Functional and binding activity of monoclonal anti-Thy-1 antibodies: evidence for different expression of the two alleles

Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antisera selected for complement-dependent cytotoxicity have high cytotoxic and binding titers on thymocytes and peripheral T cells of mouse strains bearing the appropriate Thy-1 allele. The effect of both anti-Thy-1.1 and anti-Thy-1.2 monoclonal antisera plus complement on cytotoxic T cell effectors is to abrogate their activity. On the functional activity of precursor cytotoxic T cells, monoclonal antisera against the two alleles have different effects: anti-Thy-1.2 plus complement removes precursor activity of Thy-1.2-bearing strains, including (Thy-1.1 × Thy-1.2)F₁ heterozygotes. In contrast, six different anti-Thy-1.1 monoclonals, including four of the IgM class and two of the IgG class, failed to remove cytotoxic precursor activity from the splenic T cells of AKR, A. Thy-1.1 or (CBA × AKR)F₁ mice. Analysis by fluorescence-activated cell sorting of in vitro cultured AKR spleen cells shows that Thy-1.1 antigen appears on the cell surface during the five-day culture period.     

Neuraminidase sensitivity of Thy-1 active glycoconjugates

Three forms of Thy-1 active antigens were studied for their relative sensitivity to the enzyme neuraminidase (Nase). Those studied were Thy-1 active shed material, Thy-1 active glycolipids and Thy-1 active glycoprotein. Each expressed either Thy-1.1 or Thy-1.2 allotype according to the mouse strain of origin. Thy-1 activity was assessed by a plaque forming cell (PFC) assay specific for Thy-1 allotypes and by a solid phase non-competitive immunoradiometric assay (IRA) specific for Thy-1.2. Treatment of Thy-1 active complexes shed from either C3H (Thy-1.2) or AKR (Thy-1.1) thymocytes with Nase resulted in the loss of respective Thy-1 antigenicity in both PFC assay and the direct binding IRA. The antigenicity decreased significantly within the first few hours and the loss of Thy-1 activity was complete by 24 hr. Thy-1.1 and 1.2 activity of glycolipids was also destroyed by Nase treatment, and the Thy-1.2 activity of purified Thy-1.2 glycoprotein was also destroyed by Nase treatment as assessed by the PFC assay. An unusual finding was that both Thy-1.1 active shed complexes and Thy-1.1 glycolipid after Nase treatment exhibited Thy-1.2 activity. This conversion was confirmed using two forms of Thy-1.1 for the PFC assay (which relies on the immune response to Thy-1) and for the IRA (which depends only on direct binding of anti-Thy-1.2 to the antigen). The sensitivity of all three forms of antigen was the same regardless of the source of enzyme and three sources of Nase enzymes catalysed the conversion of Thy-1.1 into Thy-1.2 active forms. These results suggest that sialic acid may be necessary but not sufficient for the expression of Thy-1 antigenicity.     

Isolation and characterization of a molecular complex containing Thy-1 antigen from the surface of murine thymocytes and T cells

A complex containing the Thy-1 (θ) antigen has been isolated from the surface of murine thymocytes and T cells by cell surface radioiodination, lysis by freezing and thawing, and immunoprecipitation. The specificity of the immunoprecipitation was firmly established using both congenic and noncongenic anti-Thy-1 sera and thymocytes from congenic and noncongenic mice. Unlike cell surface Ig and the TL and H-2 alloantigens, the antigenicity of Thy-1 was abolished by treatment with nonionic detergent. Thy-1 was localized exclusively in a complex of cell surface molecules which has a density lower than that of protein. Moreover, Thy-1 could be readily labeled by incubating thymocytes with [³H]galactose, but not ³H-labeled amino acids. These observations suggest that the antigenicity of Thy-1 may reside in a glycolipid.     

