Transplantation in miniature swine. V. Characterization of Ia antigens.

The complexity of the I region analog associated with the MHC of miniature swine has been probed by sequential antibody precipitation studies of Ia antigens. Treatment of solubilized lymphocyte preparations from MSLA homozygotes of the DD haplotype with excess AA anti-DD alloantiserum led to precipitation of only a portion of the total Ia antigens, as determined by secondary precipitation of the remaining material with CC anti-DD serum. The presumed I region of miniature swine must therefore code for more than one Ia antigen-bearing polypeptide chain. In addition, certain mouse alloantisera that had previously been shown to react with rat Ia antigens were tested for reactivity with swine Ia antigens. Anti-Iak mouse alloantisera precipitated Ia molecules from every swine extract tested, regardless of MHC type, precluding genetic mapping studies. However, sequential precipitation studies demonstrated that the cross-reactive mouse alloantisera reacted only with a subclass of swine Ia antigens, again suggesting genetic complexity of the pig Ia locus.     

The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni

Five low m.w. polypeptide antigens are expressed on the surface of freshly transformed schistosomula of Schistosoma mansoni, and were reproducibly identified by surface labeling with 125I by using IODOGEN and immunoprecipitating with immune mouse sera. These molecules have approximate m.w. of 38,000, 32,000, 20,000, 17,000, and 15,000. They correspond to antigens recognized previously by lactoperoxidase-catalyzed iodination. Analysis of the surface of developing schistosomulum demonstrated that the 38,000 and 17,000 dalton antigens were lost from the parasite surface during 48 hr of in vitro culture. This process was not dependent on the presence of host serum. The two antigens were not lost due to shedding into the culture medium but were apparently sequestered to a site where they were no longer available for surface labeling. The 32,000, 20,000, and 15,000 dalton antigens, however, remained exposed on the schistosomulum surface for up to 2 days of in vitro culture. The expression of two new antigens was also induced by culture in vitro: a doublet of approximately 45,000 daltons and an antigen of approximately 11,000 daltons. The expression of the former was dependent on the presence of serum. These results demonstrate that the development of the schistosomula surface is a complex process, with events both dependent and independent of the presence of serum. In addition, the expression of polypeptide antigens is not coordinated, and antigens are lost, retained, or appear on the schistosomulum surface during the early stages of maturation.     

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.     

Transplantation in miniature swine. XII. N-terminal sequences of class I histocompatibility antigens (SLA) and beta 2-microglobulin

Purified class I histocompatibility antigens (SLA) from three haplotypes were prepared by papain treatment of lymphoid cell membranes obtained from spleens and lymph nodes of miniature swine homozygous at their major histocompatibility complex. Antigens were purified by ion-exchange chromatography followed by gel filtration. Purity was analyzed by SDS-PAGE, and antigenic specificity by inhibition of complement-dependent, alloantiserum-mediated cytotoxicity. The SLA antigens were reduced and alkylated, and the component heavy and light chains were isolated by gel filtration under dissociating conditions. N-terminal amino acid sequences were obtained for SLAaa, SLAcc, and SLAdd heavy chains, as well as for the light chain, beta 2-microglobulin. The swine antigens showed high levels of homology with class I antigens from other animal species. Heterogeneity was observed among the swine haplotypes, and several of the positions at which substitutions were found are apparently invariant in other animal species. In contrast, only minimal sequence heterogeneity was detected within haplotypes, the basis of which may be of relevance to understanding the evolutionary development of these molecules.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Monoclonal antibodies to hamster class II MHC molecules distinguish T and B cells

Monoclonal antibodies were prepared against cell surface antigens present on Syrian hamster lymphocytes and a hamster B cell lymphoma line, GD-36. One of these antibodies, S11, precipitated glycoproteins of 29,000 and 39,000 m.w. These glycoproteins were shown to be identical to or a subset of la-like glycoproteins precipitated by hamster alloantisera; however, molecules identified by S11 differed from the predominant hamster la homologues detected with a cross-reactive monoclonal antibody to murine la.7. The immunofluorescence pattern of both anti-la reagents, S11 and anti-la.7, on hamster lymphoid cells is similar by fluorescence-activated cell sorter analysis. A subpopulation of spleen and lymph node cells stains brightly with these antibodies. By two-color fluorescence, this peripheral lymphocyte subpopulation, identified with monoclonal anti-hamster la, also bears surface immunoglobulin (IgM). These data strongly suggest that hamster resting peripheral B cells, and not T cells, express la antigens and can be identified and isolated differentially by using this marker.     

