Ia subregion gene products.     

Suppression of anti-hapten antibody responses by hapten-specific cytolytic T lymphocytes: role of I-A-associated antigen on target B lymphocytes

Cytolytic T lymphocytes (CTL) specific for 2,4,6-trinitrophenyl (TNP) determinants suppress the effector phase of a secondary anti-TNP antibody responses of murine syngeneic spleen cells in vitro. The cells mediating this suppression are hapten-specific, H-2-restricted, and possess properties typical of CTL. Moreover, the targets of the suppression appear to be antigen-primed B lymphocytes that are recognized by CTL via soluble antigen bound noncovalently to their Ig receptors. The effect of the CTL can be blocked by the addition of monoclonal antibodies directed against I-A molecules but not I-E or H-EK-encoded molecules on the target B cells, even in strain combinations in which the CTL-B cell interaction is restricted only by the H-2K and I regions of the MHC. This result suggests that B lymphocyte-bound antigen tends to associate preferentially with I-A rather than H-2K/D-encoded determinants, and that the suppressive effect of the CTL population is attributable to the minor subset that recognizes hapten-modified Ia antigens. These findings are also discussed in terms of the possible immunoregulatory function of Ia-restricted CTL.     

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.     

The Ia.2 epitope defines a subset of lipid raft-resident MHC class II molecules crucial to effective antigen presentation

Previous work established that binding of the 11-5.2 anti-I-A(k) mAb, which recognizes the Ia.2 epitope on I-A(k) class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-A(k) mAbs that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-A(k) molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2-bearing subset of I-A(k) class II molecules is critically necessary for effective B cell-T cell interactions, especially at low Ag doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-A(k) class II molecules possessing a β-chain-tethered hen egg lysosome peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2(-) tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous Ag to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II conformer vital to the initiation of MHC class II-restricted B cell-T cell interactions.     

Expression of the I-E target antigen for T-cell killing requires two genes

The H-2Ik region encodes at least two different target antigens for unrestricted T-cell mediated killing. The first is controlled by the I-A region alone and the second depends on a pair of alleles, one located to the left of I-B (presumably in I-A) and the other to the right of I-J (presumably in I-E). Hence, effector cells nominally specific for a product of the I-E region do not kill target cells with the same I-E region as the stimulator unless the I-A region is also shared. Some effectors specific for H-2Ik, such as A.TH anti-A.TL and B10.A(4R) anti-B10.A(2R), cross-react with B10.A(3R) and B10.A(5R) target cells. A product of the H-2b haplotype was shown to complement products of the H-2d or H-2k haplotypes in forming this cross-reactive determinant. The results are consistent with recent biochemical data on the component chains of Ia antigens.     

Xenopus MHC class II molecules. I. Identification and structural characterization

Class II antigens from the Xenopus laevis MHC (f haplotype) were identified by using a rabbit antihuman class II beta-chain serum (anti-p29boost). This xenoantiserum inhibits bidirectional Xenopus MLR (but not PHA-stimulation), recognizes the same molecules as certain MHC-linked Xenopus alloantisera, and immunoprecipitates class II molecules from Xenopus cells consistent with the tissue distribution of mammalian class II molecules. The Xenopus class II molecules are composed of two different chains, both of which are 30 to 35kD transmembrane glycoproteins. The alpha-chains have some N-terminal sequence homology with mammalian class II alpha-chains (both I-E/DR and I-A/DC); the beta-chains are directly recognized by anti-p29boost and have a markedly increased SDS gel mobility under nonreducing conditions. During biosynthesis, they are noncovalently associated with a number of other chains, including ones at 25kD, 33kD, and 40 to 45kD. The alpha-chains bear three N-linked glycans (two Endo H insensitive in mature material) and the beta-chains bear two (one Endo H insensitive). Unlike most mammalian class II molecules, the deglycosylated beta-chains are significantly larger and more acidic than the alpha-chains.     

The immunobiology of the T cell response to Mls-locus-disparate stimulator cells. I. Unidirectionality, new strain combinations, and the role of Ia antigens

The primary mixed lymphocyte reaction of T cells to Mls-locus-disparate stimulator cells differs from that to non-self Ia antigens in several respects. In the present experiments, the unidirectional nature of this response is shown in several strain combinations, including the newly detected Mlsa and Mlsa-like alleles expressed by strains PL/J, RF/J, and SM/J. All of these strains stimulate MHC-identical T cells strongly. In addition, they stimulate a variety of cloned T cell lines specific for Mlsa,d, which can thus be shown to respond to Mlsa,d stimulators of the H-2b,d,k,u, and v haplotypes. Although these results suggest that primary T cell responses to Mlsa,d are unlikely to be MHC restricted, these primary responses are readily inhibited by monoclonal antibodies specific for the I-A and especially the I-E products borne by the stimulator cells, as well as by monoclonal antibodies specific for L3T4a on the responding T cells. This effect of anti-Ia antibodies is not overcome by exogenous interleukin 1. Thus, I-A and especially I-E molecules are centrally involved in the unidirectional primary T cell response to the potently stimulating Mlsa and Mlsd alleles expressed by cells of several different MHC haplotypes.