Evidence that the Thy-1 molecule is the target for T cell mitogenic antibody against brain-associated antigens

Rabbit anti-mouse brain (RaMBr) antiserum can induce Lyt-1⁺, Lyt-2^?, T cells to proliferate and stimulates the same T cell subset to induce B cell proliferation. The aim of this report is to demonstrate that the mitogenic determinant recognized on the T cell surface by RaMBr antiserum is located on the Thy-1 molecule expressing the products of the Thy-1^a and Thy-1^b alleles. Evidence is drawn from serological and genetic experiments. The brain T cell cross-reactive, mitogenic determinant is not expressed on Thy-1^? mutants of the BW5147 T cell lymphoma that fail to express the Thy-1 molecule but do express other T cell surface proteins such as T-200 and gp 69, 71. Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antibodies block the binding to the appropriate T cells of the majority of the serum antibody from RaMBr antiserum. The absorption of mitogenic antibody was blocked in a similar fashion, thus demonstrating the close association of the determinant and the Thy-1 antigen defined by monoclonal alloantibodies. The mitogenic and Thy-1.1 determinants are probably located on the same molecule because of the data obtained with the BW5147 Thy-1^? mutants and the observation that Thy-1^a T cells, which express a lower level of surface Thy-1 than Thy-1^b T cells, also express lower levels of the determinant recognized by RaMBr antiserum. Furthermore, in (AKR × DBA/2)F₁ mice (Thy-1^a/b) which express less Thy-1.1 antigen than Thy-1.2 at the surface, the mitogenic determinant was found to be prefentially associated with Thy-1.2. The coordinated genetic control of the surface levels of the Thy-1 determinant and the mitogenic determinant suggests that both determinants are situated on the same molecule in the T cell membrane.     

Intact and subcellular form of thymocytes of rats are exceptionally immunogenic in mice for inducing anti-Thy-1 T cell-independent class 2 antibody responses

Results of the present study show that the primary anti-Thy-1.1 antibody response to rat antigen in Thy-1.2 mice is induced exclusively by thymocyte antigen. Thy-1 antigens of brain and bone marrow, which expressed much Thy-1 antigen, were poorly immunogenic if at all. Brain Thy-1 antigen considerably inhibited the immunogenicity of thymocyte Thy-1. Moreover, we found that subcellular form of thymocytes induce as high antibody responses as intact thymocytes do. The subcellular thymocyte Thy-1 antigen behaved as TI-2 antigen, inducing a good response in athymic nude mice but not in CBA/N mice with a B cell defect. The significance of these findings is discussed in relation to the possible physiological activity of Thy-1 or Thy-1-linked molecules on thymocytes specifically mediating lymphocyte differentiation.     

Independent mobility of cholera toxin binding sites and Thy-1 alloantigen on mouse thymocytes.

The hypothesis that Thy-1.2 carries a carbohydrate antigenic determinant with the same specificity as the monosialoganglioside GM1 was tested by attempting to co-cap Thy-1.2 and GM1 in CBA thymocytes using anti-Thy-1.2 alloantiserum and cholera toxin. Co-capping of these determinants was not observed suggesting that they are on separate molecules. We do not therefore confirm the previous reported association between Thy-1 and GM1 (Thiele, Arndt & Stark, 1977).     

Identification of autoanti-Thi-1 antibody in the sera of BALB/c mice immune to P1798 lymphoma.

The specificity was established for the anti-thymocyte autoantibody which appeared in BALB/c mice (BAA) during immunization to the P 1798 lymphoma. The results indicate that BAA is specific for the Thy-1 alloantigen of murine T cells. By immunoprecipitation of 125I-labeled A/J thymocyte surface antigens from freeze-thaw lysates and electrophoretic resolution of the immunoprecipitates on sodium dodecyl sulfate polyacrylamide gels, the BAA and congenic anti-Thy-1.1 or 1.2 were shown to be specific for determinants on the same antigens. Specificity of BAA for Thy-1.2 was shown also quantitative absorption studies. Coating P 1798 lymphoma cells with either BAA or anti-Thy-1.2 reduced the absorptive capacity of these cells for the same or the reciprocal antiserum. Coating P 1798 with an antiserum to P 1798 which was indifferent to the Thy-1.2 alloantigen did not diminish the absorptive capacity of these cells for either BAA or anti-Thy-1.2. These studies have provided firm evidence that BAA is specific for determinants on the Thy-1 alloantigen.     