Structural characterization of antigens encoded by rabbitRLA-11 histocompatibility genes

Products of rabbitRLA-11 histocompatibility genes were isolated from rabbit lymphoid tumor cells (RL-5) and characterized. The tumor cells were grown in culture with radioactive amino acids and were lysed by treatment with the detergent NP-40. Glycoprotein molecules were isolated by affinity chromatography using immobilized lentil lectin. Chromatography on purified sheep anti-rabbit beta-2-microglobulin yielded a 43,000 dalton glycoprotein fraction, designated RLA-11gp, which was noncovalently associated with beta-2-microglobulin. N-terminal amino acid sequence analysis using radiochemical methods allowed assignment of 28 of the N-terminal 35 residues. The data revealed 89% homology of the RLA-11gp N-terminus with that of the human HLA-B7 and 82% with the mouse H-2K^b histocompatibility antigens. Comparison of the RLA-11 gp N-terminal sequence data obtained in this work to sequence data reported for major histocompatibility complex antigens of other species revealed no amino acid substitutions unique to the rabbit antigens.     

Biochemical evidence that equine leucocyte antigens W13, W22 and W23 are present on horse major histocompatibility complex class II molecules

A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens precipitated from horse lymphocyte lysates using (cross-reacting) antibodies to human class I and class II MHC molecules confirmed the results obtained.     

Murine lymphocyte and embryonal carcinoma cell surface antigens recognised by rabbit anti-murine embryonal carcinoma serum

Some recent studies have suggested that murine embryonal carcinoma (EC) cells which lack classical H-2 molecules must express some novel class-I-MHC (major histocompatibility complex) type antigens. We have investigated whether rabbit anti-EC sera may recognize such components. Molecules of 40 and 48 kd were immunoprecipitated from EC cells and lymphocytes. The possibility that these molecules may be coded for by genes in the Qa-Tla locus which contains many MHC class-I genes or pseudogenes was investigated. The 40 and 48 kd molecules were not associated with beta 2-microglobulin on EC cells, nor with each other or other molecules through S-S bonds, although they appeared to have intradisulphide structures. Peptide map analysis suggested that the lymphocyte 40 and 48 kd molecules were related to the EC cell 40 and 48 kd molecules. However, these molecules were different from H-2, Ia or Qa-2 molecules from the same mouse strain.     

The thymic differentiation markers T6 and M241 are two unusual MHC class I antigens

The human beta 2-microglobulin (beta-2m)-associated human thymocyte differentiation antigens T6 and M241 were compared using biochemical techniques. T6 and M241 antigens reside on different molecules with apparent m.w. of 49,000 and 43,000, respectively. Here we show that both proteins have a protein backbone m.w. of 33,000. In addition, T6 and M241 have a large portion of their peptides in common. When we compared the protein backbone m.w. of T6 and M241 with the murine beta-2m-associated thymus leukemia (TL) antigens, we found a considerable difference in size, suggesting that T6 and M241 may not be human homologues of TL antigens and constitute a novel type of major histocompatibility (MHC) class I antigens.     

Human macromolecular insoluble cold globulin (MICG). II. Immunologic definition of T cell and null cell MICG and the biologic effect of antiserum to MICG.

Thymus cell-derived macromolecular insoluble cold globulin (T-MICG) is a 225,000-dalton protein, selectively synthesized in human T cells. Null cell-derived macromolecular insoluble cold globulin (N-MICG) is a 185,000-dalton protein, synthesized in null cells, and antigenically distinct from T-MICG. Evidence to support these conclusions was provided by using isolated cell preparations that were radiolabeled, lysed in desoxycholate, and precipitated with monospecific antiserum to each component. These studies demonstrated that antiserum to T-MICG precipitated a 225,000 dalton protein from PBL and T cells, but not from B or null cells. Antiserum to N-MICG reacted with a 185,000 dalton protein present in PBL and null cells, but not with lysates from either T or B cells. The plasma membrane distribution of these proteins was shown by absorption of antiserum to T + N-MICG with either isolated T or null cells. Antibody-induced cytotoxicity and immunofluorescence confirmed the cell surface location of T and N-MICG. Divergent biologic effects of these antisera were also noted. Antiserum to T-MICG inhibited T cell rosette formation and the one-way mixed lymphocyte reaction, although anti-N-MICG antiserum had no such effect. The potential importance of these proteins is discussed.     

