Isolation, purification, and amino acid composition of the tunicate hemocyte Thy-1 homolog

A serologic cross-reacting homolog to rodent Thy-1 glycoproteins has been isolated from hemocyte cell surfaces of the advanced invertebrate group of tunicates. The Thy-1.1 cross-reacting antigenic activity was followed during purification by inhibiting the binding of MRC OX7 monoclonal antibody to pure rat brain Thy-1 in a soluble phase radioimmunoassay. After solubilization in deoxycholate, tunicate hemocyte Thy-1.1 antigenic activity was purified by affinity chromatography using an MRC OX7 monoclonal antibody affinity column, followed by gel filtration. A 602-fold enrichment in the Thy-1.1 antigenic activity, with a yield of 55.6% compared to the starting crude membrane fraction, was obtained. The antigenic activity was associated with a single glycoprotein of molecular size of 3.1 nm and molecular weight estimated at 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% gels). Amino acid composition of the purified molecule was compared by the S delta Q index of differences in composition to mammalian and non-mammalian Thy-1 glycoproteins, Ig, major histocompatibility complex-encoded polypeptides, beta 2-microglobulin, and other recognition molecules. With this parameter, the tunicate hemocyte Thy-1 homology revealed significant relatedness to avian and mammalian Thy-1 molecules and was interestingly more related to mu chains of primitive vertebrates and to HLA class I and II encoded polypeptides than to Thy-1 molecules of higher vertebrates. Based upon these observations, the tunicate hemocyte Thy-1 homolog seems to represent an ancestral Thy-1 molecule which, in structural terms, may represent an invertebrate member of the Ig superfamily.     

Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1

It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.     

A monoclonal antibody detecting unusual Thy-1 determinants

20-10-5S is a monoclonal antibody produced by the fusion of C3H anti-C3H.SW splenocytes with the SP2/0 cell line. The antibody appears to react with Thy-1 determinants by several criteria including cytotoxicity patterns, functional assays, genetic analyses, and competitive binding experiments. However, the antibody and the determinants it detects are unusual in that: 1) 20-10-5S is autoreactive; 2) the antibody shows allospecificity for Thy-1.2 vs Thy-1.1 antigens only on peripheral lymphocytes and not on thymocytes; and 3) the antibody reacts only with determinants on murine T cells and not with antigens on brain tissue or on rat thymocytes. It therefore seems that 20-10-5S reacts with murine T cell-specific Thy-1 determinants that are lost or modified during maturation of the cells on which they are expressed.     

A Thy-1.1-specific monoclonal alloantibody activates both mouse and rat T cells

In an attempt to further evaluate the role of Thy-1 in the antigen-independent triggering of mouse T cells, we have examined the activating properties of two Thy-1.1-specific mouse monoclonal antibodies (mAb). These reagents were established from an (A.TH X A.TL)F1 hybrid mouse (Thy-1b) immunized with IL-2 producing (BALB/c (Thy-1b) X BW5147 (Thy-1a)) T hybridoma cells. Although both mAb recognized the same Thy-1.1 determinant, one mAb of the gamma 3,kappa class (H171-146) was found to induce several T hybridoma cells to produce IL-2, and AKR thymocytes or cloned helper T cells to proliferate, whereas another mAb of the gamma 1,kappa class (H171-112) failed to do so even in the presence of phorbol myristic acetate (PMA). Increased IL-2 responses of T hybridoma cells were observed when the cell bound Thy-1.1-specific mAb were crosslinked by goat anti-mouse Ig (GaMIg) antibodies. Both a T-cell activating rat anti-Thy-1.2 mAb and the anti-Thy-1.1 mAb H171-146, although directed at distinct cell surface molecules, synergistically stimulated IL-2 production by T hybridoma cells. In addition, the mouse mAb H171-146 was found to stimulate LOU/M rat thymocytes to proliferate in the presence of exogenous IL-2. These data demonstrate that T cells can use Thy-1 as a signal-transducing molecule in both mouse and rat species, and support the notion that the activating properties of Thy-1.1-specific mAb are influenced by their heavy chain isotypes.     

Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents

1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s(20,w) values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen-detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).     

Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen.

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.     

Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain, recognised by monoclonal antibody MRC OX 2

The MRC OX 2 monoclonal antibody recognises antigens present on rat thymocytes, brain, follicular dendritic cells in lymphoid organs, vascular endothelium, some smooth muscle and B-lymphocytes. The OX 2 antigens recognised by this antibody were purified from brain and thymus, by solubilisation with sodium deoxycholate, affinity chromatography with MRC OX 2 antibody and gel filtration. The purified brain and thymocyte OX 2 antigens were glycoproteins with apparent Mr 41000 and 47000 respectively as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Rabbit antisera raised against the purified antigens were analysed by radioimmunoassay and immunoperoxidase-staining of tissue sections. The brain and thymocyte OX 2 antigens were antigenically very similar to those on the other tissues. This indicates that the unusual pattern of distribution was not the result of fortuitous cross-reaction of the MRC OX 2 antibody, as the rabbit sera would be expected to recognise more determinants on the antigen than that recognised by the monoclonal antibody. The amino acid compositions of brain and thymus OX 2 antigens were very similar but with no distinguishing features. Carbohydrate compositions showed that the OX 2 antigens were highly glycosylated, with brain OX 2 antigen containing 24% and thymocyte OX 2 antigen 33% by weight of carbohydrate. Both OX 2 antigens contained carbohydrate residues typical of structures N-linked to asparagine but lacked galactosamine, indicating the absence of O-linked structures. Thymocyte OX 2 contained higher levels of galactose and sialic acid but less fucose than brain OX 2. Similar differences had been observed for brain and thymocyte Thy-1 antigens and were also observed in pooled glycoproteins purified by lentil affinity chromatography from these tissues, reflecting overall differences in the patterns of glycosylation in the two tissues. The OX 2 antigens showed many similarities to Thy-1 antigens in their odd patterns of distribution, characteristic migration on polyacrylamide gels in sodium dodecyl sulphate, and carbohydrate compositions. It is possible that OX 2 antigens, like Thy-1 antigens, have homologies with immunoglobulin domains. A possible role for OX 2 antigens in cell interactions necessary for tissue organisation is discussed.     

Purification and characterization of the mouse thymocyte Thy-1 glycoprotein.

The thymocyte Thy-1 glycoprotein was purified from mouse strain NMRI (Thy 1.2) thymus. Crude membranes were prepared in Tween 20 and solubilized in deoxycholate. The glycoproteins were isolated by affinity chromatography on concanavalin A-Sepharose, and Thy-1 was further purified by two gel-filtration cycles on Sephacryl S-200. The concentration of Thy-1 in fractions obtained during purification was measured by a solid-phase radioimmunoassay with pure mouse brain Thy-1 as standard. Analysis of the purified preparation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed at least five distinct bands in the apparent-Mr region 25000-30000, the polymorphism probably being due to carbohydrate heterogeneity. Amino-acid-analysis data were compatible with the previously published sequence of mouse brain Thy-1. Sugar content was determined at 31% (w/w), and the carbohydrate composition indicates the presence of 'complex-type' oligosaccharide chains. The mean Mr of mouse thymocyte Thy-1 was calculated to be 18 100.     

Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.     

Viral antigens act as helper determinants for antibody responses to cell surface antigens

Thy-1 alloantigens on murine thymus cells are weak immunogens in vivo for PFC responses in the absence of other antigenic disparities between the donor and recipient. Our previous work showed that non-H-2 alloantigens acted as helper determinants to augment anti-Thy-1 PFC responses. In this report we demonstrate that strong helper antigens are also produced by infection of donor thymus cells with viruses such as HSV-1, NDV, or vaccinia. This helper effect (as much as 30-fold) for a cellular antigen, requires linked recognition (expression of Thy-1 and virus in the same cell membrane), is T-dependent, antigen- (virus) specific, and is Thy-1-specific. The recognition of the viral helper sites is not restricted by the MHC genotype of the thymus cell donor, indicating that host reprocessing of antigen occurs. These are the first results that show that adventitious antigens may function as helper determinants for antibody responses to native membrane antigens and may be the mechanism that initiates several forms of acute post-viral autoimmune disease.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Serological survey of T-lymphocyte differentiation antigens in wild mice