Anti-Thy-1 antibody responses evoked by Thy-1 antigen expressed in transfected mouse mastocytoma cells and rat fibroblast.

The mouse genomic Thy-1.1 gene was isolated from a phage library constructed from AKR/J (Thy-1.1) mouse DNA. Partial nucleotide sequence analysis of the coding region showed that it has only a single nucleotide difference from the Thy-1.2 gene, namely that amino acid 89 reads CGA (Arg) in Thy-1.1 and CAA (Glu) in Thy-1.2, corresponding to the amino acid substitutions previously identified. It was subcloned into an SV-40 derived vector for transfection. Transient transfection into HeLa cells gave 2% positive staining by immunofluorescence. The gene in this vector was also co-transfected into L cells and mastocytoma cells (both of Thy-1.2 strain origin) together with the Agpt gene. L-cell clones selected for transformation proved almost negative for Thy-1.1 expression, and any positive clones gradually lost Thy-1.1 antigen expression in culture. On the contrary, all clones of mastocytoma transformants gave a high level of expression after more than 3 months in culture. The mastocytoma transformants were used to study the immunogenicity of Thy-1.1 molecules expressed on transfected cells. They evoked clear anti-Thy-1.1 plaque-forming cell (PFC) responses both in vivo and in vitro. The mastocytoma transformants also proved able to induce a T-dependent anti-Thy-1.1 antibody response in a cell transfer experiment. The immunogenicity of Thy-1.2 molecules on rat fibroblasts was also studied after transfection with a Thy-1.2 gene cosmid. Although Thy-1.2 expression was very low, these transfectants elicited a clear anti-Thy-1.2 PFC response from AKR spleen cells hyperimmunized against CBA thymocytes.     

Metabolism of H-2 and Thy-1 (Θ) alloantigens in murine thymocytes

The metabolism of Thy-1 and H-2 alloantigens was studied in murine thymocytes radiolabeled with ³H precursors or surface radioidinated. It was found that ¹²⁵I-labeled Thy-1 is rapidly shed from cultured thymocytes, whereas H-2 alloantigens are not. [³H]galactose-labeled Thy-1 is also released from cultured thymocytes, whereas [³H]leucine-labeled H-2 alloantigens are not. These results support the concept that H-2 alloantigens are integral macromolecules of the plasma membrane and suggest further that Thy-1 is a peripheral one. Possible mechanisms underlying shedding are discussed.     

Flow microfluorometric analysis of alloantigen expression during T cell development

The fluorescence-activated cell sorter (FACS) was used to examine the expression of several alloantigens on T cells: Thy-1, Lyt-1.1, Lyt-2.1, Ly-5, Ly-6 and Ly-7. Antibodies secreted by monoclonal cell lines were used to characterize the Thy-1.2, Lyt-1.1 and Lyt-2.1 antigenic determinants, whereas Ly-5.1, Ly-6.2 and Ly-7.2 determinants were identified with alloantisera raised by conventional hyperimmunization. Using indirect immunofluorescence with fluorescein isothiocyanate-conjugated reagents, the profiles obtained with the FACS demonstrated that: (a) the expression of Thy-1 is greater on normal thymocytes than on cortisone-resistant thymocytes (CRT), lymph node and spleen cells; (b) in contrast to Thy-1, the expression of Ly-1 on normal thymocytes is less than on CRT, lymph node and spleen cells; (c) about 90% of normal thymocytes but < 50% of CRT, spleen and lymph node T cells are Lyt-2⁺ and (d) although weakly expressed on normal thymocytes, the amount of Ly-6 and Ly-7 present on peripheral T cells was considerably increased. The significance of these findings and the potential for further analysis of T cell developmental pathways is discussed.     