Early events in lymphocyte activation as defined by three new monoclonal antibodies

Three new lymphocyte activation antigens are described whose kinetics of appearance place them very early in the activation pathway. The 78,000 dalton early antigen (Ea) 1 is present at low levels on resting lymphocytes, and its expression is enhanced twofold to threefold within 3 hr of stimulation. Ea2, a nondisulfide-bonded 86,000 and 73,000 dalton heterodimer, is first detectable 3 hr after activation and peaks by 9 hr. Its presence on all but a few cell lines, plus the variable association with a lower m.w. (28,000) structure, suggest that it may serve as a receptor for a growth factor. Neither Ea1 nor Ea2 are restricted to lymphocytes. The 31,000 dalton Ea3 antigen is induced only by PHA but not by other means of activation, and may pre-exist within the cell. The Ea3 antibody blocks PHA-induced but not OKT3-induced mitogenesis, suggesting differences in the pathways of activation by these two stimuli. These reagents, and OKT3, were used to define the cyclosporine A (CSA)-sensitive stage of lymphocyte activation. CSA blocks at a point before the biosynthesis of Ea1 and after that of T3/T cell receptor loss from the cell surface, at a point close to Ea2 biosynthesis.     

Guinea pig homologues of TL and QA-2 antigens.

The H-2, thymus-leukemia (TL), and Qa-2 antigens of mice are encoded by closely linked genes on murine chromosome 17, and have structural similiarity in that each antigen is borne on a approximately 44,000 dalton molecule associated with beta2 microglobulin (beta2mu). The extensive homology of major histocompatibility complex (MHC) products that exists for the mouse and guinea pig suggested that a similar homology might exist for products of genetic regions closely linked to the MHC. By taking advantage of the selective association of beta2mu with H-2, Qa-2, and TL antigens, and by using the technique of sequential immunoprecipitation, we demonstrated two previously undescribed guinea pig molecules reactive with anti-guinea pig beta2mu. The first molecule was composed of a 36,000 dalton glycoprotein associated with beta2mu and was found on guinea pig thymocytes, but not lymphocytes. The second molecule was composed of a 40,000 dalton glycoprotein associated with beta2mu, and was found on both guinea pig thymocytes and lymphocytes. By structure, chemical composition, association with beta2mu, and tissue distribution, the first molecule is an attractive candidate for the guinea pig homologue of TL antigen, whereas the second fits the criteria for the guinea pig homologue of Qa-2 antigen.     

Identification of a rabbit class I-like thymocyte-specific antigen

The availability of a monoclonal antibody, 5E2, has made it possible to characterize a new class I-like thymocyte-specific antigen in the rabbit. Flow cytometry and cyto-fluorescent microscopy show that approximately 70 to 80% of the cells reacting with 5E2 are located throughout the thymic cortex. Structural studies reveal that the cell surface molecule recognized by 5E2 is a non-covalently associated heterodimer consisting of a 45,000 dalton glycoprotein and a low m.w. beta-2-microglobulin protein. Certain properties of the 5E2 antigen were similar to the murine TL and human T6 antigens. Therefore we have tentatively assigned to the 5E2 antigen the designation R-Ta on the premise that the structural gene(s) encoding this molecule will be associated with the rabbit major histocompatibility complex.     