Allelic distributions of Thy-1, Ly-l, and Ly-2 antigens in wild mice are characteristic of each Mus musculus subspecies. Eastern mice (M.m.molossinus, M.mmusculus, M.m.castaneus, M.m.bactrianus) express the Thy-1.1 antigen, whereas Western mice (M.m. domesticus, M.m.brevirostris) express the Thy-1.2. All mice from wild populations examined in this survey express the Ly-1.2. The Ly-2.1 is distributed in Eastern mice and some Western mice, and the Ly-2.2 is found in the remaining Western mice. Allelic distributions of these antigens were also examined in two other species, Mus spretus and Mus spicilegus. Allelic constitutions of Thy-1 and Ly-1 in these species are similar to those of Eastern mice. Some M.spicilegus, however, express the Ly-1.1 antigen. This antigenic type is not found in M.musculus. Some Eastern mice related to M.m.castaneus react weakly to Ly-1.2-specific and Ly-2.1-specific monoclonal antibodies in both the complement-mediated cytotoxicity test and the absorption test. These results suggest that M.m.castaneus has unique alleles in the Ly-1 and Ly-2 loci.     

A mouse plasma cell surface antigenic determinant (PLKB) recognized by xenoantiserum. Its relationship to the PC.1 antigenic determinant.

The specificities of the xenoantisera made against mouse myeloma cells have been compared to those recognized by alloantiserum by studying patterns of cytotoxicity on both normal and malignant plasma cells. Goat antiserum obtained by immunization with Balb/c mouse myeloma ADJ-PC-22A cells and purified by in vivo absorption could detect cell surface antigenic determinants present on plasma cells and on cells of liver, kidney, and brain (PLKB antigen), as we had previously reported for a similarly prepared rabbit antiserum. In spite of an apparent similarity between the tissue representation of the PLKB determinant and that of PC.1 antigenic determinants which were detected by DBA/2 anti-ADJ-PC-22A cell alloantiserum, the PLKB antigenic determinant is not identical with the PC.1 antigenic determinant, since the former is found on the tissues of PC.1-negative as well as PC.1-positive strains of mice. However, it was deduced that the PLKB antigenic determinant and the PC.1 antigenic determinant reside in close proximity on the cell surface or maybe even on the same molecule, since Fab fragments of antiserum against either PLKB or PC.1 blocked the cytotoxicity against both antigens. On the other hand, these Fab fragments did not inhibit the cytotoxicity of anti-H-2 antiserum, indicating that neither PLKB nor PC.1 antigenic determinants are in close proximity to H-2 antigens. Association of PLKB and PC.1 determinants was further supported by the finding that the loss of the PLKB determinant in a variant of myeloma MOPC-70A corresponds to the loss of PC.1 determinant on the same cells.     

Regulation of Thy-1 gene expression in transgenic mice

Genomic DNA fragments encompassing the human Thy-1 or mouse Thy-1.1 gene have been microinjected into pronuclei of mouse embryos homozygous for the Thy-1.2 allele. In the resulting transgenic mice, the human gene is expressed in a pattern characteristic of normal human tissues, and is not influenced by the pattern of endogenous mouse Thy-1 expression. The mouse Thy-1.1 gene fragment is expressed in a pattern typical of mouse Thy-1, although it is more limited in its distribution. The results indicate the presence of multiple cis-acting regulators of Thy-1 gene expression that have changed in both their character and arrangement over the course of Thy-1 gene evolution.     

Demonstration of a hematopoietic stem cell antigen (SC-1) on a murine lymphoma and isolation of variants lacking the antigen

Murine multipotential hematopoietic stem cells (CFU-s) bear an antigen (SC-1) which is recognized by heterologous antisera to mouse brain. We have found that cloned Thy-1 negative variants of the T-cell lymphoma RL ♂1 are sensitive to complement-mediated cytolysis by anti-brain serum and can absorb the anti-stem cell activity from the antiserum. We have isolated several subclones derived from a primary Thy-1 negative variant which are not susceptible to anti-brain serum. The surface of the resistant lines has little or no antigen capable of binding anti-mouse brain antibodies as measured by either immunofluorescence or a radioimmunoassay. These lines are also unable to absorb the anti-bodies responsible for the cytotoxic effect of rabbit anti-mouse brain serum against CFU-s. We conclude that the predominant antigen, serologically detectable on Thy-1 negative variants of RL ♂1, is SC-1.     