Characterization of hepatitis B virus (Dane particle)

Hepatitis B virions (Dane particles) were purified from the sera of chronic HBsAg carriers by consecutive rate-zonal and isopycnic centrifugations in sucrose gradients using HBsAg, HBcAg and endogenous DNA polymerase activities as specific markers. Purified Dane particles, radiolabelled with Na ¹²⁵I by the chloramine-T procedure, had a higher buoyant density in CsCl (1.28 g/cm³) than unlabelled particles (1.26 g/cm³) and an estimated sedimentation coefficient of 280 s. ¹²⁵I-Dane particles were fully precipitated by anti-HBs and not by anti-HBc sera. Heavy and light density core particles were purified from heavy and light density populations of Dane particles and radioiodinated. The iodinated polypeptides of Dane particles and HBcAg were compared with those of the iodinated 22-nm form of HBsAg by SDS-PAGE. Iodinated Dane particles contained seven polypeptides with molecular weights of 18,000, 23,000, 26,000, 34,000, 43,000, 48,000 and 115,000. Heavy and light core particles contained three polypeptides with molecular weights of 18,000, 25,000 and 37,000.     

Estimation of the amount and tissue distribution of rat Thy-1.1 antigen

The distribution and amount of Thy-1.1 antigen expressed in rat and mouse tissues have been studied. When anti-Thy-1.1 antisera were absorbed with various tissues and assayed by cytotoxicity or radioactive binding assays, it was found that antigen was expressed in similar amounts on thymocytes and brain of both species. Lymphocytic leukemia cells from PVG/c rats also expressed as much Thy-1.1 as rat thymocytes. In contrast, Thy-1.1 was only detected in very small amounts on rat lymph node cells or thoracic duct lymphocytes; the level was 20-fold less than the AKR mouse equivalent. Rat spleen cells had larger amounts than other peripheral rat lymphocytes, and had only 2–3 times less antigen than AKR spleen cells. These results were confirmed by direct binding assays which showed that AKR mouse and PVG/c rat thymocytes bound more than 500000 molecules of anti-Thy-1.1 antibody per cell. Autoradiographic analysis showed 90 % of thymocytes, 12 % of spleen cells, and 2 % of lymph node or thoracic duct lymphocytes of the rat to be specifically labeled.     

Homotypic aggregation of murine T lymphocytes induced by anti-Thy-1 monoclonal antibodies.

During the course of studies of anti-Thy-1-mediated T-cell activation, we found that anti-Thy-1 monoclonal antibodies (mAb) could induce strong homotypic aggregation of murine T-lineage cells. We demonstrated that anti-Thy-1 mAb-mediated T-cell aggregation started at 10 min and reached maximum level 1 hr after addition of antibody. It was temperature dependent, requiring metabolic energy and cytoskeletal integrity similar to that mediated by phorbol myristate acetate (PMA). But the striking difference between anti-Thy-1 mAb-mediated cell aggregation and PMA-mediated cell aggregation was that the latter but not the former was blocked by anti-LFA-1 mAb. This indicates that, unlike treatment with PMA, anti-CD2 mAb or anti-CD3 mAb, anti-Thy-1 mAb treatment of T lymphocytes does not induce LFA-1 activation for cell adhesion. Murine neuroblastoma cells were not induced to aggregate by anti-Thy-1 mAb treatment, although murine T lymphoma cells were aggregated by anti-Thy-1 mAb. The T-lineage cell specificity of anti-Thy-1-mediated aggregation was further shown by the Thy-1 gene transfection into non-Thy-1 expressing cell lines. Thy-1.1 gene transfected mastocytoma cells were not aggregated by anti-Thy-1.1 antibody.     

Analysis of the Thy-1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy-1 epitopes