H-2-specific antibodies induced by injection of syngeneic leukemia cells

AKR/J mice immunized with several syngeneic leukemia cells contained antibodies in their sera which reacted with certain AKR leukemia cell lines, depending on their H-2 expression, and precipitated H-2K antigens from lysates of leukemia cells. Precipitation of H-2K was not due to virus-specific antibodies: it could not be blocked by prior absorption with H-2-negative leukemias, but was blocked by certain allogeneic lymphocytes. Tumor-specific H-2K antibodies did not react with H-2K from normal AKR lymphocytes either on the cell surface or after detergent solubilization; however, they did react with H-2K from mitogen-activated AKR and BALB.K lymphoblasts. Since both these latter cells were also lysed by AKR-Gross/MuLV-specific and H-2K^k-restricted cytotoxic T lymphocytes, we consider the possibility that antibodies detecting conformational alterations induced in H-2K^k molecules by viral association may be present in syngeneic AKR antileukemia sera.Abbreviations used in this paper GCSA Gross-virus-induced cell-surface antigen - MCF mink cell focus-forming virus - MuLV murine leukemia virus - Th T helper     

Mechanisms of lymphocyte-mediated cytotoxicity. I. The effects of anti-human lymphotoxin antisera on the cytolysis of allogeneic B cell lines by MLC-sensitized human lymphocytes in vitro

Goat and rabbit anti-human lymphotoxin sera, IgG and F(ab')2 reagents were investigated for their capacity to effect a specific alloimmune lymphocyte-mediated cytotoxic reaction. The cytotoxic reaction employed human peripheral blood or adenoid lymphocytes sensitized in MLC to allogeneic B lymphocyte cell lines and lysis was measured in a short-term 51Cr-release assay. A polyspecific anti-LT sera (anti-WS), made against unfractionated whole supernatants from lectin-activated lymphocytes and its IgG and F(ab')2 fragments, was found to be a potent inhibitor of this reaction when the anti-WS reagent was present throughout the assay period. Absorption studies indicated the anti-WS was inhibiting cytolysis at the level of effector cell or its products. Two broadly defined antibody specificities were involved in the cytolytic-inhibitory activity of the polyspecific anti-LT; i) antigens present on the normal lymphocyte cell surface; and ii) lymphocyte surface antigens associated with activated cells. These results correlate with the previously defined antigenic structure of the LT Cx and alpha H classes. Anti-LT sera reactive with the smaller m.w. alpha and beta classes and subclasses were not inhibitory, although the anti-beta sera showed a moderate enhancing activity. The results indicated that several anti-LT antibody specificities may be required to inhibit alloimmune cytolysis. The results suggest LT molecules may mediate lymphocyte-induced alloimmune cytolysis as a multi-component toxin system, rather than as an individual toxin.     

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

Genetic variability at the pig SLA complex in U.S. breeds of pigs

Lymphocytes from 90 mature pigs and 548 growing pigs from 65 litters were screened with cytotoxic antibodies against swine lymphocyte antigens. Pigs included in the survey were from the Chester White, Duroc, Hampshire, Landrace and Yorkshire breeds. Significant differences between breeds were found in the frequency of the three haplotypes tested. Such differences may provide the genetic variability needed to determine associations between disease susceptibility and the pig's major histocompatibility gene complex.     

A physicochemical and immunologic comparison of the cell growth inhibitory activity of human lymphotoxins and interferons in vitro.

The physicochemical, immunologic, and biologic relationships between humam lymphotoxins (LT) and interferons (IF) present in supernatant fluids from lectin-stimulated peripheral blood lymphocytes (PBL) and a continuous B-lymphoblastoid cell line (PGLC-33h) were analyzed. LT activity obtained from lectin-activated PBL could not be resolved from IF activity by gel filtration chromatography. LT activity eluted in multiple peaks of activity at 70 to 90,000, and 40 to 50,000 m.w., characteristic of alpha and beta LT, respectively. IF activity in these supernatant fluids eluted as a broad band between 35 and 80,000 m.w., also suggestive of molecular heterogeneity. In contrast, this m.w. heterogeneity was not observed in LT and IF activities obtained from the PGLC-33h cell line. LT and IF eluted as separate peaks of activity at 90,000 and 25,000 m.w., respectively. In addition, acid and heat lability of PGLC-33h IF suggested similarity to type II IF. Immunologic studies, with a rabbit anti-alpha class serum that neutralized LT activity from both PBL and PGLC-33h, did not affect IF activity from either of these sources. Supernatant fluids from PGLC-33h cultures were also capable of inhibiting the proliferation of HeLa cells in vitro. The growth inhibitory activity was attributed to LT- and IF-like molecules. This evidence suggests that although cytotoxic and anti-viral activities were due to separate molecules, LT and IF have overlapping biologic activities in their ability to inhibit the proliferation of cells in vitro.