Changes in cell-surface expression of MHC and Thy-1.2 determinants following treatment with lipid modulating agents

Treatment of normal mouse spleen cells with lipid fluidity modulators changes the expression of cell-surface H-2 determinants. BALB/c spleen cells treated for 1 to 2 hr with cholesteryl hemisuccinate (CHS) displayed reduced levels of all tested H-2 determinants (H-2L, H-2K, and H-2D) as evaluated by flow microfluorometry and increased membrane lipid packing density as determined by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence polarization. In contrast, decreasing membrane lipid packing density by phosphatidylcholine treatment decreased DPH fluorescence polarization and increased the expression of MHC determinants. The effects were selective in that expression of Thy-1.2 determinants was decreased by the latter treatment and not increased by CHS. The results are discussed in terms of passive modulation of antigenic expression.     

Ectopic expression of Thy-1 in the kidneys of transgenic mice induces functional and proliferative abnormalities

Hybrid human--mouse Thy-1.1 genes were injected into pronuclei of Thy-1.2 mice to produce transgenic animals. A hybrid gene composed of the 5' part of the mouse Thy-1.1 gene combined with the 3' human untranslated regions was expressed abnormally in the kidney podocytes, which resulted in severe protein-uria and subsequent death in several founder mice. A hybrid Thy-1 gene composed of the human coding region with the 5' and 3' flanking regions of the mouse gene was expressed abnormally in a different part of the kidney (the tubular epithelia), which resulted in a proliferative kidney disorder. In addition, a neoplasm was found in the brain of one of these mice. These results show that the Thy-1 protein can play an important role in the activation, proliferation, and differentiation of many different cell types.     

Coexistence of Intraspecies and Interspecies Specific Antigenic Determinants on the Major Structural Polypeptide of Mammalian C-type Viruses

THE report of a shared viral antigen (termed gs-3) among mammalian C-type viruses from four species¹, extending an earlier report of cross reactivity between mouse and cat viral antigens², has far reaching implications in the search for human cancer viruses or their gene products. The report is confirmed both by the data presented here and also by the data obtained by another laboratory³. Our gel diffusion assays using various selected sera against mouse, hamster and cat crude and purified C-type viral antigens indicate that the cross reactive antigenic determinants are specifically present on the major structural polypeptide of C-type viruses. The polypeptide also carries species specific determinants. These conclusions are drawn from complement fixation and gel diffusion tests using six types of antisera (either individual sera or pools) prepared as described in Table 1.     

Differential expression of mouse embryonic antigens TEC-1 and TEC-2 in the epididymis of four rodent species

The expression, properties and relationship of two mouse embryonic antigens (TEC-1 and TEC-2), which are defined by monoclonal antibodies, were investigated in the epididymis of four rodent species. Absorption analysis, indirect immunofluorescence microscopy and immunohistochemistry revealed that all the species studied contained in their epididymides, but not in testes, either TEC-1 (Chinese hamster), TEC-2 (guinea pigs, rats) or both TEC-1 and TEC-2 (mice) antigens. In an indirect immunofluorescence assay, the antigens were found on spermatozoa isolated from caudae epididymides of guinea pigs, rats and Chinese hamsters but not mice. On the other hand, the TEC-2 antigen, which is expressed on mouse eggs, was not detected on eggs from the other species studied. Immunolabeling of epididymal extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both epididymal antigens have apparent molecular weights of greater than 200,000. In guinea pigs, rats and mice, the antigens were detected by a two-site sandwich radioantibody-binding assay in which the antigen is immobilized and detected with the same antibody; this indicates that several antigenic determinants were present on the same carrier. In mice, some carriers seem to express both TEC-1 and TEC-2 epitopes. In Chinese hamsters, TEC-1 antigen was only detected by the solid-phase assay, suggesting that in this species there are markedly fewer antigenic determinants per carrier molecule. Interspecies differences in the activities of epididymal glycosyltransferases and/or glycosidases appear to be the biochemical mechanism of the species-specific expression of these antigens.     

T cell independent Thy-1 allo-antibody response with the use of transgenic mice

We have introduced a mouse Thy-1.1 gene into the germline of Thy-1.2 mice. The introduced gene was shown to be expressed at very high levels in thymocytes when compared with the endogenous gene. Transgenic thymocytes were shown to evoke a higher than normal primary anti-Thy-1.1 antibody response in plaque-forming cell (PFC) assays. This result suggests that a direct quantitative interaction of the Thy-1 antigen activates the B cell response.