Seventeen monoclonal anti-Thy-1 antibodies (mAb) derived from LOU/M rats immunized with mouse T cell clones were used to study the role of Thy-1 in antigen-independent T cell activation. These mAb identified Thy-1.2 or monomorphic determinants and immunoprecipitated a molecule of 25-28 kDa from detergent-solubilized, 125I-labeled T cell surface proteins. Competitive cross-inhibition binding assays demonstrated that these reagents defined 3 epitope groups including either Thy-1.2 (group A) or Thy-1 monomorphic (groups B and C) determinants. Experiments using high titered culture supernatants revealed that all 6 IgG mAb defining the epitope group C, and one IgG2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid60-L-alanine33-Ltyrosine10 (GAT) plus I-Ad-reactive BALB/c T cell hybridoma T14-117.9 to produce interleukin 2 (IL2) in the absence of accessory cells. Cross-linking of cell-bound rat mAb by a BALB/c anti-rat kappa chain mAb, or the presence of B cell lymphomas in the culture resulted in an increase of the Thy-1-mediated IL2 responses of this hybridoma. Some mAb from group B required antibody doses exceeding 80 micrograms/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in mAb-mediated T cell activation was observed when nonmitogenic doses of mAb group groups A and C were mixed in the same culture. Analysis of a panel of GAT plus I-Ad-specific T cell hybridomas revealed that these cells markedly differed in the magnitude of their IL2 responses induced by a given amount of stimulating anti-Thy-1 mAb. Such reagents also stimulated normal thymocytes to express IL2 receptor on their surface. These studies show that the epitopic specificity and the amount of anti-Thy-1 mAb, and the susceptibility of the T cell examined represent important parameters for the triggering of the Thy-1 pathway of T cell activation.     

Simultaneous detection of two different cell surface antigens by electron microscopy by means of multivalent hybrid antibody with double specificity

Multivalent hybrid antibody (MHA) complexes with dual specificity were prepared by combining two antibodies of different specificities, one against ferritin (Fer) the other against horseradish peroxidase (HRP), with protein A of Staphylococcus aaureus (SpA).Electron microscopy of mouse spleen lymphocytes and thymocytes (previously coated with mouse IgG anti-Thy-1 antibody) treated with IgG anti-Fer/SpA/IgG anti-mouse Ig complex and Fer gave better resolution and higher accuracy than previously obtained with IgG anti-HRP/SpA/IgG anti-mouse Ig complex and HRP (Mandache et al., 1980). Surface Thy-1 alloantigen and Fc receptor (charged with human IgG) treated with a mixture of IgG anti-Fer/SpA/IgG anti-Thy-1 and IgG anti-HRP/SpA/IgG anti-human Fab could be simultaneously detected on the thymocyte surface by either light or electron microscopy using Fer and HRP. The concomitant visualization of Thy-1 alloantigen (with Fer) and FcR (with HRP) on mouse thymocyte clearly shows that their distribution is largely independent and that the amount of Thy-1 antigen is greater.These results show that electron microscopy with a mixture of MHA is a useful technique for simultaneous location of two antigenic markers on the cell surface.     

Many cells in rat bone marrow have cell-surface Thy-1 antigen.

Thy-1.1 and Thy-1 xenoantigenic determinants were detected at the cell surface of many rat bone marrow cells. The absorptive capacity of bone marrow cells was 6-10% of that of thymocytes for Thy-1 antigenic determinants, and 30-45% of rat bone marrow cells were specifically labeled with anti-Thy-1 antibody as detected by autoradiography. Thus, while mice and rats are similar in having large amounts of Thy-1 in brain and thymocytes, they differ in that the rat lacks the antigen in most peripheral T cells and expresses it in a large number of bone marrow cells; the opposite is true in the mouse.     

Spontaneous clustering of Thy-1 antigens on CD4+ CD8+ thymocytes lacking TCR engagement by MHC/peptide complexes

While much is known concerning CD4+ CD8+ thymocytes positively or negatively selected through interaction of their TCR with self peptides bound to self-MHC molecules, little is known of the majority of CD4+ CD8+ thymocytes lacking this interaction. We developed a monoclonal antibody (mAb) 1D11, the ligand of which (1D11-L) is expressed on 60-70% of CD4+ CD8+ thymocytes but not on other subsets of thymocytes and peripheral T cells. 1D11-L expression on CD4+ CD8+ thymocytes reversely correlates with their TCR engagement, in vitro and in vivo. In addition, 1D11-L+ CD4+ CD8+ thymocytes were more susceptible than 1D11-L- CD4+ CD8- thymocytes to apoptosis. We also found that T lineage cells other than CD4+ CD8+ thymocytes and a Thy-1-expressing fibroblast cell line became positive for 1D11-L by cross-linking their Thy-1 antigens with anti-Thy-1 mAb but not with their Fab fragment, suggesting that 1D11 recognizes multimerized Thy-1 antigens. Confocal laser scanning microscopy revealed that Thy-1 antigens as well as 1D11-L are clustered on some CD4+ CD8+ thymocytes but not on the other subsets of thymocytes. Taken together, we suggest that clustering of Thy-1 antigens spontaneously and specifically occurs on CD4+ CD8+ thymocytes lacking TCR engagement by MHC/peptide complexes.     

Dissociation of signal transduction via Thy-1 and CD3 antigens in murine T cells

To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1+ CD3-TCR- cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1+ CD3+ cells from normal thymus or spleen did show a response to them. Since Thy-1-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and anti-CD3-epsilon (2C11). Under the presence of PMA or IL-2, accessory cell-depleted splenic T cells were cultured with G7 or 2C11. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2C11. IL-2, however, showed reciprocal effect on the proliferation of G7 and 2C11-treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-epsilon may provide a distinct intracellular pathway for T cell activation.     

Busulfan and chloramphenicol induced T cell lymphoma: cell surface characteristics and functional properties.

We report the immunological studies on three transplantable lymphoma lines that developed when CAF1 mice were injected with busulfan and chloramphenicol. The lymphoma cells displayed Thy-1.2, brain associated antigen, and H-2d alloantigen. They were negative for surface IgM and Ia antigens. Expression of T cell differentiation antigens differed among the three lines. The 508 tumour line displayed only Thy-1.2: 408 tumour line displayed Thy-1.2, Lyt-2.2 and TL; and 808 tumour line was positive for Thy-1.2, Lyt-1.2, Lyt-2.2 and TL antigens. We established in vitro culture lines from 508 and 808 lymphoma cells. The lymphoma cells did not respond to mitogens and antigens. The splenic cells from mice bearing 508 or 808 had decreased phytohaemagglutinin (PHA), concanavalin A (Con A) and mixed leucocyte responses (MLR). When mitomycin-C treated lymphoma cells from the tumour bearing mice were cocultured with normal splenic mononuclear cells, the 808 lymphoma cells suppressed the mitogenic responses of the normal cells more profoundly than 508 lymphoma cells. Adherent cells from both tumours suppressed the Con A responses of normal spleen cells. Cells from in vitro 508 or 808 cell lines had no effect on mitogenic responses of normal cells. Plasma from tumour bearing mice, but not the supernatants taken from cultures of these lymphoma cells, suppressed the mitogenic responses of normal lymphocytes. Spleen cells from normal CAF1 mice responded in mixed leucocyte tumour reactions (MLTR) when cocultured with lymphoma cells. Mice immunized with mitomycin-C treated tumour cells had greater response. Responder cells taken from mice with established 508 or 808 tumors had suppressed MLTR responses. Although prior immunization with tumor antigen increased the MLTR response, injection of live tumour cells into immunized mice resulted in a more rapid tumour growth and suppression of MLTR response.     

Evidence for the presence of Thy-1 on cultured thymic epithelial cells of mice and rats.

Thymic tissue of C57BL/6 mice and DA rats was cultured. After 6--8 weeks, cultures were analyzed for their capacity to absorb anti-Thy-1 serum, and for the expression of Thy-1 on the surface of different cell types by means of the indirect peroxidase labeling method. Three main cell types were identified: epithelium-like cells, fibrocyte-like cells and macrophages, but no lymphocytes were found. The presence of Thy-1 on cultured nonlymphocytic thymus-derived cells was demonstrated by their ability to absorb the cytotoxic activity of the appropriate anti-Thy-1 sera. Electron microscopical analyses of labeling experiments revealed that Thy-1 was predominantly expressed on epithelium-like cells with preference for their cell protrusions. The possible role of Thy-1 expression, both on thymocytes and thymus epithelium for cellular interaction, is discussed.